SARS-CoV-2 protein-derived peptides and vaccines containing them

Abstract

This invention provides an epitope peptide derived from SARS-CoV-2 protein that can induce cytotoxic T cells (CTLs) in the human body. [Solution] The present invention provides an epitope peptide derived from the SARS-CoV-2 protein having CTL-inducing ability. It also provides a polynucleotide encoding the peptide, an antigen-presenting cell that presents the peptide, a CTL that targets the peptide, and a method for inducing the antigen-presenting cell or the CTL. Furthermore, it provides a composition containing these as active ingredients. Furthermore, it provides a method for treating or preventing coronavirus infection and / or suppressing its severity using the peptide, polynucleotide, antigen-presenting cell, cytotoxic T cell, or the pharmaceutical composition of the present invention. It also provides a method for inducing an immune response to coronavirus infection. Furthermore, it provides a method for investigating a history of coronavirus infection by detecting the target TCR sequence.

Description

[Technical Field]

[0001] This invention relates to the field of biological science, more specifically to the field of viral infection prevention. In particular, this invention relates to a novel peptide effective as an infection prevention vaccine, a method for either or both the prevention and / or treatment of an infection using the peptide, and a pharmaceutical composition containing the peptide. This application claims the benefits of Japanese Patent Application No. 2020-164630 filed on 30 September 2020, International Application PCT / JP2021 / 017159 filed on 30 April 2021, and U.S. Provisional Application US63 / 236,927 filed on 25 August 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Coronavirus Disease 2019 (COVID-19), a respiratory infection caused by infection with the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2: SARS-CoV-2), has spread worldwide since being first reported in Wuhan, China. According to the World Health Organization (WHO), as of September 2020, the number of infected individuals exceeded 27 million, with 890,000 deaths. Furthermore, as of April 2021, the number of infected individuals exceeded 120 million, with 2.8 million deaths. Vaccination is considered an effective means of curbing the spread of infection, but there is currently no vaccine for COVID-19 that has been put into practical use.

[0003] It has long been known that the BCG vaccine is effective in preventing infectious diseases other than tuberculosis. Recent studies have also reported that BCG vaccination in elderly people aged 65 and over has shown an inhibitory effect on infectious diseases (Non-patent document 1: Giamarellos-Bourboulis EJ et al., Cell 2020, Online ahead of print). Studies suggest a link between BCG vaccination and the suppression of the mortality rate or the rate of increase in deaths in COVID-19 (Non-Patent Literature 2: Toyoshima Y et al., J Hum Genet 2020, Online ahead of print; Non-Patent Literature 3: Berg MK et al., Science Advances 2020, Online ahead of print). In the human defense mechanism against viral infection, an innate immune response, primarily involving immune cells (macrophages, NK cells, neutrophils, etc.), is the first to act, followed by an antigen-specific immune response by adaptive immunity (humoral and cellular immunity), primarily involving B cells and T cells. After BCG vaccination, the transcriptional activity of inflammatory cytokines in immune cells is promoted, and when infected with a virus in this trained immune state, inflammatory cytokines are rapidly released in the body. As a result, it is hypothesized that the humoral immune response by activated B cells and the cellular immune response by T cells suppress viral replication (Non-patent Literature 4: Netea MG et al., Cell 2020, 181(5):969-977).

[0004] The spike protein plays a central role in the entry of SARS-CoV-2 into human cells (Non-patent Literature 5: Hoffmann M et al., Cell 2020, 181(2):271-280). Many of the COVID-19 vaccines currently under research and development aim primarily to induce neutralizing antibodies against the SARS-CoV-2 spike protein (induction of humoral immunity) (Non-patent Literature 6: Jeyanathan M et al., Nat Rev Immunol 2020, Online ahead of print). However, mutations in the viral genome can occur as SARS-CoV-2 replicates. If a mutation occurs in the gene encoding the spike protein, the neutralizing antibodies induced by the vaccine may become ineffective. Furthermore, studies of COVID-19 survivors suggest that the survival time of neutralizing antibodies induced in the human body may be short (Non-patent Literature 7: Ibarrondo FJ et al., N Engl J Med 2020, 383(11):1085-1087; Non-patent Literature 8: Long QX et al., Nat Med 2020, 26(8):1200-1204). Therefore, there are concerns that challenges remain in vaccine development aimed at inducing humoral immunity through neutralizing antibodies.

[0005] Cellular immunity mediated by T cells is also extremely important as a defense response against infectious diseases. Cytotoxic T lymphocytes (CTLs) are CD8-positive T cells that are induced by the presentation of viral antigens (epitope peptides derived from viral proteins) by dendritic cells (DCs). Subsequently, they recognize the epitope peptides presented on human leukocyte antigen (HLA) class I molecules expressed on the surface of virus-infected cells and kill the virus-infected cells. This action destroys the infected cells, which are the replication sites for viral particles, and as a result, viral proliferation is suppressed. Compared to neutralizing antibodies, which mainly inhibit the infection of new cells by replicated viral particles, the action of CTLs, which target the replication process of viral particles itself, can be said to play a more direct role in preventing viral proliferation. Therefore, CTLs are thought to play a role in eliminating viruses from the body and suppressing the severity of infectious diseases. Reports indicating a significant decrease in CD8-positive T cells in the peripheral blood of severely ill COVID-19 patients compared to healthy individuals suggest that CTLs are an important factor in suppressing the severity of COVID-19 (Non-patent document 9: Zheng M et al., Cell Mol Immunol 2020, 17(5):533-535). Furthermore, some CTLs are known to remain in the body for extended periods as memory T cells. In 2003, the SARS-CoV epidemic resulted in over 8,000 infections. Seventeen years later, SARS-CoV-resistant CTLs were still detected in the blood of recovered patients (Non-patent Literature 10: Le Bert N et al., Nature 2020, 584(7821):457-462), suggesting that SARS-CoV-2-resistant CTLs may also remain in the body for extended periods after induction. Therefore, vaccines aimed at inducing cellular immunity could be an effective preventive measure against COVID-19. It is thought that CTLs induced by vaccines consisting of epitope peptides derived from SARS-CoV-2 protein remain in the body as memory T cells and rapidly exhibit toxic activity against virus-infected cells after SARS-CoV-2 infection, which could lead to the suppression of severe COVID-19. Therefore, the identification of SARS-CoV-2 protein-derived epitope peptides that can induce CTLs in the human body is desirable.

[0006] While candidate epitope peptides determined based on bioinformatics have been reported, verification of whether they actually bind to HLA and possess CTL-inducing ability has not been conducted, and the epitope peptides have not yet been identified. (Non-patent document 11: Grifoni A et al., Cell Host Microbe 2020, 27(4):671-680; Non-patent document 12: Crooke SN et al., Sci Rep 2020, 10(1):14179).

[0007] Furthermore, accurately understanding each individual's infection history is crucial for implementing measures to prevent the spread of SARS-CoV-2 infection. Currently, antibody testing is the mainstream method for determining infection history, but antibody titers can be very low in asymptomatic individuals or those exhibiting only mild symptoms (Non-patent Literature 13: Marchi S et al., PLoS One 2021, 16(7): e0253977; Non-patent Literature 8: Long QX et al., Nat Med 2020, 26(8): 1200-1204). This fact may impose limitations on antibody testing, and new testing methods are desired. In addition to humoral immunity centered on antibodies, cellular immunity centered on T cells also plays an important role in controlling viral infection. Cytotoxic T cells (CTLs) recognize viral protein-derived peptides presented on major histocompatibility complex (MHC) class I molecules on the surface of virus-infected cells via T cell receptors (TCRs), and then damage the virus-infected cells. The T-cell response to viral infection is thought to manifest as a significant increase or decrease in specific TCRs in a comprehensive analysis of T-cell-derived TCRs in peripheral blood. TCRs derived from SARS-CoV-2-specific CTLs may serve as important markers for understanding SARS-CoV-2 infection. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Giamarellos-Bourboulis EJ et al., Cell 2020, Online ahead of print (Volume 183, Issue 2, 2020, Pages 315-323.e9) [Non-Patent Document 2] Toyoshima Y et al., J Hum Genet 2020, Online ahead of print (65, pages 1075-1082, 2020) [Non-Patent Document 3] Berg MK et al., Science Advances 2020, Online ahead of print (Vol 6, No. 32, : eabc1463, 2020) [Non-Patent Document 4] Netea MG et al., Cell 2020, 181(5):969-977 [Non-Patent Document 5] Hoffmann M et al., Cell 2020, 181(2):271-280 [Non-Patent Document 6] Jeyanathan M et al., Nat Rev Immunol 2020, Online ahead of print (20, 615-632, 2020) [Non-Patent Document 7] Ibarrondo FJ et al., N Engl J Med 2020, 383(11):1085-1087 [Non-Patent Document 8] Long QX et al., Nat Med 2020, 26(8):1200-1204 [Non-Patent Document 9] Zheng M et al., Cell Mol Immunol 2020, 17(5):533-535 [Non-Patent Document 10] Le Bert N et al., Nature 2020, 584(7821):457-462 [Non-Patent Document 11] Grifoni A et al., Cell Host Microbe 2020, 27(4):671-680 [Non-Patent Document 12] Crooke SN et al., Sci Rep 2020, 10(1):14179 [Non-Patent Document 13] Marchi S et al., PLoS One 2021, 16(7): e0253977 [Summary of the Invention]

[0009] This invention relates to HLA-A peptides selected from among the four structural proteins and six non-structural proteins of SARS-CoV-2 (reference sequence: GenBank accession number MN908947 (SEQ ID NO: 16)). * 24:02 or HLA-A * The objective is to provide an epitope peptide that binds to 02:01 and has CTL-inducing ability. The four structural proteins refer specifically to the following proteins: spike protein (Reference array: GenBank accession number QHD43416 (Sequence ID: 17)); Envelope protein (Reference array: GenBank accession number QHD43418 (Sequence ID: 18)); Matrix protein (Reference array: GenBank accession number QHD43419 (Sequence ID: 19)); and nuclear protein (Reference array: GenBank accession number QHD43423 (Sequence ID: 20) On the other hand, the six non-structural proteins specifically refer to the following proteins: ORF1ab (Reference array: GenBank accession number QHD43415 (Sequence ID: 21)); ORF3a (Reference array: GenBank accession number QHD43417 (Sequence ID: 22)); ORF6 (Reference array: GenBank accession number QHD43420 (Sequence ID: 23)); ORF7a (Reference array: GenBank accession number QHD43421 (Sequence ID: 24)); ORF8 (Reference array: GenBank accession number QHD43422 (Sequence ID: 25)); and ORF10 (Reference array: GenBank accession number QHI42199 (Sequence ID: 26)) The epitope peptide of the present invention is HLA-A, which is prevalent in Japan and other Asian countries. * 24:02 Positive cases or HLA-A, which is common in Western countries * In individuals who tested positive for 02:01 (Cao K et al., Hum Immunol 2001, 62(9): 1009-1030; Gonzalez-Galarza FF et al., Nucleic Acids Res 2020, 48(D1):D783-D788), it was demonstrated that a specific and robust immune response to COVID-19 could be induced. Because the amino acid sequence of the epitope peptide provided in this invention has low similarity to the amino acid sequence derived from human proteins, it is expected that a highly safe vaccine that is less likely to cause unexpected side effects can be created. On the other hand, since this amino acid sequence is commonly found in SARS-CoV and MERS-CoV, which have been prevalent in the past, it may also be found in new coronavirus proteins that may emerge in the future. Therefore, a vaccine consisting of this epitope peptide may be effective not only against the current SARS-CoV-2 but also against coronavirus infections that may become prevalent in the future.

[0010] The present invention also provides compositions comprising one or more peptides of the present invention, one or more polynucleotides encoding one or more peptides of the present invention, APCs of the present invention, exosomes presenting the peptides of the present invention, and / or CTLs of the present invention. The compositions of the present invention are preferably pharmaceutical compositions. Pharmaceutical compositions of the present invention can be used for the treatment and / or prevention of coronavirus infection, and for suppression of severe disease progression. They can also be used to induce an immune response to coronavirus infection. When administered to a subject, the peptides of the present invention are presented on the surface of APCs, thereby inducing CTLs that target the peptides. Therefore, it is a further object of the present invention to provide a composition that induces CTLs, comprising one or more peptides of the present invention, one or more polynucleotides encoding one or more peptides of the present invention, APCs of the present invention, and / or exosomes presenting the peptides of the present invention.

[0011] A further object of the present invention is to provide a method for inducing an APC having CTL-inducing ability, comprising the steps of contacting the APC with one or more types of peptides of the present invention, or introducing a polynucleotide encoding any one of the peptides of the present invention into the APC.

[0012] The present invention also provides a method for inducing CTLs, comprising the steps of: co-culturing CD8-positive T cells with an APC that presents a complex of an HLA antigen and the peptide of the present invention on its surface; co-culturing CD8-positive T cells with an exosome that presents a complex of an HLA antigen and the peptide of the present invention on its surface; or introducing a vector containing polynucleotides encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of the present invention presented on the cell surface by an HLA antigen into CD8-positive T cells.

[0013] Another object of the present invention is to provide isolated APCs that present a complex of an HLA antigen and the peptide of the present invention on their surface. The present invention further provides isolated CTLs that target the peptide of the present invention. These APCs and CTLs can be used in immunotherapy for coronavirus infection.

[0014] Another object of the present invention is to provide a method for inducing an immune response to coronavirus infection in a subject, comprising the step of administering to the subject the peptide of the present invention, a polynucleotide encoding the peptide, an APC of the present invention, an exosome presenting the peptide of the present invention, and / or a CTL of the present invention. Furthermore, another object of the present invention is to provide a method for treating and / or preventing coronavirus infection and suppressing its severity in a subject, comprising the step of administering to the subject the peptide of the present invention, a polynucleotide encoding the peptide, an APC of the present invention, an exosome presenting the peptide of the present invention, and / or a CTL of the present invention.

[0015] This invention also relates to the TCR expressed by T cells that recognize SARS-CoV-2 protein-derived peptides. The TCR is a protein molecule consisting of an α-chain and a β-chain dimer. Human T cells recognize peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules through the TCR. As a result, T cell proliferation, differentiation, cytokine production, or secretion of cytotoxic substances (perforin and granzyme) are induced. The TCRα gene includes the Vα, Jα, and Cα genes. The TCRβ gene includes the Vβ, Dβ, Jβ, and Cβ genes. The region that determines the specificity of the TCR to peptides is called the Complementarity Determining Region (CDR), and there are CDR1, CDR2, and CDR3. In particular, CDR3 is in direct contact with the peptide, so its amino acid sequence is extremely important in determining specificity. In the TCRα chain, the CDR3 is between V and J, and in the TCRβ chain, it is between V, D, and DJ, and diversity arises from base insertions and deletions. SARS-CoV-2 infection or vaccination may selectively increase T cells that recognize SARS-CoV-2 protein-derived peptides. For the purpose of investigating such immune responses in the form of changes in the detection frequency of specific TCRs in peripheral blood, the amino acid sequences of TCRs identified from T cells that recognize SARS-CoV-2 protein-derived peptides as shown herein (particularly the amino acid sequence in CDR3) are of great value. In other words, the present invention aims to provide a method for determining a target immune response to SARS-CoV-2, comprising the step of determining the detection frequency of TCRs in the target peripheral blood.

[0016] In addition to the foregoing, other objects and features of the present invention will become more fully apparent by reading the following detailed description in conjunction with the accompanying figures and examples. However, it should be understood that both the above summary of the invention and the following detailed description are illustrative embodiments and do not limit the present invention or other alternative embodiments thereof. In particular, the present invention will be described herein with reference to several specific embodiments, but it should be understood that these descriptions are illustrative and not intended to limit the present invention. Various modifications and applications can be conceived by those skilled in the art without departing from the spirit and scope of the invention as described by the accompanying claims. Similarly, other objects, features, benefits, and advantages of the present invention will become apparent from this summary and the specific embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits, and advantages will become apparent from the above in conjunction with the accompanying examples, data, figures, and any reasonable inferences drawn therefrom, either alone or in consideration of the references incorporated herein. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 consists of images showing the results of an IFN-γ enzyme-coupled immunosorbent spot (ELISPOT) assay performed using cells induced with peptides derived from SARS-CoV-2 protein. In the figure, "(+)" indicates IFN-γ production in HLA-A*24:02-expressing target cells (TISI cells) pulsed with the target peptide, and "(-)" indicates IFN-γ production in TISI cells that were not pulsed with any peptide (negative control). Comparison with the negative control shows that peptide-specific IFN-γ production was observed for peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), 11 (SEQ ID NO: 11), 12 (SEQ ID NO: 12), 13 (SEQ ID NO: 13), and 15 (SEQ ID NO: 15) (Figure 1a). On the other hand, peptide 6 (SEQ ID NO: 6) is shown as a typical example of negative data in which peptide-specific IFN-γ production was not observed (Figure 1b).

[0018] [Figure 2]Figure 2 is a line graph showing the results of enzyme-linked immunosorbent assay (ELISA) measurements of IFN-γ produced from cells stimulated with peptide 1 (SEQ ID NO: 1), peptide 2 (SEQ ID NO: 2), peptide 4 (SEQ ID NO: 4), peptide 5 (SEQ ID NO: 5), peptide 7 (SEQ ID NO: 7), peptide 9 (SEQ ID NO: 9), peptide 10 (SEQ ID NO: 10), or peptide 13 (SEQ ID NO: 13). These results indicate the establishment of HLA-A*24:02-restricted CTL lines that produce IFN-γ specifically after peptide induction. In the figure, "(+)" indicates IFN-γ production by the CTL line against HLA-A*24:02-expressing target cells (TISI cells) pulsed with the target peptide, and "(-)" indicates IFN-γ production by the CTL line against TISI cells that were not pulsed with any peptide. The R / S ratio represents the ratio of the number of responder cells (CTLs) to the number of target cells (stimulators) that stimulate them.

[0019] [Figure 3] Figure 3 consists of images showing the results of an IFN-γ enzyme-coupled immunosorbent spot (ELISPOT) assay performed using cells induced with peptides derived from SARS-CoV-2 protein. In the figure, "(+)" indicates IFN-γ production in HLA-A*02:01-expressing target cells (T2 cells) pulsed with the target peptide, and "(-)" indicates IFN-γ production in T2 cells that were not pulsed with any peptide (negative control). Comparison with the negative control shows that peptide-specific IFN-γ production was observed for peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 4 (SEQ ID NO: 4), 7 (SEQ ID NO: 7), 10 (SEQ ID NO: 10), 12 (SEQ ID NO: 12), and 13 (SEQ ID NO: 13) (Figure 3a). On the other hand, peptide 5 (SEQ ID NO: 5) is shown as a typical example of negative data where peptide-specific IFN-γ production was not observed (Figure 3b).

[0020] [Figure 4]Figure 4 is a line graph showing the results of measuring IFN-γ produced by cells stimulated with peptide 1 (SEQ ID NO: 1), peptide 2 (SEQ ID NO: 2), peptide 10 (SEQ ID NO: 10), or peptide 13 (SEQ ID NO: 13) using enzyme-linked immunosorbent assay (ELISA). These results indicate the establishment of HLA-A*02:01-restricted CTL lines that produce IFN-γ specifically after peptide induction. In the figure, "(+)" indicates IFN-γ production by the CTL line against HLA-A*02:01-expressing target cells (T2 cells) pulsed with the target peptide, and "(-)" indicates IFN-γ production by the CTL line against T2 cells that were not pulsed with any peptide. The R / S ratio represents the ratio of the number of responder cells (CTL line cells) to the number of stimulator cells that stimulate them.

[0021] [Figure 5] Figure 5 is a line graph showing IFN-γ production of SARS-CoV-2 protein-derived peptide-specific CTL clones established by limiting dilution from PBMCs after in vitro CTL induction. IFN-γ production was observed in CTL clones in response to target cells (+) pulsed with SARS-CoV-2 protein-derived peptide, while no significant IFN-γ production was observed in CTL clones in response to target cells (-) not pulsed with the peptide. This confirms that the CTL clones recognized the peptide derived from the SARS-CoV-2 protein presented in HLA. The R / S ratio represents the ratio of the number of responder cells (CTL clones) to the number of target cells (stimulator cells) that stimulate them.

[0022] [Figure 6-1]Figure 6 consists of (a) and (b) the results of tetramer assays performed on privately-derived microcells (PBMCs) collected from COVID-19 recovered individuals or SARS-CoV-2 uninfected individuals. A tetramer-positive CD8-positive T cell population that recognizes a SARS-CoV-2 protein-derived peptide presented on HLA-A*24:02 was detected from PBMCs derived from COVID-19 recovered individuals (Figure 6a). A tetramer-positive CD8-positive T cell population was also detected from PBMCs derived from SARS-CoV-2 uninfected individuals (Figure 6b). [Figure 6-2] The continuation of Figure 6-1 is shown below. [Modes for carrying out the invention]

[0023] In carrying out or testing aspects of the present invention, any methods and materials similar to or equivalent to those described herein may be used, but preferred methods, apparatus, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited thereto, as the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc., described herein can be modified according to conventional experimental methods and optimizations. It should also be understood that the technical terms used herein are for the purpose of describing only specific types or aspects and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0024] I. Definition In this specification, the words “a,” “an,” and “the” mean “at least one” unless otherwise specified. The terms “isolated” and “purified” used in reference to substances (e.g., peptides, antibodies, polynucleotides, etc.) indicate that the substance substantially does not contain at least one substance that might otherwise be present in its natural source. Therefore, an isolated or purified peptide refers to a peptide that substantially does not contain other cellular material from the cell or tissue source from which the peptide originates, such as carbohydrates, lipids, and other contaminating proteins. Furthermore, if the peptide is chemically synthesized, an isolated or purified peptide refers to a peptide that substantially does not contain precursor substances or other chemicals. The term “substantially cellular-free” includes peptide preparations from which the peptide has been isolated from cellular components of the isolated cell or recombinantly produced cell. Therefore, a substantially cellular-free peptide includes peptide preparations containing less than approximately 30%, 20%, 10%, or 5%, 3%, 2%, or 1% (on a dry weight basis) of other cellular material. When peptides are recombinantly produced, the isolated or purified peptides are substantially free of culture medium, and peptides substantially free of culture medium include peptide preparations containing less than about 20%, 10%, or 5%, 3%, 2%, or 1% (on a dry weight basis) of the volume of the peptide preparation. When peptides are chemically synthesized, the isolated or purified peptides are substantially free of precursor substances and other chemicals, and peptides substantially free of precursor substances and other chemicals include peptide preparations containing less than about 30%, 20%, 10%, 5%, 3%, 2%, or 1% (on a dry weight basis) of the volume of the peptide preparation. The fact that a particular peptide preparation is an isolated or purified peptide can be confirmed, for example, by the appearance of a single band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining of the gel. In preferred embodiments, the peptides and polynucleotides of the present invention are isolated or purified.

[0025] The terms “polypeptide,” “peptide,” “protein,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms apply to both natural amino acid polymers and unnatural amino acid polymers containing one or more unnatural amino acid residues. Unnatural amino acids include amino acid analogs and amino acid mimes.

[0026] As used herein, the term "amino acid" refers to natural amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids are amino acids encoded by the genetic code, and amino acids modified post-translation within cells (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). The term "amino acid analog" refers to compounds that have the same basic chemical structure as natural amino acids (hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group), but have a modified R group or a modified skeleton (e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium). The term "amino acid mime" refers to compounds that have a different structure from common amino acids but have similar functions. The amino acids may be either L-amino acids or D-amino acids, but the peptides of the present invention are preferably polymers of L-amino acids.

[0027] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides.

[0028] As used herein, the term “composition” is intended to encompass products containing specific amounts of specific components, and any products arising directly or indirectly from specific amounts of specific components in combination. Where the composition is a pharmaceutical composition, the term “composition” is intended to encompass products containing active and inactive components, and any products arising directly or indirectly from any combination, complex formation, or aggregation of any two or more components, from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Accordingly, the pharmaceutical compositions of the present invention encompass any compositions prepared by mixing the compounds or cells of the present invention with a pharmaceutically or physiologically acceptable carrier. As used herein, the terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” mean a pharmaceutically or physiologically acceptable material, composition, substance, or medium, including but not limited to liquid or solid fillers, diluents, excipients, solvents, and encapsulating materials.

[0029] Unless otherwise specified, the term "viral infection" refers to coronavirus infection, and examples of coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. In an exemplary manner, "coronavirus infection" refers to SARS-CoV-2 infection in HLA-A24 or HLA-A02 positive subjects.

[0030] Unless otherwise specified, the terms “cytotoxic T lymphocyte,” “cytotoxic T cell,” and “CTL” are used interchangeably herein and, unless otherwise defined, refer to a subgroup of T lymphocytes capable of recognizing non-self cells (e.g., tumor / cancer cells, virus-infected cells) and inducing the death of such cells.

[0031] Unless otherwise specified, the term "HLA-A24" refers to HLA-A * 24:01, HLA-A * 24:02, HLA-A * 24:03, HLA-A* 24:04, HLA-A * 24:07, HLA-A * 24:08, HLA-A * 24:20, HLA-A * 24:25, HLA-A * Refers to HLA-A24 type including subtypes such as 24:88.

[0032] Unless otherwise specified, the term "HLA-A02 (or HLA-A2)" refers to HLA-A * 02:01, HLA-A * 02:02, A * 02:03, A * 02:04, A * 02:05, A * 02:06, A * 02:07, A * 02:10, A * 02:11, A * 02:13, A * 02:16, A * 02:18, A * 02:19, A * 02:28, and A * Refers to HLA-A02 type including subtypes such as 02:50.

[0033] Unless otherwise specified, the term "coronavirus protein" refers to a protein consisting of the full-length amino acid sequence of each protein encoded by the genomic sequence of the coronavirus. Preferred examples include the structural proteins and non-structural proteins of SARS-CoV-2. Examples of coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. The genomic base sequences (reference sequences) of each of these coronaviruses and the corresponding amino acid sequences of the coronavirus proteins encoded thereby can be obtained, for example, using the following Genbank accession numbers: SARS-CoV-2: MN908947 MERS-CoV: JX869059 SARS-CoV: Tor2: AY274119, BJ01: AY278488, or GZ02: AY390556

[0034] Unless otherwise specified, the term "SARS-CoV-2 protein" refers to the protein consisting of the full-length amino acid sequence of each protein encoded by the SARS-CoV-2 genome sequence, and includes the four structural proteins and six non-structural proteins of SARS-CoV-2. The four structural sequences and six non-structural proteins are summarized in Table 1. Note that in the table, for the non-structural protein orf1ab, two ORFs in the reference sequence, 266..13468 and 13468..21555, are joined together to form a single ORF through ribosomal frameshift.

[0035] [Table 1]

[0036] Unless otherwise specified, the terms “coronavirus-infected cell,” “virus-infected cell,” and “infected cell” are used interchangeably herein and refer to cells infected with coronavirus unless otherwise specified. Examples of coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0037] In this specification, the term “infection” includes the invasion and / or replication of a virus in a cell or body tissue, and the pathological conditions resulting from the invasion and replication of the virus. The invasion and replication stages of the viral life cycle include, but are not limited to, the binding of a viral particle to a cell, the introduction of viral genetic information into the cell, the expression of viral proteins, the production of new viral particles, and the release of viral particles from the cell.

[0038] In relation to a subject or patient, the expression "the subject's (or patient's) HLA antigen is HLA-A24 or HLA-A02" as used herein means that the subject or patient possesses the HLA-A24 or HLA-A02 antigen gene as a major histocompatibility complex (MHC) class I molecule in a homozygous or heterozygous manner, and that the HLA-A24 or HLA-A02 antigen is expressed as an HLA antigen in the subject's or patient's cells.

[0039] To the extent that the methods and compositions of the present invention are useful in relation to the "treatment" of coronavirus infection, the treatment is considered "effective" if it brings about clinical benefits, such as alleviation of clinical symptoms of coronavirus infection in the subject, or suppression of the progression to severe illness. The main symptoms of coronavirus infection are generally known to include fever, cough, chills, rigidity, and muscle pain. The symptoms of many infected patients are mild and are said to recover in about 1-2 weeks. However, some patients experience respiratory distress, and may be accompanied by significant dyspnea, hypoxia, and even acute respiratory distress syndrome (ARDS). Recent reports also indicate that, particularly in patients infected with SARS-CoV-2, unique symptoms such as abnormalities in smell and taste have been observed in relatively mild cases. Alternatively, thrombosis and cytokine storms in severely ill patients are known to contribute to the progression of the disease. When the treatment is applied preventively, "effective" means that the treatment delays or prevents the onset of coronavirus infection, or prevents or alleviates the clinical symptoms of coronavirus infection. Effectiveness is determined in relation to any known method for diagnosing or treating coronavirus infection. For example, if any of the specific symptoms mentioned above are suppressed, it can be said that the treatment or prevention is effective.

[0040] To the extent that the methods and compositions of the present invention are useful in connection with the “prevention” of coronavirus infection, the term “prevention” herein includes any action that reduces the burden of mortality or morbidity due to the disease. Prevention may be carried out at “primary, secondary, and tertiary levels of prevention.” Primary prevention aims to avoid the onset of the disease, while secondary and tertiary levels of prevention include actions aimed at reducing the adverse effects of a pre-existing disease by preventing disease progression and the appearance of symptoms, as well as by restoring function and reducing disease-related complications. Alternatively, prevention may include a wide range of prophylactic treatments aimed at mitigating the severity of a particular disorder, such as reducing clinical symptoms such as fever, shortness of breath, cough, and upper respiratory tract infections.

[0041] In connection with the present invention, the treatment and / or prevention of coronavirus infection, and / or the suppression of severe disease, includes delaying the onset of at least one symptom of coronavirus infection, or improving them. It also includes inhibiting viral replication in coronavirus-infected cells. Effective treatment and / or prevention of coronavirus infection reduces mortality, improves the prognosis of individuals infected with coronavirus infection, and alleviates the symptoms associated with coronavirus infection. For example, symptom reduction or improvement constitutes effective treatment and / or prevention and includes a 10%, 20%, 30%, or greater reduction or a state of symptom stabilization.

[0042] In this invention, inhibition of viral replication includes inhibition of the viral replication process within infected cells. When CTLs induced by the peptide of this invention present antigens to the viral proteins expressed during the viral replication process in virus-infected cells, their cytotoxic activity damages the cellular functions of the infected cells (gene replication and protein translation). Alternatively, the CTLs destroy the infected cells themselves. As a result, the replication process of viral particles, which depends on the cellular functions of the infected cells, is inhibited, and viral replication is suppressed. In this invention, suppression of severe illness means suppression of the worsening or progression of any symptoms associated with coronavirus infection. Specifically, the progression of respiratory distress symptoms requiring interventional treatment such as oxygen inhalation or the use of a ventilator or extracorporeal membrane oxygenation (ECMO) is a typical example of severe illness in coronavirus infection. Therefore, preventing, avoiding, or mitigating the progression (worsening) of these symptoms is included in the suppression of severe illness in this invention.

[0043] In relation to the present invention, the term “antibody” refers to an immunoglobulin and its fragments that specifically react with a designated protein or its peptide. Antibodies may include human antibodies, primate antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabeled molecules, and antibody fragments. Furthermore, “antibody” is used in a broad sense herein and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from two or more intact antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity. An “antibody” may be an antibody of any class (e.g., IgA, IgD, IgE, IgG, and IgM).

[0044] This invention relates to the TCR expressed by T cells that recognize SARS-CoV-2-derived peptides. The TCR is a protein molecule consisting of an α-chain and a β-chain dimer. Human T cells recognize peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules through the TCR. As a result, T cell proliferation, differentiation, cytokine production, or secretion of cytotoxic substances (perforin and granzyme) are induced. The TCR-α gene includes the Vα, Jα, and Cα genes. The TCR-β gene includes the Vβ, Dβ, Jβ, and Cβ genes. The region that determines the specificity of the TCR to peptides is called the Complementarity Determining Region (CDR), and there are CDR1, CDR2, and CDR3. In particular, CDR3 is in direct contact with the peptide, so its amino acid sequence is extremely important in determining the antigen recognition specificity of the TCR. In the TCR-α chain, the CDR3 is between V and J, and in the TCR-β chain, it is between V, D, and DJ, and diversity arises from base insertions and deletions. In this invention, TCR refers to a protein molecule expressed on T cells and consisting of an α-chain and a β-chain dimer. In this invention, T cells refer to human T cells, and may be human T cells that recognize SARS-CoV-2 derived peptides. Furthermore, in this invention, TCR refers to a molecule that is expressed on T cells and, through itself, recognizes peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules, thereby inducing T cell proliferation, differentiation, cytokine production, or secretion of cytotoxic substances (perforin and granzyme). The TCR-α gene includes the Vα, Jα, and Cα genes. The TCR-β gene includes the Vβ, Dβ, Jβ, and Cβ genes. The region that determines the specificity of the TCR to peptides is called the Complementarity Determining Region (CDR), and there are CDR1, CDR2, and CDR3. In particular, CDR3 is in direct contact with the peptide, so its amino acid sequence is extremely important in determining the antigen recognition specificity of the TCR. In the TCR-α chain, the CDR3 is between V and J, and in the TCR-β chain, it is between V, D, and DJ, and diversity arises from base insertions and deletions. In the present invention, the TCR may have a CDR sequence that is expressed on T cells and specifically recognizes SARS-CoV-2-derived peptides.

[0045] In this invention, "history of coronavirus infection" refers to the fact that the subject has been infected with a coronavirus, particularly SARS-CoV-2, in the past. Even if the subject was infected in the past but was asymptomatic, the past infection can be confirmed by detecting the TCR of this invention. Furthermore, a subject who is currently infected means that the infection was established previously. Therefore, a subject who is currently infected has a history of coronavirus infection. COVID-19 refers to "novel coronavirus infection" confirmed by a physician's diagnosis.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this invention pertains.

[0047] II. Peptides HLA-A24 is a common allele in Asians, while HLA-A02 is a common allele in Caucasians (Sette A, Sidney J., Immunogenetics 1999, 50:201-12; Cao K et al., Hum Immunol 2001, 62(9):1009-1030; Gonzalez-Ga larza FF et al., Nucleic Acids Res 2020, 48(D1):D783-D788). Therefore, by providing a CTL-inducing peptide derived from SARS-CoV-2 protein that is constrained by HLA-A24 or HLA-A02, an effective treatment method for coronavirus infection can be provided to many Asians or Caucasians. Accordingly, the present invention provides a peptide derived from SARS-CoV-2 protein that can induce CTLs in an HLA-A24 or HLA-A02-restricted manner.

[0048] The peptides of the present invention are SARS-CoV-2 protein-derived peptides that can induce CTLs in an HLA-A24 or HLA-A02-restricted manner. Examples of peptides that can induce CTLs in an HLA-A24-restricted manner include peptides having amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15. Examples of peptides that can induce CTLs in an HLA-A02-restricted manner include peptides having amino acid sequences selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13.

[0049] In vitro stimulation of T cells with dendritic cells pulsed with these peptides can establish CTLs that possess specific cytotoxic activity for these peptides. The established CTLs exhibit specific cytotoxic activity against target cells pulsed with each peptide.

[0050] CTLs are induced by the presentation of coronavirus antigen (epitope peptide derived from coronavirus protein), and subsequently recognize the epitope peptide presented on human leukocyte antigen (HLA) class I molecules expressed on the surface of coronavirus-infected cells, thereby killing the virus-infected cells. Therefore, coronavirus antigen is an excellent target for immunotherapy. Accordingly, the peptides of the present invention can be suitably used for immunotherapy of coronavirus infection. Preferred peptides are nonapeptides (peptides consisting of 9 amino acid residues) or decapeptides (peptides consisting of 10 amino acid residues), with peptides consisting of amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15 being more preferred. For example, peptides having the amino acid sequences described in SEQ ID NOs: 1, 2, 4, 7, 10, 12, or 13 are suitable for inducing CTLs that exhibit specific cytotoxic activity against coronavirus-infected cells having HLA-A24 or HLA-A02, and can be suitably used for immunotherapy of coronavirus infection in HLA-A24 or HLA-A02 positive patients. In a more preferred embodiment, the peptide of the present invention is a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15 for HLA-A24 positive patients, and a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 for HLA-A02 positive patients.

[0051] The peptides of the present invention may have additional amino acid residues adjacent to the amino acid sequence of the peptide, as long as the resulting peptide retains the CTL-inducing ability of the original peptide. The additional amino acid residues may consist of any type of amino acid, as long as they do not impair the CTL-inducing ability of the original peptide. Therefore, the peptides of the present invention encompass peptides having CTL-inducing ability, comprising amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15. Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, and usually less than about 15 amino acids. Therefore, if the original peptide is a nonapeptide, the peptides of the present invention encompass peptides of 10 amino acid length, or 11-40 amino acid length, resulting from the addition of additional amino acids to the original peptide. Also, if the original peptide is a decapeptide, the peptides encompass peptides of 11-40 amino acid length. Such peptides can be, for example, 11-20 amino acid length or 11-15 amino acid length. Preferred examples of additional amino acid residues are those adjacent to the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of each protein encoded by the SARS-CoV-2 genome sequence (e.g., SEQ ID NOs: 17-26). Therefore, the peptide of the present invention includes peptide fragments of the SARS-CoV-2 protein having CTL-inducing ability, comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15.

[0052] Generally, modifications to one, two, or more amino acids in a peptide do not affect the function of that peptide, and in some cases may even enhance the desired function of the original peptide. In fact, modified peptides (i.e., peptides consisting of amino acid sequences in which one, two, or several amino acid residues have been modified (i.e., substituted, deleted, inserted, and / or added) compared to the original reference sequence) are known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Accordingly, in one embodiment, the peptide of the present invention may be a peptide having CTL-inducing activity, comprising an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted, and / or added to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15.

[0053] Those skilled in the art can recognize individual substitutions to an amino acid sequence that alter a single amino acid or a small proportion of amino acids, which tend to result in the preservation of the properties of the original amino acid side chains. Therefore, they are often referred to as “conservative substitutions” or “conservative modifications,” and modifications of proteins by “conservative substitutions” or “conservative modifications” can produce modified proteins with similar functions to the original protein. Tables of conservative substitutions listing functionally similar amino acids are well known in the art. Examples of functionally similar amino acid side chain properties include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains that share the following functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W). In addition, the following eight groups each include amino acids that are recognized in the art as having conserved substitutions with one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); and 8) Cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins 1984).

[0054] Such conserved modified peptides are also included in the peptides of the present invention. However, the peptides of the present invention are not limited to these, and may include non-conservative modifications as long as the modified peptide retains the CTL-inducing ability of the original peptide. Furthermore, modified peptides do not exclude CTL-inducing peptides derived from polymorphic variants, interspecific homologs, and alleles of the SARS-CoV-2 protein.

[0055] As long as the CTL-inducing ability of the original peptide is preserved, a small number (e.g., one, two, or several) or a small percentage of amino acids can be modified (i.e., substituted, deleted, inserted, and / or added). In this specification, the term “several” means five or fewer amino acids, e.g., four or three or fewer. The percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, or 1–5%.

[0056] When used in conjunction with immunotherapy, the peptide of the present invention should preferably be presented on the surface of cells or exosomes as a complex with an HLA antigen. Therefore, it is preferable that the peptide of the present invention has a high binding affinity to the HLA antigen. For this reason, the peptide may be modified by substitution, deletion, insertion and / or addition of amino acid residues to obtain a modified peptide with improved binding affinity. Since the regularity of the sequence of peptides presented by binding to HLA antigens is known (Falk, et al., Immunogenetics 1994 40 232-41; Chujoh, et al., Tissue Antigens 1998: 52: 501-9; Takiguchi, et al., Tissue Antigens 2000: 55: 296-302), modifications based on such regularity can be introduced into the peptide of the present invention.

[0057] For example, in peptides that bind to HLA Class I, the second amino acid from the N-terminus and the C-terminus are generally anchor residues involved in binding to HLA Class I (Rammensee HG, et al., Immunogenetics. 1995;41(4):178-228). For example, peptides with high HLA-A24 binding affinity tend to have the second amino acid from the N-terminus substituted with phenylalanine, tyrosine, methionine, or tryptophan. Similarly, peptides with the C-terminal amino acid substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine also tend to have high HLA-A24 binding affinity. Therefore, to increase HLA-A24 binding affinity, it may be desirable to substitute the second amino acid from the N-terminus with phenylalanine, tyrosine, methionine, or tryptophan, and / or substitute the C-terminal amino acid with phenylalanine, leucine, isoleucine, tryptophan, or methionine. Therefore, peptides having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15, in which the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, methionine, or tryptophan, and / or the C-terminus is substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine, are included in the present invention.

[0058] Similarly, the present invention includes peptides comprising amino acid sequences in SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 in which one, two, or several amino acids are substituted, deleted, inserted, and / or added, and which have one or both of the following characteristics: (a) the second amino acid from the N-terminus is phenylalanine, tyrosine, methionine, or tryptophan; and (b) the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, or methionine. In a preferred embodiment, the peptides of the present invention include amino acid sequences in SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15, comprising either or both of the substitution of the second amino acid from the N-terminus with phenylalanine, tyrosine, methionine, or tryptophan, and the substitution of the C-terminal amino acid with phenylalanine, leucine, isoleucine, tryptophan, or methionine.

[0059] Similarly, peptides possessing high HLA-A02 binding affinity tend to have a second amino acid from the N-terminus substituted with leucine or methionine and / or a C-terminal amino acid substituted with valine or leucine. Therefore, it may be desirable to substitute the second amino acid from the N-terminus with leucine or methionine and / or the C-terminal amino acid with valine or leucine to enhance HLA-A02 binding affinity. Accordingly, peptides having amino acid sequences selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, in which the second amino acid from the N-terminus is substituted with leucine or methionine and / or the C-terminus is substituted with valine or leucine, are included in the present invention.

[0060] Similarly, the present invention includes peptides comprising amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 in which one, two, or several amino acids are substituted, deleted, inserted, and / or added, and which have one or both of the following characteristics: (a) the second amino acid from the N-terminus is leucine or methionine, and (b) the C-terminal amino acid is valine or leucine. In a preferred embodiment, the peptides of the present invention include amino acid sequences in SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, which include either or both of the substitution of the second amino acid from the N-terminus with leucine or methionine, and the substitution of the C-terminal amino acid with valine or leucine.

[0061] Substitutions can be introduced not only at the terminal amino acids but also at the potential T cell receptor (TCR) recognition sites of peptides. Several studies have shown this, for example, with CAP1, p53. (264-272) Her-2 / neu (369-377) , or gp100 (209-217) Studies have demonstrated that peptides with amino acid substitutions may have activity equivalent to or even superior to that of the original peptide (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al. J Immunol. (2002) Feb 1;168(3):1338-47, SO Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206, and SO Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-14).

[0062] The present invention also intends to enable the addition of one, two, or several amino acids to the N-terminus and / or C-terminus of the peptides of the present invention (for example, peptides consisting of amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15). Such modified peptides that retain CTL-inducing ability are also included in the present invention. For example, a peptide to which one, two, or several amino acids have been added to the N-terminus and / or C-terminus of a peptide consisting of the amino acid sequence described in SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, or 15, upon contact with an APC, is taken up into the APC and processed to become a peptide consisting of the amino acid sequence described in SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, or 15. Subsequently, it can induce CTLs by being presented on the cell surface of the APC via the antigen presentation pathway.

[0063] However, if the amino acid sequence of a peptide is identical to a portion of the amino acid sequence of an endogenous or exogenous protein with a different function, side effects such as autoimmune disorders and / or allergic reactions to certain substances may be induced. Therefore, it is preferable to perform a homology search using available databases to avoid situations where the amino acid sequence of a peptide matches the amino acid sequence of another protein. If the homology search reveals that there are no peptides that differ from the target peptide by even one or two amino acids, the target peptide can be modified to increase its binding affinity to the HLA antigen and / or its CTL-inducing ability without the risk of such side effects.

[0064] Peptides in which one, two, or several amino acids of the peptide of the present invention have been modified are expected to retain the CTL-inducing ability of the original peptide, but it is preferable to confirm the CTL-inducing ability of the modified peptide. In this specification, "peptide having CTL-inducing ability" refers to a peptide in which CTLs are induced by APC stimulated by that peptide. "CTL induction" includes induction of differentiation into CTLs, induction of CTL activation, induction of CTL proliferation, induction of cytotoxic activity of CTLs, induction of lysis of target cells by CTLs, and induction of increased IFN-γ production by CTLs.

[0065] The ability to induce CTLs can be confirmed by inducing APCs (e.g., B lymphocytes, macrophages, and dendritic cells) that possess HLA antigens, stimulating them with peptides, mixing them with CD8-positive T cells, and then measuring the IFN-γ released by CTLs to target cells. Preferably, human peripheral blood mononuclear leukocyte-derived dendritic cells can be used as the APCs. Transgenic animals prepared to express HLA antigens can also be used as the reaction system. Alternatively, for example, target cells can be... 51 By radiolabeling with Cr or similar, the cytotoxic activity of peptide-induced CTLs can be calculated from the radioactivity released from target cells. Alternatively, the CTL-inducing ability can be evaluated by measuring the IFN-γ produced and released by CTLs in the presence of peptide-stimulated APCs and visualizing the inhibition zone on the culture medium using an anti-IFN-γ monoclonal antibody.

[0066] In addition to the modifications described above, the peptides of the present invention can also be linked to other peptides, as long as the resulting linked peptide retains its CTL-inducing ability. Examples of suitable peptides to be linked with the peptides of the present invention include other CTL-inducing peptides derived from coronavirus proteins. Furthermore, the peptides of the present invention can also be linked to each other. Suitable linkers that can be used for linking peptides are known in the art, and linkers such as AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO: 27) (RPM Sutmuller et al., J Immunol. 2000, 165: 7308-15), or K (S. Ota et al., Can Res. 62, 1471-6, KS Kawamura et al., J Immunol. 2002, 168: 5709-15) can be used. The peptides can be linked in various configurations (e.g., chain-like, overlapping, etc.), and three or more peptides can be linked.

[0067] The peptides of the present invention can also be linked to other substances, as long as the resulting linked peptide retains its CTL-inducing ability. Suitable substances for linking with the peptides of the present invention include, for example, peptides, lipids, sugars or glycans, acetyl groups, and natural or synthetic polymers. The peptides of the present invention can also be modified, such as by glycosylation, side-chain oxidation, or phosphorylation, as long as their CTL-inducing ability is not impaired. Such modifications can impart additional functions (e.g., targeting and delivery functions) or stabilize the peptides.

[0068] For example, it is known in the art that D-amino acids, amino acid mimes, or non-natural amino acids can be introduced to enhance the in vivo stability of peptides, and this concept can also be adapted to the peptides of the present invention. The stability of peptides can be assayed in several ways. For example, stability can be tested using peptidases, as well as various biomediums such as human plasma and serum (see, for example, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

[0069] Furthermore, as described above, modified peptides in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added can be screened or selected to have the same or higher activity compared to the original peptide. Therefore, the present invention also provides a method for screening or selecting modified peptides that have the same or higher activity compared to the original. Specifically, the present invention provides a method for screening peptides having CTL-inducing ability, comprising the following steps: (a) A step of creating candidate amino acid sequences in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added to an original amino acid sequence consisting of amino acid sequences selected from Sequence IDs 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15; (b) The step of selecting candidate sequences from the candidate sequences created in (a) that do not have significant homology (sequence identity) with any known human gene product; (c) The step of contacting the peptide consisting of the candidate sequence selected in (b) with the APC; (d) The step of bringing the APCs in (c) into contact with CD8-positive T cells; and (e) Selecting a peptide having equivalent or higher CTL-inducing ability than the peptide consisting of the original amino acid sequence. In one embodiment, the APC for contacting the peptide is an APC that is positive for either HLA-A02 and / or HLA-A24.

[0070] In this specification, the peptide of the present invention is also referred to as "SARS-CoV-2 peptide" or "SARS-CoV-2 polypeptide."

[0071] In one embodiment of the present invention, a portion of the TCR may include one, two, or three complementarity determining regions (CDRs) on either the α-chain and / or β-chain of the TCR. In a preferred embodiment, a portion of the TCR includes CDR3 on either or both of the α-chain and / or β-chain of the TCR. The amino acid sequences of preferred CDR3s identified in the present invention are as follows: CDR3 of the human T cell receptor α chain, identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, and 40, and CDR3 of the human T cell receptor β chain, identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41. In one aspect of the present invention, the amino acid sequences of the CDR3 of the T cell receptor α chain and the T cell receptor β chain can be combined, for example, as follows: CDR3 of the T cell receptor α chain, CDR3 of the T cell receptor β chain Sequence ID: 32 Sequence ID: 33, Sequence ID: 34 Sequence ID: 35, Sequence ID: 36 Sequence ID: 37, Sequence ID: 38, Sequence ID: 39, and Sequence ID: 40 Sequence ID: 41. Furthermore, CDR3 including conservative modifications is also included in the CDR3 of the present invention. However, the peptides of the present invention are not limited to these and may include non-conservative modifications as long as the modified CDR3 retains the function of the original CDR3.

[0072] In this invention, when the TCR from which the original CDR3 is transplanted is conferred with the same antigen recognition specificity as the TCR from which the original CDR3 originates, it means that the original function of CDR3 is retained in the modified CDR3. Such specific recognition can be confirmed by any known method, and preferred methods include, for example, tetramer assays using HLA molecules and peptides administered to subjects from which the TCR was obtained (e.g., Altman et al. Science. 1996, 274, 94-6; McMichael et al. J Exp Med. 1998, 187, 1367-71), as well as the ELISPOT assay. By performing the ELISPOT assay, it is possible to confirm that T cells expressing the TCR on the cell surface recognize the cell by the TCR, and that a signal is transmitted intracellularly, followed by the release of cytokines such as IFN-γ from the T cells. The cytotoxic activity of T cells against target cells can be investigated using methods well known in the art. Preferred methods include, for example, chromium release assays using coronavirus-infected HLA-positive cells as target cells.

[0073] III. Preparation of the Peptide of the Present Invention The peptides of the present invention can be prepared using well-known techniques. For example, the peptides of the present invention can be prepared using recombinant DNA technology or chemical synthesis. The peptides of the present invention can be synthesized individually or as longer polypeptides containing two or more peptides. After production in host cells using recombinant DNA technology or chemical synthesis, the peptides of the present invention can be isolated from host cells or synthetic reaction products. That is, the peptides of the present invention can be purified or isolated so as to be substantially free of other host cell proteins and their fragments, or any other chemical substances.

[0074] The peptides of the present invention may include modifications such as glycosylation, side-chain oxidation, or phosphorylation, provided that the modifications do not impair the biological activity of the original peptide. Other exemplary modifications include the incorporation of D-amino acids or other amino acid mimetic molecules, which can be used, for example, to extend the serum half-life of the peptide.

[0075] The peptides of the present invention can be obtained by chemical synthesis based on a selected amino acid sequence. Examples of conventional peptide synthesis methods that can be adapted to this synthesis include the methods described in the following literature: (i)Peptide Synthesis, Interscience, New York, 1966; (ii)The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) "Peptide Synthesis" (in Japanese), Maruzen, 1975; (iv) "Fundamentals and Experiments of Peptide Synthesis" (in Japanese), Maruzen, 1985; (v) "Pharmaceutical Development" (in Japanese), Continued Vol. 14 (Peptide Synthesis), Hirokawa Shoten, 1991; (vi)WO99 / 67288; and (vii) Barany G. & Merrifield RB, Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-18.

[0076] Alternatively, the peptide of the present invention can be obtained by adapting any known genetic engineering method for producing the peptide (e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector containing a polynucleotide encoding the peptide of the present invention in an expressible form (e.g., downstream of a regulatory sequence corresponding to a promoter sequence) is prepared and transformed into a suitable host cell. The host cell is then cultured to produce the peptide of the present invention. Alternatively, the peptide of the present invention can be produced in vitro using an in vitro translation system.

[0077] IV. Polynucleotides The present invention also provides polynucleotides encoding any of the peptides of the present invention. These include polynucleotides derived from the natural SARS-CoV-2 gene (e.g., GenBank accession number MN908947 (SEQ ID NO: 16)) and polynucleotides having the same conservatively modified nucleotide sequence. In this specification, the term “conservatively modified nucleotide sequence” refers to a sequence encoding the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, numerous functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position where alanine is specified by a certain codon, the codon can be changed to any of the corresponding codons without altering the encoded polypeptide. Such a mutation in nucleic acid is a “silent mutation” and is a type of conservatively modified mutation. Any nucleic acid sequence in this specification encoding a peptide also represents any possible silent mutation of that nucleic acid. Those skilled in the art will recognize that functionally identical molecules can be obtained by modifying each codon in a nucleic acid (with the exception of AUG, which is usually the sole codon for methionine, and TGG, which is usually the sole codon for tryptophan). Therefore, each silent mutation in the nucleic acid encoding the peptide is implicitly described in each disclosed sequence.

[0078] The polynucleotides of the present invention may consist of DNA, RNA, and their derivatives. DNA is appropriately composed of bases such as A, T, C, and G, while in RNA, T is replaced with U.

[0079] The polynucleotides of the present invention can encode multiple peptides of the present invention, with or without intervening amino acid sequences. For example, the intervening amino acid sequences may provide cleavage sites (e.g., enzyme recognition sequences) for the polynucleotide or the translated peptide. Furthermore, the polynucleotide may include any additional sequences to the coding sequence encoding the peptide of the present invention. For example, the polynucleotide may be a recombinant polynucleotide containing regulatory sequences necessary for peptide expression, or it may be an expression vector (e.g., a plasmid) having a marker gene or the like. Generally, such recombinant polynucleotides can be prepared by manipulating polynucleotides using conventional recombination techniques, for example, polymerases and endonucleases.

[0080] The polynucleotides of the present invention can be prepared using either recombinant or chemical synthesis techniques. For example, the polynucleotides can be prepared by insertion into a suitable vector, which can then be expressed when transfected into competent cells. Alternatively, the polynucleotides can be amplified using PCR techniques or expression in a suitable host (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). Alternatively, the polynucleotides can be synthesized using solid-phase techniques described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

[0081] V. exosome The present invention further provides intracellular vesicles called exosomes that present a complex formed between the peptide of the present invention and an HLA antigen on their surface. Exosomes can be prepared, for example, by methods detailed in Japanese Patent Publication No. 11-510507 and WO99 / 03499, and can be prepared using APCs obtained from patients subject to treatment and / or prevention. Exosomes of the present invention can be administered as a vaccine in a manner similar to that of the peptide of the present invention.

[0082] The type of HLA antigen contained in the complex must match that of the target requiring treatment and / or prevention. For example, in Asian countries including Japan, HLA-A24 (e.g., HLA-A * 24:02), in Europe and America, HLA-A02 (for example, HLA-A * 02:01) is a widely observed allele, and these HLA antigen types are considered suitable for treating Asian or Caucasian patients. Typically, in a clinic setting, pre-determining the HLA antigen type of a patient requiring treatment allows for the appropriate selection of peptides that have a high binding affinity to a specific HLA antigen or that have the ability to induce CTLs through antigen presentation mediated by a specific HLA antigen.

[0083] The exosome of the present invention presents a complex of the peptide of the present invention with HLA-A24 or HLA-A02 on its surface. When the HLA that forms a complex with the peptide of the present invention is HLA-A24, the peptide of the present invention is preferably a peptide or a modified peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15, and more preferably a peptide or a modified peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. When the HLA that forms a complex with the peptide of the present invention is HLA-A02, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof.

[0084] VI. Antigen presenting cells (APC) The present invention also provides an APC that presents a complex formed between an HLA antigen and the peptide of the present invention on its surface. Alternatively, the present invention provides an APC having a complex formed between an HLA antigen and the peptide of the present invention on its cell surface. The APC of the present invention may be an isolated APC. When used in reference to cells (APCs, CTLs, etc.), the term “isolated” means that the cell has been separated from other types of cells. The APC of the present invention may be derived from an APC from a patient who is the target of treatment and / or prevention, and may be administered as a vaccine, alone or in combination with other drugs including the peptide of the present invention, exosomes, or CTLs.

[0085] The APCs of the present invention are not limited to any particular type of cell, but include cells known to present protein antigens on their cell surface so as to be recognized by lymphocytes, such as dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells. DCs are representative APCs with the most potent CTL-inducing activity among APCs, and therefore DCs can be preferably used as the APCs of the present invention.

[0086] For example, APCs of the present invention can be obtained by inducing DCs from peripheral blood monocytes and then stimulating them in vitro, ex vivo, or in vivo with the peptide of the present invention. When the peptide of the present invention is administered to a subject, APCs that present the peptide of the present invention are induced in the subject's body. Therefore, APCs of the present invention can be obtained by administering the peptide of the present invention to a subject and then recovering the APCs from the subject. Alternatively, APCs of the present invention can also be obtained by contacting APCs recovered from a subject with the peptide of the present invention.

[0087] In subjects, the APC of the present invention can be administered alone or in combination with other agents including the peptide, exosome, or CTL of the present invention to induce an immune response against coronavirus-infected cells. For example, ex vivo administration may include the following steps: (a) The step of recovering the APC from the first target, (b) The step of contacting the APC from step (a) with the peptide, and (c) The stage in which the APC from stage (b) is administered to the second target.

[0088] The first and second subjects may be the same individual or different individuals. If the first and second subjects are different individuals, it is preferable that the HLA types of the first and second subjects are the same. The APC obtained by step (b) above may serve as a vaccine for treating and / or preventing coronavirus infection. In some embodiments, the method of the present invention may further include a step of recovering the APC after step (b).

[0089] The APC of the present invention, obtained by the method described above, has CTL-inducing ability. The term "CTL-inducing ability" as used in relation to the APC refers to the ability of the APC to induce CTLs when brought into contact with CD8-positive T cells. The CTLs induced by the APC of the present invention are SARS-CoV-2 protein-specific CTLs and exhibit specific cytotoxic activity against SARS-CoV-2 infected cells.

[0090] In addition to the methods described above, the APC of the present invention can also be prepared by introducing a polynucleotide encoding the peptide of the present invention into the APC in vitro. The polynucleotide to be introduced may be in the form of DNA or RNA. Examples of introduction methods include, but are not limited to, various methods conventionally practiced in the art, such as lipofection, electroporation, and calcium phosphate methods. More specifically, methods such as those described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; and published Japanese Patent Publication No. 2000-509281 can be used. By introducing a polynucleotide encoding the peptide of the present invention into the APC, the polynucleotide undergoes transcription, translation, etc., in the cell, and the resulting peptide is then processed by MHC class I, and the peptide of the present invention is presented on the cell surface of the APC via the presentation pathway.

[0091] In a preferred embodiment, the APC of the present invention is HLA-A24 (more preferably HLA-A * This is an APC that presents the complex formed between 24:02) and the peptide of the present invention on its own cell surface. When the HLA that forms a complex with the peptide of the present invention is HLA-A24, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15.

[0092] Alternatively, in a preferred embodiment, the APC of the present invention is HLA-A02 (more preferably HLA-A *This is an APC that presents the complex formed between 02:01) and the peptide of the present invention on its own cell surface. When the HLA that forms a complex with the peptide of the present invention is HLA-A02, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13.

[0093] Furthermore, the APC of the present invention is preferably an APC induced by a method comprising the steps described in (a) or (b) below: (a) HLA-A24 (more preferably HLA-A * 24:02) or HLA-A02 (more preferably HLA-A * A step of contacting an APC expressing 02:01) with the peptide of the present invention; (b) HLA-A24 (more preferably HLA-A * 24:02) or HLA-A02 (more preferably HLA-A * The step of introducing a polynucleotide encoding the peptide of the present invention into an APC expressing 02:01).

[0094] The peptide of the present invention, which is contacted with an APC expressing HLA-A24, is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15. The peptide of the present invention, which is contacted with an APC expressing HLA-A02, is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13.

[0095] Furthermore, the present invention provides the use of the peptide of the present invention for producing a pharmaceutical composition that induces APCs having CTL-inducing ability. In addition, the present invention provides a method or process for producing a pharmaceutical composition that induces APCs having CTL-inducing ability. Furthermore, the present invention also provides the peptide of the present invention for inducing APCs having CTL-inducing ability.

[0096] VII. Cytotoxic T lymphocytes (CTL) CTLs induced by the peptide of the present invention can be used as vaccines, similar to the peptide of the present invention, to enhance the immune response targeting coronavirus-infected cells in vivo. Therefore, the present invention provides CTLs induced or activated by the peptide of the present invention. The CTLs of the present invention are CTLs that target the peptide of the present invention and are capable of binding to a complex of the peptide of the present invention with an HLA antigen. Binding of the CTL to the complex occurs via a T cell receptor (TCR) present on the cell surface of the CTL. The CTLs of the present invention may be isolated CTLs.

[0097] The CTLs of the present invention can be obtained by (1) administering the peptide of the present invention to a target, or (2) stimulating target-derived APCs and CD8-positive T cells or peripheral blood mononuclear cells (PBMCs) with the peptide of the present invention in vitro, or (3) contacting CD8-positive T cells or PBMCs in vitro with APCs or exosomes that present a complex of HLA antigen and the peptide of the present invention on their surface, or (4) introducing a vector containing polynucleotides encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of the present invention presented on the cell surface by an HLA antigen into CD8-positive T cells. The exosomes and APCs used in method (2) or (3) above can be prepared by the methods described in chapters "V. Exosomes" and "VI. Antigen-Presenting Cells (APCs)", respectively, and details of method (4) above are described in chapter "VIII. T Cell Receptors (TCRs)". In some embodiments, the methods of the present invention may further include a step of recovering the induced CTLs after each step.

[0098] The CTLs of the present invention can be administered alone to patients who are eligible for treatment and / or prevention, or in combination with other drugs containing the peptides, APCs, or exosomes of the present invention for the purpose of modulating the effect. Furthermore, the CTLs of the present invention may be CTLs derived from CD8-positive T cells from patients who are eligible for administration of the CTLs. The CTLs of the present invention act specifically on target cells that present the peptides of the present invention, for example, the same peptide used to induce the CTLs of the present invention. Target cells may be cells that endogenously express the SARS-CoV-2 protein, such as coronavirus-infected cells, or cells transfected with the SARS-CoV-2 gene. Cells that present the peptides on their cell surface upon stimulation with the peptides of the present invention can also be targets of attack by the CTLs of the present invention. Furthermore, the target cells of the CTLs of the present invention are preferably HLA-A24 (more preferably HLA-A * 24:02) or HLA-A02 (more preferably HLA-A* 02:01) This is a positive cell.

[0099] In a preferred embodiment, the CTL of the present invention is HLA-A24 (more preferably HLA-A * 24:02) or HLA-A02 (more preferably HLA-A * The CTL specifically targets cells expressing both HLA-A24 (more preferably HLA-A24) and SARS-CoV-2 protein. In this specification, "targeting" a cell means that the CTL recognizes a cell that presents a complex of HLA and the peptide of the present invention on its cell surface and exhibits cytotoxic activity against that cell. Furthermore, "specifically targeting" means that the CTL exhibits cytotoxic activity against the cell in question but not against other cells. In relation to the CTL, the term "recognizing a cell" means that the CTL binds to the complex of HLA and the peptide of the present invention presented on the cell surface via its TCR and exhibits specific cytotoxic activity against that cell. Therefore, the CTL of the present invention preferably expresses HLA-A24 (more preferably HLA-A24) presented on the cell surface. * 24:02) or HLA-A02 (more preferably HLA-A * This is a CTL that can bind via a TCR to the complex formed between (02:01) and the peptide of the present invention. Furthermore, the CTL of the present invention is preferably a CTL derived by a method comprising the steps described in (a) or (b) below: (a) CD8-positive T cells are HLA-A24 (more preferably HLA-A * 24:02) or HLA-A02 (more preferably HLA-A * 02:01) A step of bringing the complex of the peptide of the present invention into contact in vitro with an APC or exosome that presents itself on its surface; (b) CD8-positive T cells have HLA-A24 (more preferably HLA-A) on their cell surface. * 24:02) or HLA-A02 (more preferably HLA-A *A step of introducing polynucleotides that encode each subunit of the TCR capable of binding to the peptide of the present invention as presented in 02:01).

[0100] VIII. T cell receptor (TCR) The present invention also provides a composition comprising polynucleotides encoding each subunit of the TCR capable of binding to the peptide of the present invention presented on the cell surface by an HLA antigen, and a method of using the same. The polynucleotides confer specificity to coronavirus-infected cells to CD8-positive T cells by expressing a TCR capable of binding to the peptide of the present invention presented on the cell surface by an HLA antigen. By using methods known in the art, the polynucleotides encoding the α and β chains as TCR subunits of CTLs induced by the peptide of the present invention can be identified (WO2007 / 032255, and Morgan et al., J Immunol, 171, 3288 (2003)). For example, PCR is preferred for analyzing the TCR. PCR primers for analysis may, for example, be a 5'-R primer (5'-gtctaccaggcattcgcttcat-3') (SEQ ID NO: 28) as a 5'-side primer, and as 3'-side primers, they may be, but are not limited to, a 3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 29) specific to the TCRα chain C region, a 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3') (SEQ ID NO: 30) specific to the TCRβ chain C1 region, or a 3-TRβ-C2 primer (5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO: 31) specific to the TCRβ chain C2 region. The TCR formed by introducing the identified polynucleotides into CD8-positive T cells can bind with high affinity to target cells presenting the peptide of the present invention and mediate the efficient killing of target cells presenting the peptide of the present invention both in vivo and in vitro.

[0101] The polynucleotides encoding each subunit of the TCR can be incorporated into a suitable vector, such as a retroviral vector. These vectors are well known in the art. The polynucleotides, or vectors containing them in an expressible form, can be introduced into CD8-positive T cells, such as patient-derived CD8-positive T cells. The present invention provides a ready-made composition that enables the rapid and easy production of modified T cells with superior coronavirus-infected cell-killing properties by rapidly modifying the patient's own T cells (or T cells derived from another subject).

[0102] In this specification, a specific TCR is a TCR that, when present on the surface of CD8-positive T cells, specifically recognizes a complex of the peptide of the present invention and an HLA antigen presented on the surface of target cells, thereby conferring specific cytotoxic activity to target cells. The specific recognition of the complex can be confirmed by any known method, preferred examples of which include HLA multimer staining analysis using HLA molecules and the peptide of the present invention, and the ELISPOT assay. By performing the ELISPOT assay, it can be confirmed that T cells to which the polynucleotide has been introduced specifically recognize target cells by the TCR, and that the signal is transmitted intracellularly. Confirmation that the TCR, when present on the surface of CD8-positive T cells, can confer target cell-specific cytotoxic activity to CD8-positive T cells can also be confirmed by known methods. Preferred methods include, for example, measuring the cytotoxic activity against target cells by a chromium release assay.

[0103] Furthermore, in relation to HLA-A24, the present invention provides CTLs prepared by transduction into CD8-positive T cells of polynucleotides encoding each subunit of the TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15. In relation to HLA-A02, the present invention provides CTLs prepared by transduction into CD8-positive T cells of polynucleotides encoding each subunit of the TCR that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13.

[0104] Transduced CTLs can be homed in vivo and propagated by well-known in vitro culture methods (e.g., Kawakami et al., J Immunol., 142, 3452-61 (1989)). The CTLs of the present invention can be used to form immunogenic compositions useful for the treatment or prevention of disease in patients in need of treatment or prevention (see WO2006 / 031221, which is incorporated herein by reference).

[0105] This invention relates to the TCR expressed by T cells that recognize SARS-CoV-2-derived peptides. The TCR is a protein molecule consisting of an α-chain and a β-chain dimer. Human T cells recognize peptides presented on MHC class I (also known as HLA, Human Leukocyte Antigen) molecules through the TCR. As a result, T cell proliferation, differentiation, cytokine production, or secretion of cytotoxic substances (perforin and granzyme) are induced. The TCR-α gene includes the Vα, Jα, and Cα genes. The TCR-β gene includes the Vβ, Dβ, Jβ, and Cβ genes. The region that determines the specificity of the TCR to peptides is called the Complementarity Determining Region (CDR), and there are CDR1, CDR2, and CDR3. In particular, CDR3 is in direct contact with the peptide, so its amino acid sequence is extremely important in determining the antigen recognition specificity of the TCR. In the TCR-α chain, the CDR3 is between V and J, and in the TCR-β chain, it is between V, D, and DJ, and diversity arises from base insertions and deletions.

[0106] Alternatively, in one embodiment of the present invention, a portion of the TCR may include one, two, or three complementarity determining regions (CDRs) on either or both of the α-chain and / or β-chain of the TCR. In a preferred embodiment, a portion of the TCR includes CDR3 on either or both of the α-chain and / or β-chain of the TCR. The amino acid sequences of preferred CDR3s identified in the present invention are as follows: CDR3 of the human T cell receptor α chain, identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, and 40, and CDR3 of the human T cell receptor β chain, identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41. In one aspect of the present invention, the amino acid sequences of the CDR3 of the T cell receptor α chain and the T cell receptor β chain can be combined, for example, as follows: CDR3 of the T cell receptor α chain, CDR3 of the T cell receptor β chain Sequence ID: 32 Sequence ID: 33, Sequence ID: 34 Sequence ID: 35, Sequence ID: 36 Sequence ID: 37, Sequence ID: 38, Sequence ID: 39, and Sequence ID: 40 Sequence ID: 41. In one embodiment, the α-chain, β-chain, TCR comprising them, and the encoding polynucleotide of the present invention can be isolated.

[0107] IX. Pharmaceutical Compositions The present invention also provides a composition or pharmaceutical composition comprising at least one active ingredient selected from the following: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC of the present invention; (d) Exosome of the present invention; (e) The CTL of the present invention.

[0108] The pharmaceutical compositions of the present invention may, in addition to the above-mentioned active ingredients, contain carriers, excipients, etc., commonly used in pharmaceuticals, as needed and without particular limitation. Examples of carriers that can be used in the pharmaceutical compositions of the present invention include sterile water, physiological saline, phosphate buffer, and culture medium. Accordingly, the present invention also provides a pharmaceutical composition comprising at least one active ingredient selected from (a) to (e) below and a pharmaceutically acceptable carrier: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC of the present invention; (d) Exosome of the present invention; (e) The CTL of the present invention.

[0109] Furthermore, the pharmaceutical composition of the present invention may optionally contain stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, flocculation inhibitors, and the like. SARS-CoV-2 protein expression is significantly increased in coronavirus-infected cells compared to coronavirus-uninfected cells. Therefore, the peptide of the present invention or the polynucleotide encoding said peptide can be used for any or a combination of purposes selected from the treatment, prevention, and suppression of severe coronavirus infection. Accordingly, the present invention provides a pharmaceutical composition for any or a combination of purposes selected from the treatment, prevention, and suppression of severe coronavirus infection, comprising one or more of the peptide or polynucleotide of the present invention as an active ingredient. Alternatively, the peptide of the present invention can be presented on the surface of an exosome or APC for use as a pharmaceutical composition. In addition, CTLs of the present invention that target any one of the peptides of the present invention can also be used as an active ingredient in the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention may contain a therapeutically effective amount or a pharmaceutically effective amount of the above active ingredient.

[0110] The pharmaceutical compositions of the present invention may also be used as vaccines. In connection with the present invention, the term "vaccine" (also referred to as "immunogenic composition") refers to a composition that, when inoculated into an animal, has the function of inducing an immune response that provides anti-infective activity against coronavirus. Therefore, the pharmaceutical compositions of the present invention can be used to induce an immune response that provides anti-infective activity against coronavirus. The immune response induced by the peptides, polynucleotides, APCs, CTLs and pharmaceutical compositions of the present invention is not particularly limited as long as it provides anti-infective activity against coronavirus, but exemplifies the induction of CTLs specific to coronavirus-infected cells and the induction of cytotoxic activity specific to coronavirus-infected cells. The pharmaceutical compositions of the present invention can be used in human subjects or patients for any selection of the following purposes: treatment, prevention, and suppression of severe coronavirus infection, or a combination thereof. The pharmaceutical compositions of the present invention can be preferably used in subjects that are positive for HLA-A24 or HLA-A02. Furthermore, the pharmaceutical compositions of the present invention can be preferably used to treat and / or prevent coronavirus infection in subjects having HLA-A24 or HLA-A02, and / or to suppress the progression of the disease.

[0111] In another embodiment, the present invention also provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition for either the treatment and / or prevention of coronavirus infection: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0112] Alternatively, the present invention further provides active ingredients selected from the following for use in either the treatment and / or prevention of coronavirus infection: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0113] Alternatively, the present invention further provides a method or process for producing a pharmaceutical composition for either or both the treatment and prevention of coronavirus infection, comprising the step of formulating at least one active ingredient selected from the following and a pharmaceutically or physiologically acceptable carrier: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0114] In another embodiment, the present invention also provides a method or process for producing a pharmaceutically acceptable composition for either or both the treatment and / or prevention of coronavirus infection, comprising the step of mixing an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0115] In another embodiment, the present invention also provides a method for either or both the treatment and / or prevention of coronavirus infection, comprising the step of administering at least one active ingredient selected from the following: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0116] In the present invention, peptides having amino acid sequences selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15 were found to be HLA-A24-restricted epitope peptides capable of inducing a potent and specific immune response. Therefore, the pharmaceutical composition of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15 is an HLA antigen that targets HLA-A24 (e.g., HLA-A24). * It is particularly suitable for administration to subjects having HLA-A24 (i.e., HLA-24 positive subjects). The same applies to pharmaceutical compositions comprising a polynucleotide encoding any of these peptides (i.e., the polynucleotide of the present invention), an APC or exosome presenting these peptides (i.e., the APC or exosome of the present invention), and a CTL targeting these peptides (i.e., the CTL of the present invention). That is, a pharmaceutical composition comprising an active ingredient relating to a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15 is suitable for administration to subjects having HLA-A24 (i.e., HLA-24 positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NOs: 1, 2, 4, 5, 7, 9, 10, or 13.

[0117] Similarly, in the present invention, peptides having amino acid sequences selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 were found to be HLA-A02-restricted epitope peptides capable of inducing a potent and specific immune response. Therefore, the pharmaceutical composition of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 is an HLA antigen that can induce an HLA-A02 (e.g., HLA-A) *It is particularly suitable for administration to subjects having HLA-A02. The same applies to pharmaceutical compositions comprising a polynucleotide encoding any of these peptides (i.e., the polynucleotide of the present invention), an APC or exosome presenting these peptides (i.e., the APC or exosome of the present invention), and a CTL targeting these peptides (i.e., the CTL of the present invention). That is, a pharmaceutical composition comprising an active ingredient relating to a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13 is suitable for administration to subjects having HLA-A02 (i.e., HLA-A02 positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequences of SEQ ID NOs: 1, 2, 10, and 13.

[0118] The viral infections treated and / or prevented by the pharmaceutical compositions of the present invention are not particularly limited as long as they are coronavirus infections, and coronaviruses include SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0119] In addition to the above-mentioned active ingredients, the pharmaceutical composition of the present invention may include other peptides having the ability to induce CTLs in coronavirus-infected cells (e.g., CTL-inducing peptides derived from other coronavirus proteins), other polynucleotides encoding the other peptides, and other cells that present the other peptides.

[0120] The pharmaceutical compositions of the present invention may optionally contain other therapeutic substances as active ingredients, provided that the anti-infective effect of the above-mentioned active ingredients, such as the peptides of the present invention, against coronaviruses is not inhibited. For example, the pharmaceutical compositions of the present invention may optionally contain anti-inflammatory compositions, analgesics, antiviral agents, and other chemotherapeutic agents. In addition to including other therapeutic substances in the pharmaceutical compositions of the present invention themselves, the pharmaceutical compositions of the present invention may be administered sequentially or simultaneously with one or more other pharmaceutical compositions. The dosage of the pharmaceutical compositions of the present invention and other pharmaceutical compositions depends, for example, on the type of pharmaceutical composition used, the disease being treated, and the schedule and route of administration.

[0121] In addition to the components specifically mentioned herein, it should be understood that the pharmaceutical compositions of the present invention may also include other components that are conventional in the art, depending on the type of formulation.

[0122] The present invention also provides a product or kit comprising the pharmaceutical composition of the present invention. The product or kit of the present invention may include a container containing the pharmaceutical composition of the present invention. Suitable containers include, but are not limited to, bottles, vials, and test tubes. Containers may be made from a variety of materials, such as glass or plastic. Containers may be labeled, and the label may indicate the disease or disease condition for which the pharmaceutical composition of the present invention is intended. The label may also indicate instructions regarding administration, etc.

[0123] The product or kit of the present invention may further include, optionally, a second container containing a pharmaceutically acceptable diluent, in addition to a container containing the pharmaceutical composition of the present invention. The product or kit of the present invention may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, injection needles, syringes, and accompanying leaflets containing instructions for use.

[0124] The pharmaceutical composition of the present invention may be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient, as needed. The pack may include metal foil or plastic foil, for example, as in a blister pack. Instructions for administration may be attached to the pack or dispenser device.

[0125] (1) A pharmaceutical composition containing a peptide as an active ingredient The pharmaceutical composition containing the peptide of the present invention can be formulated by conventional formulation methods if necessary. In addition to the peptide of the present invention, the pharmaceutical composition of the present invention may contain carriers, excipients, etc., commonly used in pharmaceuticals, as needed and without particular limitations. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, Tris-buffered saline, 0.3% glycine, culture medium, etc. Furthermore, the pharmaceutical composition of the present invention may optionally contain stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, flocculation inhibitors, etc. Since the pharmaceutical composition of the present invention can induce specific immunity against coronavirus-infected cells, it can be used for the treatment or prevention of coronavirus infection.

[0126] For example, the pharmaceutical composition of the present invention can be prepared by dissolving the peptide solution in a pharmaceutically or physiologically acceptable water-soluble carrier such as sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, or Tris-buffered saline, and then sterilizing the peptide solution after adding stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, flocculation inhibitors, etc., as needed. The method of sterilizing the peptide solution is not particularly limited, but filtration sterilization is preferred. Filtration sterilization can be performed, for example, using a filtration sterilization filter with a pore size of 0.22 μm or less. The peptide solution after filtration sterilization can be administered to a target, for example, as an injectable agent, although this is not limited to the above. Furthermore, the pharmaceutical composition of the present invention may be prepared as a lyophilized formulation by freeze-drying the above peptide solution. The lyophilized formulation can be prepared by filling the peptide solution prepared as described above into an appropriate container such as an ampoule, vial, or plastic container, freeze-drying it, and then sealing the container with a sterilized rubber stopper after repressurization. Lyophilized formulations can be administered to a subject after being redissolved in a pharmaceutically or physiologically acceptable water-soluble carrier such as sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, or Tris-buffered saline, prior to administration. Preferred examples of the pharmaceutical compositions of the present invention include injectable formulations of such filtered and sterile peptide solutions, and lyophilized formulations of said peptide solutions. Furthermore, kits comprising such lyophilized formulations and redissolving solutions are also included in the present invention. Moreover, kits comprising a container containing a lyophilized formulation which is the pharmaceutical composition of the present invention, and a container containing its redissolving solution are also included in the present invention.

[0127] The pharmaceutical compositions of the present invention may also include combinations of two or more types of the peptides of the present invention. The peptide combinations may take the form of a cocktail of mixed peptides, or the peptides may be conjugated to each other using standard techniques. For example, the peptides may be chemically conjugated or expressed as a single fusion polypeptide sequence. By administering the peptides of the present invention, the peptides are presented at high density on APCs by the HLA antigen, and then CTLs that specifically react to the complex formed between the presented peptides and the HLA antigen are induced. Alternatively, APCs (e.g., DCs) can be isolated from a subject and then stimulated with the peptides of the present invention to obtain APCs that present one of the peptides of the present invention on their cell surface. These APCs can be administered again to the subject to induce CTLs in the subject, thereby increasing their aggression against coronavirus-infected cells. Neutralizing antibodies may lose their protective function against infection due to mutations in their target epitopes (viral immune escape). Generally, the effect of antigenic mutations is particularly large in monoclonal antibodies, which depend on a single epitope for antigen-binding specificity. On the other hand, if multiple CTL epitopes are present (a cocktail), even if one epitope mutates, the CTLs that recognize the other epitopes remain effective. Mixing epitopes derived from multiple proteins can be considered an effective strategy to avoid a decrease in therapeutic efficacy due to viral immune escape.

[0128] The pharmaceutical compositions of the present invention may also include adjuvants known to efficiently establish cellular immunity. An adjuvant is a compound that, when administered together with (or in succession with) an immunologically active antigen, enhances the immune response to that antigen. As adjuvants, known ones described in literature such as Clin Microbiol Rev 1994, 7: 277-89 can be used. Examples of suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), ISCO Matrix, GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG7909, etc.), oil-in-water emulsions, saponins or their derivatives (QS21, etc.), lipopolysaccharides such as lipid A or its derivatives (MPL Examples of adjuvants include, but are not limited to, RC529, GLA, E6020, lipopeptides, lactoferrin, flagellin, double-stranded RNA or its derivatives (e.g., poly-IC), bacterial DNA, imidazoquinolines (e.g., imiquimod, R848), C-type lectin ligands (e.g., trehalose-6,6'-dibehenate (TDB)), CD1d ligands (e.g., α-galactosylceramide), squalene emulsions (e.g., MF59, AS03, AF03), PLGA, etc. In some embodiments, the pharmaceutical compositions of the present invention may contain an amount of adjuvant sufficient to stimulate an immune response. The adjuvant may be contained in a separate container from the pharmaceutical composition containing the peptide of the present invention in a kit containing the pharmaceutical composition of the present invention. In this case, the adjuvant and the pharmaceutical composition may be administered to the subject sequentially or may be mixed immediately before administration to the subject. A kit containing such a pharmaceutical composition containing the peptide of the present invention and an adjuvant is also provided by the present invention. If the pharmaceutical composition of the present invention is a lyophilized formulation, the kit may further include a redissolving solution. The present invention also provides a kit containing a container containing the pharmaceutical composition of the present invention and a container containing an adjuvant. The kit may further include a container containing a redissolving solution, if necessary.

[0129] When an oily adjuvant is used as the adjuvant, the pharmaceutical composition of the present invention may be prepared as an emulsion. The emulsion can be prepared, for example, by mixing and stirring the peptide solution prepared as described above with the oily adjuvant. The peptide solution may be redissolved after lyophilization. The emulsion may be either a W / O type emulsion or an O / W type emulsion, but a W / O type emulsion is preferred to obtain a high immune response enhancing effect. IFA can be preferably used as the oily adjuvant, but is not limited to it. The emulsion may be prepared immediately before administration to the subject, in which case the pharmaceutical composition of the present invention may be provided as a kit containing the peptide solution and the oily adjuvant of the present invention. If the pharmaceutical composition of the present invention is a lyophilized formulation, the kit may further include a redissolving solution.

[0130] Furthermore, the pharmaceutical composition of the present invention may be a liposome formulation containing the peptide of the present invention, a granular formulation in which the peptide is bound to beads with a diameter of several micrometers, or a formulation in which lipids are bound to the peptide.

[0131] In another aspect of the present invention, the peptides of the present invention may also be administered in the form of pharmaceutically acceptable salts. Preferred examples of salts include salts with alkali metals (such as lithium, potassium, and sodium), salts with alkaline earth metals (such as calcium and magnesium), salts with other metals (such as copper, iron, zinc, and manganese), salts with organic bases, salts with amines, salts with organic acids (such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, and methanesulfonic acid), and salts with inorganic acids (such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, and nitric acid). Thus, pharmaceutical compositions containing pharmaceutically acceptable salts of the peptides of the present invention are also encompassed by the present invention. Furthermore, "peptides of the present invention" includes not only the free peptides but also their pharmaceutically acceptable salts.

[0132] In some embodiments, the pharmaceutical compositions of the present invention may further contain components that stimulate CTLs. Lipids have been identified as substances that can stimulate CTLs in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε-amino and α-amino groups of lysine residues and then linked to the peptides of the present invention. The lipid-modified peptides can then be administered directly in the form of micelles or particles, administered via liposomes, or emulsified in an adjuvant. As another example of lipid stimulation of the CTL response, E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used to stimulate CTLs when covalently bound to a suitable peptide (see, for example, Deres et al., Nature 1989, 342: 561-4).

[0133] Examples of methods for administering the peptides or pharmaceutical compositions of the present invention include, but are not limited to, oral, intradermal, subcutaneous, intramuscular, intraosseous, peritoneal, and intravenous injections, as well as systemic administration or local administration near the target site. Preferred administration methods include subcutaneous injection near lymph nodes such as the axilla or groin. Administration may be performed as a single dose or as a booster dose. The peptides of the present invention can be administered to a target in a therapeutic or pharmaceutically effective amount for treating coronavirus infection or for inducing immunity (more specifically, CTLs) against coronavirus-infected cells. The dose of the peptides of the present invention can be appropriately adjusted depending on the disease to be treated or prevented, the patient's age, weight, method of administration, etc., and for each peptide of the present invention, it can be typically 0.001 mg to 1000 mg, for example 0.01 mg to 100 mg, for example 0.1 mg to 30 mg, for example 0.1 mg to 10 mg, or for example 0.5 mg to 5 mg. Furthermore, the administration interval can be once every few days to several months, for example, once a week. Those skilled in the art can appropriately select the appropriate dosage.

[0134] In a preferred embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of the peptide of the present invention and a pharmaceutically or physiologically acceptable carrier. In another embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of the peptide of the present invention, a pharmaceutically or physiologically acceptable carrier, and an adjuvant. The pharmaceutical composition of the present invention may contain the peptide of the present invention in an amount of 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 30 mg, even more preferably 0.1 mg to 10 mg, for example 0.5 mg to 5 mg. Furthermore, if the pharmaceutical composition of the present invention is an injectable preparation, the peptide of the present invention may be contained at a concentration of 0.001 mg / ml to 1000 mg / ml, preferably 0.01 mg / ml to 100 mg / ml, more preferably 0.1 mg / ml to 30 mg / ml, even more preferably 0.1 mg / ml to 10 mg / ml, for example 0.5 mg / ml to 5 mg / ml. In this case, for example, 0.1 to 5 ml, preferably 0.5 to 2 ml, of the pharmaceutical composition of the present invention can be administered to the subject by injection. On the other hand, if the pharmaceutical composition of the present invention contains an adjuvant, the adjuvant can be included in an amount effective in enhancing the subject's immune response to the peptide.

[0135] Alternatively, the present invention provides a method for any or more purposes selected from the treatment, prevention, and suppression of severe coronavirus infection, comprising administering a therapeutically effective amount of the peptide or pharmaceutical composition of the present invention to a subject. The peptide of the present invention is typically 0.001 mg to 1000 mg, for example 0.01 mg to 100 mg, for example 0.1 mg to 30 mg, for example 0.1 mg to 10 mg, and for example 0.5 mg to 5 mg can be administered to a subject in a single dose. In a preferred embodiment, the peptide of the present invention is administered to the subject together with an adjuvant. The administration interval can be once every few days to several months, preferably once every few days to one month, for example, once a week or once every two weeks. On the other hand, in the method of the present invention, when an adjuvant is administered together with the peptide, the adjuvant can be administered in an amount effective in enhancing the subject's immune response to the peptide.

[0136] (2) Pharmaceutical composition containing polynucleotide as an active ingredient The pharmaceutical compositions of the present invention may also comprise a polynucleotide encoding the peptide of the present invention in an expressible form. In this specification, the phrase "in an expressible form" means that the polynucleotide, when introduced into a cell, will express the peptide of the present invention. In exemplary embodiments, the sequence of the polynucleotide of the present invention includes regulatory elements necessary for the expression of the peptide of the present invention. The polynucleotide of the present invention may be provided with sequences necessary to achieve stable insertion into the genome of a target cell (see, for example, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12, for a description of homologous recombination cassette vectors). See, for example, Wolff et al., Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO98 / 04720. Examples of DNA-based delivery technologies include “naked DNA,” facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, for example, U.S. Patent No. 5,922,687).

[0137] The peptides of the present invention can also be expressed using viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccinia virus or fowlpox virus. For example, vaccinia virus can be used as a vector for expressing the peptides of the present invention. Upon introduction into a host, recombinant vaccinia virus expresses immunogenic peptides, thereby inducing an immune response. Vaccinia vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus calmette-Guérin). The BCG vector is described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors useful for therapeutic administration or immunization are known, such as adenovirus vectors and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, and detoxified anthrax toxin vectors. For example, see Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.

[0138] The delivery of the polynucleotides of the present invention into a patient may be direct, in which case the patient may be directly exposed to a vector containing the polynucleotides of the present invention. Alternatively, it may be indirect, in which case cells are first transformed in vitro with a vector containing the polynucleotides of the present invention, and then the cells are transplanted into the patient. These two approaches are known as in vivo and ex vivo gene therapies, respectively.

[0139] For a general review of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan, Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in Biotechnology 1993, 11(5): 155-215. Methods generally known in the field of recombinant DNA technology, which can also be used in the present invention, are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990. The administration method may be oral, intradermal, subcutaneous, or intravenous injection, and systemic administration or local administration near the target site is used. Administration can be performed as a single dose or boosted by multiple doses. The polynucleotides of the present invention can be administered to a subject in a therapeutic or pharmaceutically effective dose for treating coronavirus infection or for inducing immunity (more specifically, CTLs) against coronavirus-infected cells. The dose of polynucleotides in a suitable carrier, or in cells transformed with the polynucleotides encoding the peptides of the present invention, can be appropriately adjusted depending on the disease being treated, the patient's age, weight, method of administration, etc., and can typically be between 0.001 mg and 1000 mg, for example between 0.01 mg and 100 mg, for example between 0.1 mg and 30 mg, for example between 0.1 mg and 10 mg, for example between 0.5 mg and 5 mg. The administration interval can be once every few days to once every few months, for example, once a week. Those skilled in the art can appropriately select an appropriate dose.

[0140] Methods using X peptides, exosomes, APCs, and CTLs APCs and CTLs can be induced using the peptides and polynucleotides of the present invention. CTLs can also be induced using the exosomes and APCs of the present invention. The peptides, polynucleotides, exosomes, and APCs of the present invention can be used in combination with any other compound, as long as their CTL-inducing ability is not inhibited. Therefore, CTLs of the present invention can be induced using a pharmaceutical composition containing any of the peptides, polynucleotides, APCs, and exosomes of the present invention. Furthermore, APCs of the present invention can be induced using a pharmaceutical composition containing the peptides or polynucleotides of the present invention.

[0141] (1) Method for guiding the APC The present invention provides a method for inducing APCs having CTL-inducing ability using the peptide or polynucleotide of the present invention.

[0142] The method of the present invention includes the step of contacting the APC with the peptide of the present invention in vitro, ex vivo, or in vivo. For example, a method of contacting the APC with the peptide ex vivo may include the following steps: (a) The step of recovering the APC from the target; and (b) A step in which the APC from step (a) is brought into contact with the peptide of the present invention. The APC is not limited to any particular type of cell, but can be any cell known to present proteinaceous antigens on its cell surface so as to be recognized by lymphocytes, such as DCs, Langerhans cells, macrophages, B cells, and activated T cells. DCs are preferred because they have the most potent CTL-inducing ability among APCs. Any peptide of the present invention can be used alone or in combination with other peptides of the present invention. Alternatively, the peptide of the present invention can be used in combination with other CTL-inducing peptides (e.g., CTL-inducing peptides derived from other coronavirus proteins). In some embodiments, the method of the present invention may further include a step of recovering the APC after step (b).

[0143] On the other hand, when the peptide of the present invention is administered to a subject, the APC comes into contact with the peptide in vivo, and as a result, an APC with high CTL-inducing ability is induced in the subject's body. Therefore, the method of the present invention may include the step of administering the peptide of the present invention to a subject. Similarly, when the polynucleotide of the present invention is administered to a subject in an expressible form, the peptide of the present invention is expressed in vivo, and this comes into contact with the APC in vivo, and as a result, an APC with high CTL-inducing ability is induced in the subject's body. Therefore, the present invention may also include the step of administering the polynucleotide of the present invention to a subject.

[0144] The present invention may also include the step of introducing the polynucleotide of the present invention into an APC in order to induce an APC having CTL-inducing ability. For example, the method may include the following steps: (a) The step of recovering the APC from the target; and (b) A step of introducing a polynucleotide encoding the peptide of the present invention into the APC of step (a). Step (b) can be carried out as described above in Chapter VI, "Antigen-Presenting Cells (APCs)".

[0145] Accordingly, in one embodiment, the present invention provides a method for inducing an APC having CTL-inducing ability, comprising the steps of (a) or (b) below: (a) The step of contacting the APC with the peptide of the present invention; (b) The step of introducing a polynucleotide encoding the peptide of the present invention into the APC.

[0146] Furthermore, the present invention provides a method for preparing an APC having CTL-inducing ability, comprising the following steps: (a) or (b): (a) The step of contacting the APC with the peptide of the present invention; (b) The step of introducing a polynucleotide encoding the peptide of the present invention into the APC.

[0147] The above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo. The APC used in the above method may be derived from the subject to which the induced APC is to be administered, or it may be derived from a different subject. In one embodiment, the method of the present invention may further include a step of recovering the APC after step (b). When using an APC derived from a different subject (donor) than the target recipient, the HLA types of the recipient and the donor must be the same. In the method of the present invention, when using a peptide or a modified peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 as the peptide of the present invention, the HLA types of both the recipient and the donor must be HLA-A24 (more preferably HLA-A24). * It is preferable that the APC used in the above method is HLA-A24 (more preferably HLA-A *It is preferable that the APC expresses 24:02). When using a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof, as the peptide of the present invention, the HLA type of both the recipient and the donor is HLA-A02 (more preferably HLA-A02). * 02:01) is preferable. Alternatively, the APC used in the above method is HLA-A02 (more preferably HLA-A * It is preferable that the APC expresses 02:01). The APC can be prepared by separating the PBMC from the blood collected from the donor by specific gravity centrifugation or the like, and then preparing the PBMC using a known method.

[0148] In another embodiment, the present invention also provides a pharmaceutical composition for inducing CTL-inducing APCs, comprising the peptide of the present invention or a polynucleotide encoding the peptide.

[0149] Alternatively, the present invention further provides the use of the peptide of the present invention or the polynucleotide encoding the peptide in the preparation of a pharmaceutical composition for inducing APCs having CTL-inducing ability.

[0150] Alternatively, the present invention further provides a peptide or a polynucleotide encoding the peptide for use in the induction of APCs having CTL-inducing ability.

[0151] Alternatively, the present invention further provides a method or process for producing a pharmaceutical composition for inducing APC, comprising the step of formulating a peptide of the present invention or a polynucleotide encoding the peptide and a pharmaceutically or physiologically acceptable carrier.

[0152] In another embodiment, the present invention also provides a method or process for producing a pharmaceutical composition for inducing an APC having CTL-inducing ability, comprising the step of mixing the peptide of the present invention or a polynucleotide encoding the peptide with a pharmaceutically or physiologically acceptable carrier. APCs induced by the method of the present invention can induce CTLs specific to coronavirus proteins (i.e., the CTLs of the present invention).

[0153] (2) Methods for inducing CTLs The present invention also provides a method for inducing CTLs using the peptides, polynucleotides, exosomes, or APCs of the present invention.

[0154] When the peptides, polynucleotides, exosomes, or APCs of the present invention are administered to a target, CTLs are induced in the target's body, and the intensity of the immune response targeting coronavirus-infected cells is enhanced. Therefore, the method of the present invention may include the step of administering the peptides, polynucleotides, APCs, or exosomes of the present invention to a target.

[0155] Alternatively, CTLs can be induced by using them in vitro or ex vivo. For example, the method of the present invention may include the following steps: (a) The stage of recovering the APC from the target, (b) The step of contacting the APC from step (a) with the peptide of the present invention, and (c) The APCs from step (b) are co-cultured with CD8-positive T cells. The induced CTLs may then be returned to the subjects.

[0156] The APCs co-cultured with CD8-positive T cells in step (c) above can also be prepared by introducing a polynucleotide encoding the peptide of the present invention into the APC, as described above in Chapter VI. Antigen-Presenting Cells (APCs). However, the APCs used in the method of the present invention are not limited to these, and any APCs that present a complex of the HLA antigen and the peptide of the present invention on its surface can be used.

[0157] In the method of the present invention, instead of such APCs, exosomes that present a complex of HLA antigen and the peptide of the present invention on their surface may be used. That is, the method of the present invention may include a step of co-culturing exosomes that present a complex of HLA antigen and the peptide of the present invention on their surface. Such exosomes can be prepared by the method described above in Chapter "V. Exosomes".

[0158] Furthermore, CTLs can also be induced by introducing a vector containing polynucleotides encoding each subunit of the TCR that can bind to the peptide of the present invention presented on the cell surface by an HLA antigen into CD8-positive T cells. Such transduction can be carried out as described above in Chapter VIII, "T Cell Receptor (TCR)".

[0159] Therefore, in one embodiment, the present invention provides a method for inducing CTLs, comprising steps selected from the following: (a) A step of co-culturing CD8-positive T cells with APCs that present a complex of HLA antigen and the peptide of the present invention on their surface; (b) A step of co-culturing CD8-positive T cells with exosomes that present a complex of HLA antigen and the peptide of the present invention on their surface; and (c) A step of introducing a vector containing polynucleotides encoding each subunit of the TCR that can bind to the peptide of the present invention presented on the cell surface by an HLA antigen into CD8-positive T cells.

[0160] The above method can be performed in vitro, ex vivo, or in vivo, but is preferably performed in vitro or ex vivo. When performed in vitro or ex vivo, in one embodiment, the method of the present invention may include a step of recovering the induced CTLs after either step. The APCs or exosomes and CD8-positive T cells used in the above method may be derived from the subject to whom the induced CTLs are to be administered, or from a different subject. When using APCs or exosomes and CD8-positive T cells derived from a different subject (donor) than the subject to whom the administration is to be performed, the HLA types of the subject and the donor must be the same. For example, when using a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof as the peptide of the present invention, the HLA types of both the subject and the donor must be HLA-A24 (more preferably HLA-A * It is preferable that the APC or exosome used in the above method is HLA-A24 (more preferably HLA-A * Preferably, the APC or exosome presents a complex of HLA-A24 and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15, or a modified peptide thereof) on its surface. In this case, the induced CTL exhibits specific cytotoxic activity against cells presenting the complex of HLA-A24 and the peptide of the present invention (for example, HLA-A24-positive cells infected with coronavirus). When using a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof, as the peptide of the present invention, the HLA types of both the recipient and the donor are HLA-A02 (more preferably HLA-A02). * 02:01) is preferable. Alternatively, the APC or exosome used in the above method is HLA-A02 (more preferably HLA-A *Preferably, the APC or exosome presents a complex of HLA-A02 and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, or a modified peptide thereof) on its surface. In this case, the induced CTL exhibits specific cytotoxic activity against cells presenting the complex of HLA-A02 and the peptide of the present invention (for example, HLA-A02-positive cells infected with coronavirus).

[0161] In another embodiment, the present invention also provides a composition or pharmaceutically active ingredient for inducing CTLs, comprising at least one active ingredient selected from the following: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APCs that present the peptide of the present invention on their own surface; and (d) An exosome that presents the peptide of the present invention on its surface.

[0162] In another embodiment, the present invention also provides the use of an active ingredient selected from the following in the preparation of a composition or pharmaceutical composition for inducing CTLs: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APCs that present the peptide of the present invention on their own surface; and (d) An exosome that presents the peptide of the present invention on its surface.

[0163] Alternatively, the present invention further provides active ingredients selected from the following for use in CTL induction: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APCs that present the peptide of the present invention on their own surface; and (d) An exosome that presents the peptide of the present invention on its surface.

[0164] Alternatively, the present invention further provides a method or process for producing a composition or pharmaceutical composition for inducing CTLs, comprising the step of formulating an active ingredient selected from the following and a pharmaceutically or physiologically acceptable carrier: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APCs that present the peptide of the present invention on their own surface; and (d) An exosome that presents the peptide of the present invention on its surface.

[0165] In another embodiment, the present invention also provides a method or process for producing a composition or pharmaceutical composition for inducing CTLs, comprising the step of mixing an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APCs that present the peptide of the present invention on their own surface; and (d) An exosome that presents the peptide of the present invention on its surface.

[0166] XI. Methods for inducing an immune response Furthermore, the present invention provides a method for inducing an immune response to coronavirus infection. The coronavirus includes, but is not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. In addition, it is preferable that the coronavirus-infected cells express HLA-A24 or HLA-A02.

[0167] The present invention also provides a method for inducing an immune response against coronavirus-infected cells. The peptides of the present invention are derived from structural or non-structural proteins of SARS-CoV-2 and also share amino acid sequences commonly found in SARS-CoV and MERS-CoV proteins. Therefore, when an immune response against coronavirus-infected cells is induced, viral replication in the coronavirus-infected cells is inhibited as a result. Accordingly, the present invention also provides a method for inhibiting viral replication in coronavirus-infected cells. The method of the present invention is particularly suitable for inhibiting coronavirus replication in coronavirus-infected cells expressing HLA-A24 or HLA-A02.

[0168] The methods of the present invention may include a step of administering a composition comprising one of the peptides of the present invention or a polynucleotide encoding one of them. The methods of the present invention also intend to administer an exosome or APC presenting one of the peptides of the present invention. For further details, please refer to section IX. Pharmaceutical Compositions, in particular the section describing the use of the pharmaceutical compositions of the present invention as vaccines. In addition, exosomes and APCs that can be used in the methods of the present invention to induce an immune response are described in detail in sections V. Exosomes, VI. Antigen-Presenting Cells (APCs) and X. Methods Using Peptides, Exosomes, APCs, and CTLs (1) and (2) above.

[0169] In another embodiment, the present invention also provides a pharmaceutical composition or vaccine for inducing an immune response to coronavirus infection, comprising an active ingredient selected from the following: (a) The peptide of the present invention; (b) Polynucleotides encoding the peptide of the present invention in an expressible form; (c) APC that presents the peptide of the present invention on its own surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0170] Alternatively, the present invention further provides a pharmaceutical composition or a vaccine for inducing an immune response against a coronavirus-infected cell, the pharmaceutical composition or vaccine comprising an active ingredient selected from the following: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) An exosome presenting the peptide of the present invention on its surface; and (e) The CTL of the present invention.

[0171] Alternatively, the present invention further provides a pharmaceutical composition or a vaccine for inhibiting the growth of coronavirus in a coronavirus-infected cell, the pharmaceutical composition or vaccine comprising an active ingredient selected from the following: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) An exosome presenting the peptide of the present invention on its surface; and (e) The CTL of the present invention.

[0172] In another aspect, the present invention also provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition or a vaccine for inducing an immune response against a coronavirus infection: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) An exosome presenting the peptide of the present invention on its surface; and (e) The CTL of the present invention.

[0173] Alternatively, the present invention further provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition or vaccine for inducing an immune response against coronavirus-infected cells: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) An exosome presenting the peptide of the present invention on its surface; and (e) The CTL of the present invention.

[0174] Alternatively, the present invention further provides the use of an active ingredient selected from the following in the manufacture of a pharmaceutical composition or vaccine for inhibiting the growth of coronavirus in coronavirus-infected cells: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) An exosome presenting the peptide of the present invention on its surface; and (e) The CTL of the present invention.

[0175] The present invention also provides a method or process for manufacturing a pharmaceutical composition for inducing an immune response against coronavirus infection, which may include the step of mixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier.

[0176] Alternatively, the present invention provides a method for inhibiting the growth of coronavirus in coronavirus-infected cells or for inducing an immune response against coronavirus infection, which includes the step of administering to a subject a vaccine or pharmaceutical composition containing an active ingredient selected from the following: (a) The peptide of the present invention; (b) A polynucleotide encoding the peptide of the present invention in an expressible form; (c) An APC presenting the peptide of the present invention on its surface; (d) Exosomes that present the peptide of the present invention on their own surface; and (e) The CTL of the present invention.

[0177] In connection with the present invention, coronavirus infection can be treated by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Alternatively, an immune response to coronavirus infection can be induced by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Examples of such coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. Furthermore, an immune response to coronavirus-infected cells can be induced by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Therefore, it is also possible to confirm whether the subject to be treated is infected with coronavirus before administering a vaccine or pharmaceutical composition containing the above active ingredients.

[0178] Therefore, in one embodiment, the present invention provides a method for treating coronavirus infection in patients requiring treatment of the infection, such method comprising the following steps: i) The step of measuring the expression level of the SARS-CoV-2 gene or the protein encoded by it in a biological sample taken from a subject infected with the coronavirus; ii) A step to identify subjects infected with the coronavirus based on the SARS-CoV-2 gene, or the protein expression level encoded therein, measured in i); and iii) The step of administering at least one component selected from the group consisting of (a) to (e) above to a subject infected with coronavirus.

[0179] Alternatively, the present invention also provides a vaccine or pharmaceutical composition comprising at least one active ingredient selected from the group consisting of (a) to (e) above for administration to a subject infected with coronavirus. The present invention further provides a method for identifying or selecting a subject to be treated with at least one active ingredient selected from the group consisting of (a) to (e) above, such a method comprising the following steps: i) The step of measuring the expression level of the SARS-CoV-2 gene or the protein encoded by it in a biological sample taken from a subject infected with the coronavirus; ii) A step of identifying subjects having coronavirus-infected cells expressing the SARS-CoV-2 gene or the protein encoded therein, based on the expression level of the SARS-CoV-2 gene or the protein encoded therein, as measured in i); and iii) The step of identifying or selecting the subjects identified in ii) as subjects who can be treated with at least one active ingredient selected from the group consisting of (a) to (e) above.

[0180] The biological sample taken from a subject to measure the expression level of the SARS-CoV-2 gene or the protein encoded therein in the above method is not particularly limited, but a tissue sample containing coronavirus-infected cells collected by biopsy or the like is preferably used. Alternatively, detection of coronavirus RNA in pharyngeal swabs or saliva is commonly performed in identifying coronavirus-infected individuals. Therefore, in the present invention, the coronavirus gene includes the genomic RNA of the coronavirus or the mRNA from which it is transcribed. The expression level of the SARS-CoV-2 gene or the protein encoded therein in a biological sample can be measured by known methods, such as methods for detecting the transcript of the SARS-CoV-2 gene by probe or PCR (e.g., cDNA microarray method, Northern blotting method, RT-PCR method, etc.), or methods for detecting the translation product of the SARS-CoV-2 gene by antibody, etc. (e.g., Western blotting method, immunohistochemistry method, immunochromatography method, etc.). Furthermore, the biological sample may be a blood sample, in which case the blood level of antibodies against SARS-CoV-2 protein may be measured, and the expression level of SARS-CoV-2 protein may be evaluated based on the blood level. The blood level of antibodies against SARS-CoV-2 protein can be measured using known methods, for example, enzyme-linked immunosorbent assay (EIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA) using SARS-CoV-2 protein or the peptide of the present invention as antigens can be used. Furthermore, the expression level of SARS-CoV-2 protein in a subject may be evaluated by detecting CTLs specific to the peptide of the present invention. The level of CTLs specific to the peptide of the present invention can be measured, for example, by separating PBMCs from blood collected from the subject and measuring the cytotoxic activity against target cells pulsed with the peptide of the present invention. Cytotoxic activity can be measured, for example, by the amount of interferon-γ released. In addition, the complex of the peptide of the present invention and HLA described below can also be used to measure the CTL level. The determination of whether or not coronavirus-infected cells in a subject express SARS-CoV-2 protein may be made by comparing the measurement results with those of the same type of biomaterial collected from a subject that is not infected with coronavirus. That is, if the level of the substance measured in a biological sample collected from a coronavirus-infected subject is elevated compared to the level of the substance measured in the same type of biomaterial collected from a subject that is not infected with coronavirus (normal control level), it can be determined that the cells of the coronavirus-infected subject express SARS-CoV-2 protein.

[0181] In a preferred embodiment, it is preferable to confirm the target's HLA type before administering at least one active ingredient selected from the group consisting of (a) to (e) above. For example, when administering an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15, it is preferable to select a target that is HLA-A24 positive. When administering an active ingredient related to a peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 7, 10, 12, and 13, it is preferable to select a target that is HLA-O2 positive. Methods for determining the HLA haplotype, so-called HLA typing, are well known to those skilled in the art. Alternatively, lymphocyte cytotoxicity tests (LCTs) that determine the HLA type by the reactivity of antibodies specific to each HLA with HLA antigens on lymphocytes are also known.

[0182] The present invention also provides a complex of the peptide of the present invention with HLA. The complex of the present invention may be a monomer or a polymer. If the complex of the present invention is a polymer, the number of polymerization units is not particularly limited, and it can be a polymer with any number of polymerization units. Examples include, but are not limited to, tetramers, pentamers, hexamers, etc. Dextramers (WO2002 / 072631) and streptamers (Knabel M et al., Nat Med. 2002 Jun;8(6):631-7.) are also included as polymers of the present invention. The complex of the peptide of the present invention with HLA can be prepared according to known methods (e.g., Altman JD et al., Science. 1996, 274(5284):94-6, WO2002 / 072631, WO2009 / 003492, Knabel M et al., Nat Med. 2002 Jun;8(6):631-7., etc.). The complex of the present invention can be used, for example, to quantify CTLs specific to the peptide of the present invention. For example, a blood sample is taken from a subject administered the pharmaceutical composition of the present invention, PBMCs are separated, CD4-negative cells are prepared, and the complex of the present invention, to which a fluorescent dye has been conjugated, is brought into contact with the CD4-negative cells. Subsequently, the proportion of CTLs specific to the peptide of the present invention can be measured by flow cytometry analysis. For example, the immune response-inducing effect of the pharmaceutical composition of the present invention can be monitored by measuring CTLs specific to the peptide of the present invention before, during, and / or after administration of the pharmaceutical composition of the present invention.

[0183] XII. Antibodies The present invention further provides antibodies that bind to the peptides of the present invention. Preferred antibodies bind specifically to the peptides of the present invention and do not bind (or bind weakly) to others. The binding specificity of an antibody can be confirmed by inhibition testing. That is, if the binding between the antibody being analyzed and polypeptides (SEQ ID NOs: 17-26) consisting of the amino acid sequences of each protein encoded by the full-length SARS-CoV-2 genome sequence is inhibited in the presence of the peptides of the present invention, it is shown that this antibody binds specifically to the peptides of the present invention. Antibodies against the peptides of the present invention can be used in assays for disease diagnosis and prognosis, as well as in the selection of recipients for the pharmaceutical compositions of the present invention and the monitoring of the pharmaceutical compositions of the present invention.

[0184] The present invention also provides various immunological assay methods for detecting and / or quantifying the peptide or fragment thereof. Such immunological assay methods include, but are not limited to, radioimmunoassay, immunochromatography, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA), and the like, and are carried out within the range of various immunological assay forms known in the art.

[0185] The antibodies of the present invention can be used in immunological imaging methods capable of detecting coronavirus-infected cells, including, but not limited to, radioscintigraphy imaging using the labeled antibodies of the present invention. Such assays are used clinically in the detection, monitoring, and prognosis of coronavirus-infected cells, and examples of coronaviruses in such coronavirus infections include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0186] The antibody of the present invention can be used in any form, such as a monoclonal antibody or a polyclonal antibody, and can further include antiserum obtained by immunizing an animal such as a rabbit with the peptide of the present invention, polyclonal antibodies and monoclonal antibodies of all classes, human antibodies, and chimeric antibodies and humanized antibodies produced by genetic recombination.

[0187] The peptide or fragment thereof of the present invention used as an antigen for obtaining an antibody can be obtained by chemical synthesis or by genetic engineering techniques based on the amino acid sequences disclosed herein.

[0188] The peptide used as an immunizing antigen may be the peptide of the present invention or a fragment of the peptide of the present invention. Also, in order to enhance immunogenicity, the peptide may be conjugated or linked to a carrier. As the carrier, keyhole limpet hemocyanin (KLH) is well known. Methods for conjugating KLH and the peptide are also well known in the art.

[0189] Any mammal can be immunized with the antigen, but when producing monoclonal antibodies, it is preferable to consider the compatibility with the parental cells used for cell fusion. Generally, animals of the order Rodentia, Lagomorpha, or Primate can be used. Animals of the order Rodentia include, for example, mice, rats, and hamsters. Animals of the order Lagomorpha include, for example, rabbits. Animals of the order Primate include, for example, monkeys of the suborder Catarrhini (Old World monkeys) such as Macaca fascicularis, rhesus monkeys, baboons, and chimpanzees.

[0190] Methods for immunizing animals with antigens are well known in the art. Intraperitoneal or subcutaneous injection of antigens is a standard method for immunizing mammals. More specifically, the antigen is diluted in an appropriate amount of phosphate-buffered saline (PBS), physiological saline, etc., and suspended. If necessary, the antigen suspension can be mixed with an appropriate amount of standard adjuvant, such as Freund's complete adjuvant, emulsified, and then administered to the mammal. Subsequently, it is preferable to administer the antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. An appropriate carrier may be used for immunization. After immunization as described above, the serum can be examined by standard methods for the increase in the amount of the desired antibody.

[0191] Polyclonal antibodies against the peptide of the present invention can be prepared by collecting blood from a mammal in which an increase in the desired antibody level in the serum has been confirmed after immunization, and separating the serum from the blood by any conventional method. The polyclonal antibody may be serum containing the polyclonal antibody, or a fraction containing the polyclonal antibody may be isolated from the serum. Immunoglobulin G or M can be prepared from a fraction that recognizes only the peptide of the present invention, for example, by using an affinity column conjugated with the peptide of the present invention, and then further purifying this fraction using a protein A or protein G column.

[0192] To prepare monoclonal antibodies, immune cells are collected from mammals after confirming an increase in the desired antibody level in the serum following immunization, and then subjected to cell fusion. The immune cells used for cell fusion can preferably be obtained from the spleen. The other parent cell to be fused with the immune cells can be, for example, mammalian myeloma cells, and more preferably myeloma cells that have acquired characteristics for drug-induced cell selection.

[0193] The above-mentioned immune cells and myeloma cells can be fused according to known methods, for example, the method described by Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

[0194] Hybridomas obtained by cell fusion can be selected by culturing them in a standard selective medium such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically continued in HAT medium for a period sufficient to kill all cells except the desired hybridomas (non-fused cells) (e.g., several days to several weeks). Standard limiting dilutions can then be performed to screen and clone hybridoma cells that produce the desired antibody.

[0195] In addition to the above method of immunizing non-human animals with antigens to prepare hybridomas, human lymphocytes, such as EB virus-infected lymphocytes, can also be immunized in vitro with peptides, cells expressing the peptides, or lysates thereof. The immunized lymphocytes can then be fused with infinitely proliferative human-derived myeloma cells, such as U266, to obtain hybridomas that produce desired human antibodies capable of binding to the peptides (Japanese Patent Publication No. 63-17688).

[0196] Next, the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, and ascites fluid is extracted. The obtained monoclonal antibody can be purified, for example, by ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or affinity column conjugated with the peptide of the present invention.

[0197] Alternatively, immune cells that produce antibodies, such as immunized lymphocytes, can be immortalized using oncogenes and used in the preparation of monoclonal antibodies.

[0198] The monoclonal antibodies obtained in this manner can also be prepared by recombination using gene engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the UK by MacMillan Publishers LTD (1990)). For example, recombinant antibodies can be prepared by cloning the DNA encoding the antibody from an immune cell such as an antibody-producing hybridoma or immunized lymphocyte, inserting it into a suitable vector, and then introducing it into a host cell. The present invention also provides recombinant antibodies prepared in the manner described above.

[0199] Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody, as long as it binds to the peptide of the present invention. For example, the antibody fragment may be Fab, F(ab')2, Fv, or a single-stranded Fv(scFv) in which Fv fragments derived from the H chain and L chain are linked by a suitable linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, the antibody fragment can be prepared by treating the antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding an antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

[0200] Antibodies can be modified by binding with various molecules, such as polyethylene glycol (PEG). This invention provides such modified antibodies. Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are conventional in the art.

[0201] Alternatively, the antibodies of the present invention may be obtained as chimeric antibodies between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or as humanized antibodies comprising a complementation-determining region (CDR) derived from a non-human antibody and a framework region (FR) and a constant region derived from a human antibody. Such antibodies can be prepared according to known techniques. Humanization can be performed by substituting the corresponding sequence of the human antibody with the CDR sequence of the non-human antibody (see, for example, Verhoeyen et al., Science 239:1534-6 (1988)). Thus, such humanized antibodies are chimeric antibodies in which a substantially incomplete human variable domain is substituted with a corresponding sequence derived from a non-human species.

[0202] Complete human antibodies, including the human variable region in addition to the human framework and constant regions, can also be used. Such antibodies can be produced using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments presented on bacteriophages (e.g., Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, human antibodies can also be produced by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes are partially or completely inactivated. This approach is described, for example, in U.S. Patents 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0203] The antibodies obtained as described above may be purified until homogeneous. For example, antibodies can be separated and purified according to separation and purification methods used for common proteins. For example, antibodies can be separated and isolated by appropriately selecting and combining the use of column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing, but are not limited to these (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Protein A columns and Protein G columns can be used as affinity columns. Examples of Protein A columns that should be used include, for example, Hyper D, POROS, and Sepharose FF (Pharmacia).

[0204] Exemplary chromatography methods, in addition to affinity chromatography, include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed using liquid-phase chromatography such as HPLC and FPLC.

[0205] For example, the antigen-binding activity of the antibody of the present invention can be measured using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme-mediated immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence (IF). In the case of ELISA, the antibody of the present invention is immobilized on a plate, the peptide of the present invention is added to the plate, and then a sample containing a desired antibody, such as the culture supernatant of antibody-producing cells or purified antibody, is added. Next, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is added, and the plate is incubated. Subsequently, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, and the antigen-binding activity of the sample is evaluated by measuring the absorbance. To evaluate the antibody binding activity, a peptide fragment, such as a C-terminal or N-terminal fragment, may be used as the antigen. BIAcore (Pharmacia) may be used to evaluate the activity of the antibody of the present invention.

[0206] By exposing a sample believed to contain the peptide of the present invention to the antibody of the present invention, and detecting or measuring the immune complex formed by the antibody and the peptide, the peptide of the present invention can be detected or measured by the method described above. For example, the antibody of the present invention can be used to detect the peptide of the present invention present in a target blood sample (e.g., a serum sample). Alternatively, the antibody of the present invention present in a target blood sample (e.g., a serum sample) can be detected using the peptide of the present invention. The results of measuring the peptide of the present invention or the antibody of the present invention in a target blood sample can be used to select recipients for the pharmaceutical composition of the present invention or to monitor the effects of the pharmaceutical composition of the present invention.

[0207] XIII. Vectors and host cells The present invention also provides a vector comprising a polynucleotide encoding the peptide of the present invention and a host cell into which the vector has been introduced. The vector of the present invention can be used to retain the polynucleotide of the present invention in a host cell, to express the peptide of the present invention in a host cell, or to administer the polynucleotide of the present invention for gene therapy.

[0208] When Escherichia coli is the host cell and the vector is amplified within Escherichia coli (e.g., JM109, DH5α, HB101, or XL1-Blue) to produce large quantities, the vector must have a "replication origin" for amplification within Escherichia coli and a marker gene for selecting transformed Escherichia coli (e.g., a drug resistance gene selected by drugs such as ampicillin, tetracycline, kanamycin, or chloramphenicol). For example, M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script can be used. In addition, pGEM-T, pDIRECT, and pT7 can also be used for cloning in the same way as the above vectors. When the vector is used for the production of the peptide of the present invention, an expression vector may be used. For example, an expression vector to be expressed in Escherichia coli must have the above characteristics in order to be amplified within Escherichia coli. When using E. coli such as JM109, DH5α, HB101, or XL1-Blue as host cells, the vector needs to have a promoter capable of efficiently expressing the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1989)), the araB promoter (Better et al., Science 240: 1041-3 (1988)), or the T7 promoter. In this regard, for example, pGE X-5X-1 (Pharmacia), the "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase) can be used instead of the above vectors. Furthermore, the vector may also contain a signal sequence for peptide secretion. An exemplary signal sequence for inducing peptide secretion into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Methods for introducing the vector into target host cells include, for example, the calcium chloride method and electroporation.

[0209] In addition to Escherichia coli, expression vectors derived from mammals (e.g., pcDNA3 (Invitrogen), and pEGF-BOS (Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), insect cells (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), pBacPAK8), plants (e.g., pMH1, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIpneo), yeast (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis (e.g., pPL608, pKTH50) can be used to produce the polypeptides of the present invention.

[0210] To express a vector in animal cells such as CHO, COS, or NIH3T3 cells, the vector must have promoters necessary for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoter, and preferably a marker gene for selecting transformants (e.g., a drug resistance gene selected by a drug (e.g., neomycin, G418)). Examples of known vectors having these features include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0211] III. Methods for detecting a history of SARS-CoV-2 infection Detection of cellular responses to SARS-CoV-2 infection in the target population. Significant IFN-γ production was observed in T cells stimulated by specific SARS-CoV-2 protein-derived peptides (SEQ ID NOs: 1, 4, 5, 7, 9, 10, 13). Therefore, peptide-specific CTL clones were established, and TCR sequence analysis was performed to identify the CDR3 amino acid sequence of the TCR expressed by the SARS-CoV-2 protein-derived peptide-specific CTL clones (Table 3). If TCRα or TCRβ, or a pair thereof, containing CDR3 with an amino acid sequence as shown in Table 3 is detected in a subject, it means that a peptide-specific CTL response was induced in the subject. Thus, an increase in specific gene pairs in a T cell population stimulated by SARS-CoV-2 protein-derived peptides may be useful as a surrogate marker for detecting a CTL response in a subject after stimulation. If such a CTL response is confirmed, it means that the subject was previously infected with SARS-CoV-2. In relation to the present invention, "peptide-specific CTL response" is understood to mean that the TCR formed between the α-subunit and β-subunit pair specifically recognizes the complex formed between the peptide of the present invention and the HLA molecule. As discussed above, the CTL cell-inducing ability of the peptide defined by the specific sequence of the present invention can be maintained even after amino acid modification. Therefore, in addition to stimulation by the specific peptide, even when T cells are induced from a mutant peptide, their antigen specificity is considered "peptide-specific" insofar as their TCR specifically recognizes such a complex formed by the original peptide.

[0212] In a preferred embodiment, the present invention is a method for detecting a T cell response induced by SARS-CoV-2 infection in a subject, (a) A step of providing a sample obtained from a subject, wherein the sample contains T cells; (b) the step of detecting the presence of SARS-CoV-2 protein-derived peptide-specific T cells induced by SARS-CoV-2 infection in the sample; and (c) If the presence of T cells is indicated in (b) using the T cell receptor (TCR) as an indicator, it indicates a possible past infection with SARS-CoV-2. The present invention provides a method that includes the detection of either or both of the α and β subunits of CDR3, which consist of the amino acid sequences shown in Table 3, to reveal the possibility of past SARS-CoV-2 infection. Specifically, for example, a subject whose past SARS-CoV-2 infection history we want to investigate (HLA-A * PBMCs are collected from the blood of a person who tested positive at 24:02 and the TCR repertoire is analyzed. On the other hand, for example, a person who is not infected with SARS-CoV-2 (HLA-A * Using the 24:02 positive case as a control, similar repertoire analysis results are obtained for the PBMCs, and by comparing the two, if more SARS-CoV-2 protein-derived peptide-specific T cells are detected than in the control, it indicates that the subject has a history of SARS-CoV-2 infection. For the control, for example, human PBMCs from before the reporting of human infection with SARS-CoV-2 can be used. Specifically, human PBMCs prepared from blood collected before December 2019 can be used as a control, but are not limited to this.

[0213] In the present invention, any biological sample obtained from a subject can be used to detect a T cell response, as long as the sample contains T cells. For example, for the purposes of the present invention, blood or a blood-derived sample can be used as a biological sample. In the present invention, a blood-derived sample contains a cell population that includes T cells. Methods for obtaining a cell population that includes T cells are well known to those skilled in the art.

[0214] The T cell receptor (TCR) consists of the V, D, J, and C genes. It is believed that the rearrangement of the V and J genes, as well as the random insertion and deletion of bases between the VDJ genes (CDR3), leads to a diversity of up to 10 to the power of 18 in the TCR. Therefore, there are T cells in the human body that express various TCRs. The study of TCR diversity (what types of TCRs are detected and how frequently) within a given T cell population is called TCR repertoire analysis. When performing TCR repertoire analysis, cDNA with an adapter attached to the 5' end is synthesized from RNA derived from a T cell population in order to amplify various TCR genes without bias using PCR. Next-generation sequencing (NGS) is used to sequence the large number of DNA fragments (sequencing libraries) obtained using adapter-specific forward primers and TCR-α or TCR-β-specific reverse primers. Next-generation sequencers are instruments capable of determining the base sequences of millions of DNA fragments in parallel. In TCR repertoire analysis, it is necessary to determine long base sequences spanning the V, D, J, and C genes that make up the TCR. Therefore, among next-generation sequencers, MiSeq (Illumina), which excels at long-read analysis (determining base sequences of approximately 300 bp), is often used. By comprehensively analyzing the base sequences of TCR-encoding mRNA contained in the sample (essentially meaning analyzing the base sequences of cDNA obtained by reverse transcription of the mRNA base sequences), the detection frequency of each CDR3 in the T cell population constituting the sample can be easily determined. Examples of primer base sequences useful for determining the base sequences of CDR3 are shown below. Forward primer (adapter sequence common to TCR-α and TCR-β, SEQ ID NO: 46): 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3' Reverse primer (for TCR-α, SEQ ID NO: 48): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3' Reverse primer (for TCR-β, SEQ ID NO: 47): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3'

[0215] In other words, the present invention provides a method for detecting a T cell response, comprising the following steps: (a) The step of extracting gDNA from PBMCs derived from the subject, or synthesizing cDNA using RNA extracted from PBMCs as a template; (b) Deciphering the TCRα and TCRβ gene sequences from gDNA or cDNA and determining the frequency of each amino acid sequence in the TCR; (c) The presence of infection-induced SARS-CoV-2-specific T cells is indicated when a SARS-CoV-2-derived peptide-reactive TCR is detected in each amino acid sequence of the TCR determined in (b). In one aspect of the present invention, the detection frequency of SARS-CoV-2-derived peptide-reactive TCRs can be compared with a control group. For example, the results of TCR repertoire analysis of PBMCs derived from SARS-CoV-2 non-infected individuals can be used as a control group. Therefore, if the detection frequency of SARS-CoV-2-derived peptide-reactive TCRs is higher than that of the control group, it indicates that a SARS-CoV-2-specific T cell response was induced. The method of the present invention may include a step of collecting peripheral blood lymphocytes (PBMCs) from a subject prior to the present invention. By repeating a series of analyses over time, changes in the frequency of SARS-CoV-2-derived peptide-responsive TCRs can also be tracked. For example, changes in the frequency of SARS-CoV-2-derived peptide-responsive TCRs can be tracked after inoculation with the SARS-CoV-2-derived peptide of the present invention or after SARS-CoV-2 infection to evaluate the effect of enhancing the immune response.

[0216] Therefore, the T cell response detection method of the present invention makes it possible to know the results of induction of an immune response in subjects to whom the peptide of the present invention has been administered. In other words, the present invention provides a method for detecting a T cell response, comprising the steps of administering the peptide of the present invention to a subject and detecting the T cell response in the subject after administration. Furthermore, the present invention additionally allows for the selection of subjects in whom a sufficient T cell response was not detected and to be targeted for boost inoculation of the peptide of the present invention.

[0217] The following are examples of aspects of the present invention based on the above description, but the present invention is not limited to these. [1] Peptides of less than 15 amino acids having cytotoxic T cell (CTL) induction ability, comprising an amino acid sequence selected from the following groups: (a) Sequence ID: an amino acid sequence selected from the group consisting of 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15; and (b) Amino acid sequences in which one, two, or several amino acids are substituted, deleted, inserted, and / or added to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15. [2] A peptide according to [1] has one or both of the following characteristics for an amino acid sequence selected from the group consisting of Sequence IDs 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15: (a) The second amino acid from the N-terminus is replaced with an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, and tryptophan; and (b) The C-terminal amino acid is replaced with an amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, tryptophan, or methionine. [3] A peptide according to [1], having one or both of the following characteristics for an amino acid sequence selected from the group consisting of Sequence IDs 1, 2, 4, 7, 10, 12, and 13: (a) The second amino acid from the N-terminus is replaced with an amino acid selected from the group consisting of leucine and methionine; and (b) The C-terminal amino acid is replaced with an amino acid selected from the group consisting of valine and leucine. [4] The peptide described in [1], comprising an amino acid sequence selected from the group consisting of Sequence IDs 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. [5] A polynucleotide encoding one of the peptides described in any one of [1] to [4]. [6] A composition comprising a pharmaceutically acceptable carrier and at least one active ingredient selected from the group consisting of (a) to (e): (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4]. [7] The composition according to [6], wherein the active ingredient is at least one component selected from the group consisting of (a) to (d) below, and is a composition for inducing CTLs: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (d) An exosome that presents a complex of a peptide described in any one of [1] to [4] and an HLA antigen on its cell surface. [8] The composition described in [6], which is a pharmaceutical composition. [9] The composition according to [8], which is a pharmaceutical composition for one or more uses selected from the group consisting of (i) treatment of coronavirus infection, (ii) prevention of coronavirus infection, and (iii) suppression of severe coronavirus infection.

[10] The composition according to [8] for inducing an immune response to coronavirus infection.

[11] The composition according to [9] or

[10] , wherein the coronavirus of coronavirus infection is selected from the group consisting of SARS-CoV-2, MERS-CoV and SARS-CoV.

[12] A composition according to any one of [6] to

[11] , formulated for administration to subjects who are HLA-A24 or HLA-A02 positive.

[13] A method for inducing an APC having CTL-inducing ability, comprising a step selected from the group consisting of (a) and (b) below: (a) The step of contacting the APC with the peptide described in any one of items [1] to [4] in vitro, ex vivo, or in vivo, and (b) The step of introducing a polynucleotide encoding the peptide described in any one of the items [1] to [4] into the APC.

[14] A method for inducing CTLs, comprising a step selected from the group consisting of (a) to (c) below: (a) A step of co-culturing CD8-positive T cells with APCs that present a complex of HLA antigen and a peptide described in any one of the items [1] to [4] on their surface, (b) A step of co-culturing CD8-positive T cells with exosomes that present a complex of HLA antigen and a peptide described in any one of the items [1] to [4] on their surface, and (c) A step in which CD8-positive T cells are introduced with polynucleotides that encode each subunit of a T cell receptor (TCR) capable of binding to any of the peptides described in [1] to [4] presented on the cell surface by an HLA antigen.

[15] An APC that presents a complex of an HLA antigen and one of the peptides described in [1] to [4] on its surface.

[16] The APC described in

[15] , derived by the method described in

[13] .

[17] A CTL that targets any of the peptides described in [1] to [4].

[18] CTLs as described in

[17] , induced by the methods described in

[14] .

[19] A method for inducing an immune response to coronavirus infection, comprising the step of administering a composition comprising at least one component selected from the group consisting of (a) to (e) below: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[20] A method for any or more purposes selected from the treatment, prevention and suppression of severe coronavirus infection, comprising the step of administering at least one component selected from the group consisting of (a) to (e) below: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[21] An antibody that binds to any one of the peptides described in [1] to [4].

[22] A method for screening peptides having CTL-inducing ability, comprising the following steps: (a) A step of creating candidate amino acid sequences in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added to an original amino acid sequence consisting of amino acid sequences selected from Sequence IDs 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15; (b) The step of selecting candidate sequences from the candidate sequences created in (a) that do not have significant homology (sequence identity) with any known human gene product; (c) The step of contacting the peptide consisting of the candidate sequence selected in (b) with the APC; (d) The step of bringing the APCs in (c) into contact with CD8-positive T cells; and (e) Selecting a peptide having equivalent or higher CTL-inducing ability than the peptide consisting of the original amino acid sequence.

[23] Use of at least one component selected from the group consisting of (a) to (e) below in the preparation of a composition for inducing an immune response against coronavirus infection: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[24] Use of at least one active ingredient selected from the group consisting of (a) to (e) below in the manufacture of a pharmaceutical composition for any or more purposes selected from the treatment, prevention and suppression of severe coronavirus infection: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[25] Use of at least one active ingredient selected from the following groups (a) to (e) to induce an immune response against coronavirus infection: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[26] Use of at least one active ingredient selected from the following groups (a) to (e) for any or more purposes selected from the treatment, prevention and suppression of severe coronavirus infection: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[27] A method for inducing cytotoxic activity against coronavirus-infected cells, comprising the step of administering at least one active ingredient selected from the group consisting of (a) to (e) below: (a) One or more peptides as described in any one of the items [1] to [4]; (b) One or more polynucleotides encoding a peptide described in any one of the items [1] to [4] in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of a peptide described in any one of the items [1] to [4] and an HLA antigen on their cell surface; (d) Exosomes that present a complex of a peptide described in any one of [1] to [4] and an HLA antigen on their cell surface; and (e) A CTL that targets any one of the peptides described in [1] to [4].

[28] A lyophilized preparation containing one or more types of peptides as described in any one of the items [1] to [4].

[29] A pharmaceutical composition prepared by a method comprising dissolving one or more peptides described in any one of [1] to [4] in a water-soluble carrier and sterilizing by filtration.

[30] An aqueous solution comprising one or more types of peptides described in any one of items [1] to [4] and a water-soluble carrier, which has been filtered and sterilized.

[31] An emulsion comprising one or more peptides described in any one of items [1] to [4], a water-soluble carrier, and an oily adjuvant. A kit comprising a container containing a pharmaceutical composition described in any one of paragraphs

[32] [8] to

[12] , and a container containing an adjuvant.

[33] A kit comprising a container containing a lyophilized preparation comprising any one of the peptides described in [1] to [4], a container containing an adjuvant, and a container containing a resolving solution for the lyophilized preparation. A kit comprising a container containing a composition according to any one of items

[34] [6] to

[12] , and a container containing an adjuvant.

[35] A T cell receptor α chain containing a CDR3 identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, and 40, or a functionally equivalent CDR3.

[36] A T cell receptor β chain containing a CDR3 identified by any amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, and 41, or a functionally equivalent CDR3. A T cell receptor comprising a combination of a T cell receptor α chain from any of the T cell receptors described in

[37]

[35] and a T cell receptor β chain from any of the T cell receptors described in

[36] .

[38] The T cell receptor described in

[37] , wherein the amino acid sequences of the CDR3 of the T cell receptor α chain and the T cell receptor β chain are one of the following combinations: CDR3 of the T cell receptor α chain, CDR3 of the T cell receptor β chain Sequence ID: 32 Sequence ID: 33, Sequence ID: 34 Sequence ID: 35, Sequence ID: 36 Sequence ID: 37, Sequence ID: 38, Sequence ID: 39, and Sequence ID: 40 Sequence ID: 41.

[39] A polynucleotide encoding either a T cell receptor α chain from any of the T cell receptors described in

[35] and either a T cell receptor β chain from any of the T cell receptors described in

[36] .

[40] A TCR that recognizes one of the peptides listed in [1]-[4] that are presented on the APC by the HLA antigen.

[41] Methods for determining a history of SARS-CoV-2 infection, including the following stages: (a) The step of extracting gDNA from PBMCs derived from the subject, or synthesizing cDNA using RNA extracted from PBMCs as a template; (b) The step of comprehensively sequencing the TCRα and TCRβ gene sequences from gDNA or cDNA using NGS (next-generation sequencing) and determining the TCR repertoire; (c) This step involves profiling the TCR repertoire using SARS-CoV-2-derived peptide-reactive TCRs as indicators to evaluate the presence of infection-induced SARS-CoV-2-specific T cells.

[42] The method of

[41] in which a SARS-CoV-2-derived peptide-reactive TCR is the TCR of

[40] .

[0218] Although the present invention has been described in detail herein with respect to its particular aspects, it should be understood that the foregoing description is in fact illustrative and descriptive, and is intended to illustrate the present invention and its preferred embodiments. Those skilled in the art will readily understand, through conventional experimentation, that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Accordingly, the present invention is intended to be defined not by the foregoing description, but by the appended claims and their equivalents.

[0219] The present invention will be described in more detail below with reference to examples. However, the following materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope of the present invention, although they may be helpful to those skilled in the art in the preparation and use of certain forms of the present invention. Those skilled in the art may use similar or equivalent methods and materials in the practice or testing of the present invention. [Examples]

[0220] material and method cell line TISI cells (HLA-A), a human lymphoblastoid cell line *24:02 / -) was purchased from the International Histocompatibility Working Group. It is a human lymphoblast cell line, specifically T2 cells (HLA-A * 02:01 / -) was purchased from ATCC.

[0221] Selection of SARS-CoV-2 derived peptides HLA-A * The 9mer and 10mer peptides derived from SARS-CoV-2 protein that are expected to bind to 24:02 were determined using the binding prediction server "NetMHC 4.0" (http: / / www.cbs.dtu.dk / services / NetMHC / ) (Nielsen M et al., Protein Sci 2003, 12(5):1007-1017; Andreatta M et al., Bioinformatics 2016, 32(4):511-517). In particular, peptides commonly found in SARS-CoV Tor2 (GenBank accession number AY274119), SARS-CoV BJ01 (GenBank accession number AY278488), SARS-CoV GZ02 (GenBank accession number AY390556), and MERS-CoV (GenBank accession number JX869059) were selected as epitope candidates (Kiyotani K et al., J Hum Genet 2020, 65(7):569-575).

[0222] Peptide synthesis The peptide was synthesized by Cosmo Bio Inc. (Tokyo, Japan) according to a solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography (HPLC). The quality of the peptide (purity of 90% or higher) was guaranteed by HPLC and mass spectrometry. The peptide was dissolved in dimethyl sulfoxide (final concentration: 20 mg / ml) and stored at -80°C.

[0223] In vitro CTL induction Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells to induce specific cytotoxic T cells (CTLs) against peptides presented on human leukocyte antigens (HLA). As previously reported in the literature, DCs were generated in vitro (Nakahara S et al., Cancer Res 2003, 63(14):4112-4118). Specifically, healthy volunteers (HLA-A) were used. * 24:02 positive or HLA-A * Peripheral blood mononuclear cells (PBMCs) collected from (02:01 positive) were seeded into tissue culture dishes (Corning), and monocytes in the PBMCs were allowed to adhere to the dishes. They were cultured for 7 days in the presence of 1000 IU / ml granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml interleukin (IL)-4 (R&D System). The culture medium used was AIM-V medium (Invitrogen) containing inactivated type AB serum (MP Biomedicals) (2% ABS / AIM-V medium). DCs differentiated from monocytes by cytokines and autologous CD8-positive T cells obtained using the CD8 Positive Isolation Kit (Invitrogen) were cultured in a ratio of 1:20 (1.5 x 10⁶). 4 1 DC and 3 x 10 5The cells were mixed with 100 CD8-positive T cells and cultured in a 48-well plate (Corning). Peptides were then added (final peptide concentration: 20 μg / ml). 0.5 ml of 2% ABS / AIM-V medium was used per well, and IL-7 (R&D System) and IL-21 (Cell Genix) were added (final concentrations: IL-7 10 ng / ml, IL-21 30 ng / ml). Three days after the start of culture, DCs and peptides were added again (final peptide concentration: 20 μg / ml). DCs were prepared immediately before use using the same method as described above. Seven days after the start of culture, IL-2 (Novartis), IL-7, and IL-15 (Novoprotein) were added (final concentrations: IL-2 48 IU / ml, IL-7 5 ng / ml, and IL-15 5 ng / ml) (Wolfl M et al., Nat Protoc 2014, 9(4):950-966). From day 9 onward (after two DC stimulations), IFN-γ production in peptide-pulsed TISI or T2 cells was confirmed by enzyme-linked immunosorbent spot (ELISPOT) assay.

[0224] CTL propagation procedure CTLs were propagated using a method similar to that reported by Riddell et al. (Walter EA et al., N Engl J Med 1995, 333(16): 1038-1044; Riddell SR et al., Nat Med 1996, 2(2): 216-223). Two types of human B lymphoblastoid cell lines (5 x 10⁶ each) treated with mitomycin C were grown in tissue culture flasks (FALCON). 6CTLs were cultured in 5% ABS / AIM-V medium with IL-2 and anti-CD3 antibody (BD biosciences, final concentration: 40 ng / ml) (culture volume: 25 ml / flask). The day after the start of culture, IL-2 was added to the culture (final IL-2 concentration: 120 IU / ml). On days 5, 8, and 11, the medium was changed with 5% ABS / AIM-V medium containing 60 IU / ml of IL-2 (final IL-2 concentration: 30 IU / ml) (Yoshimura S et al., PLoS One 2014, 9(1):e85267).

[0225] Confirmation of IFN-γ production To confirm peptide-specific IFN-γ production in peptide-induced CTLs, IFN-γ ELISPOT assays and IFN-γ ELISAs were performed. TISI cells or T2 cells pulsed with the peptide were prepared as target cells. The IFN-γ ELISPOT assays and IFN-γ ELISAs were performed according to the assay kit manufacturer's recommended procedures.

[0226] result HLA-A derived from SARS-CoV-2 protein * 24:02 Selection of Binding Peptides Tables 2a and 2b show HLA-A by "NetMHC 4.0". * Table 2a lists 9mer and 10mer peptides derived from SARS-CoV-2 protein that were predicted to bind to 24:02, in order of highest binding affinity. Table 2b shows peptides common to both SARS-CoV and MERS-CoV. Table 2b shows peptides common only to SARS-CoV. HLA-A * A total of 15 peptides were selected as candidate epitope peptides that may have the ability to bind to 24:02.

[0227] [Table 2a] The position number indicates the position of the first amino acid of the peptide, counting from the N-terminus of the protein. The binding affinity (nM) was calculated using "NetMHC4.0".

[0228] [Table 2b] The position number indicates the position of the first amino acid of the peptide, counting from the N-terminus of the protein. The binding affinity (nM) was calculated using "NetMHC4.0".

[0229] HLA-A by SARS-CoV-2 protein-derived peptides * 24:02 Induction of restrictive CTL HLA-A * SARS-CoV-2 protein-derived peptide-specific CTLs were induced using 24:02-positive PBMCs according to the protocol described in "Materials and Methods". Peptide-specific IFN-γ production by cells was confirmed by the ELISPOT assay (Figure 1). Peptide-specific IFN-γ production was observed for peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), 11 (SEQ ID NO: 11), 12 (SEQ ID NO: 12), 13 (SEQ ID NO: 13), and 15 (SEQ ID NO: 15) (Figure 1a). On the other hand, no specific IFN-γ production was observed for the other peptides shown in Tables 2a and 2b. For example, no specific IFN-γ production was observed for peptide 6 (SEQ ID NO: 6) (Figure 1b). All peptides are HLA-A * There was a possibility of binding to 24:02, but the result was HLA-A * Twelve peptides that bind to 24:02 and possess CTL-inducing activity were identified.

[0230] SARS-CoV-2 protein-derived peptide-specific HLA-A * 24:02 Establishment of restrictive CTL lines HLA-A *Cells that showed specific IFN-γ production in response to peptide 1 (SEQ ID NO: 1), peptide 2 (SEQ ID NO: 2), peptide 4 (SEQ ID NO: 4), peptide 5 (SEQ ID NO: 5), peptide 7 (SEQ ID NO: 7), peptide 9 (SEQ ID NO: 9), peptide 10 (SEQ ID NO: 10), or peptide 13 (SEQ ID NO: 13) in a 24:02-restricted IFN-γ ELISPOT assay were grown to evaluate HLA-A * A 24:02-restricted CTL line was established. IFN-γ measurement by ELISA revealed HLA-A ionization when pulsed with peptide 1 (SEQ ID NO: 1), peptide 2 (SEQ ID NO: 2), peptide 4 (SEQ ID NO: 4), peptide 5 (SEQ ID NO: 5), peptide 7 (SEQ ID NO: 7), peptide 9 (SEQ ID NO: 9), peptide 10 (SEQ ID NO: 10), or peptide 13 (SEQ ID NO: 13). * IFN-γ production from the CTL line was observed against 24:02-expressing target cells (TISI cells) (Figure 2). This suggests that peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), and 13 (SEQ ID NO: 13) are HLA-A * It was clearly demonstrated that it binds to 24:02 and has CTL-inducing ability.

[0231] HLA-A by SARS-CoV-2 protein-derived peptides * 02:01 Induction of restrictive CTL HLA-A * Peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), 11 (SEQ ID NO: 11), 12 (SEQ ID NO: 12), 13 (SEQ ID NO: 13), and 15 (SEQ ID NO: 15), which have been confirmed to have 24:02-restricted CTL-inducing ability, are HLA-A * 02:01 We investigated whether or not it possesses the ability to induce restrictive CTLs. HLA-A *02:01 Using positive PBMCs, HLA-A * 02:01 Restricted CTLs were induced according to the protocol described in "Materials and Methods". Peptide-specific IFN-γ production of the cells was confirmed by ELISPOT assay (Figure 3). Peptide-specific IFN-γ production was observed in Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 4 (SEQ ID NO: 4), Peptide 7 (SEQ ID NO: 7), Peptide 10 (SEQ ID NO: 10), Peptide 12 (SEQ ID NO: 12) and Peptide 13 (SEQ ID NO: 13) (Figure 3a). On the other hand, specific IFN-γ production against Peptide 3 (SEQ ID NO: 3), Peptide 5 (SEQ ID NO: 5), Peptide 9 (SEQ ID NO: 9), Peptide 11 (SEQ ID NO: 11) and Peptide 15 (SEQ ID NO: 15) was not observed. The result of Peptide 5 (SEQ ID NO: 5) is shown as an example (Figure 3b). From the above, HLA-A * 24:02 Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 4 (SEQ ID NO: 4), Peptide 7 (SEQ ID NO: 7), Peptide 10 (SEQ ID NO: 10), Peptide 12 (SEQ ID NO: 12) and Peptide 13 (SEQ ID NO: 13) having the ability to induce restricted CTLs also bind to HLA-A * 02:01 and it was revealed that they have the ability to induce HLA-A * 02:01 restricted CTLs.

[0232] SARS-CoV-2 protein-derived peptide-specific HLA-A * 02:01 Establishment of restrictive CTL lines HLA-A * Cells showing specific IFN-γ production against Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 10 (SEQ ID NO: 10) or Peptide 13 (SEQ ID NO: 13) in the HLA-A * 02:01 restricted IFN-γ ELISPOT assay were proliferated to establish an HLA-A *IFN-γ production from the CTL line was observed against 02:01-expressing target cells (T2 cells) (Figure 4). This suggests that peptide 1 (SEQ ID NO: 1), peptide 2 (SEQ ID NO: 2), peptide 10 (SEQ ID NO: 10), and peptide 13 (SEQ ID NO: 13) are HLA-A * It also binds at 02:01, HLA-A * It was clearly demonstrated that it possesses the ability to induce restrictive CTLs at 02:01.

[0233] Peptide homology analysis Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 3 (SEQ ID NO: 3), Peptide 4 (SEQ ID NO: 4), Peptide 5 (SEQ ID NO: 5), Peptide 7 (SEQ ID NO: 7), Peptide 9 (SEQ ID NO: 9), Peptide 10 (SEQ ID NO: 10), Peptide 11 (SEQ ID NO: 11), Peptide 12 (SEQ ID NO: 12), Peptide 13 (SEQ ID NO: 13), and Peptide 15 (SEQ ID NO: 15) were confirmed to be able to induce CTLs that exhibit peptide-specific IFN-γ production. Therefore, in order to confirm the homology between the amino acid sequences of Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 3 (SEQ ID NO: 3), Peptide 4 (SEQ ID NO: 4), Peptide 5 (SEQ ID NO: 5), Peptide 7 (SEQ ID NO: 7), Peptide 9 (SEQ ID NO: 9), Peptide 10 (SEQ ID NO: 10), Peptide 11 (SEQ ID NO: 11), Peptide 12 (SEQ ID NO: 12), Peptide 13 (SEQ ID NO: 13), and Peptide 15 (SEQ ID NO: 15) and the amino acid sequences derived from human proteins, a homology analysis was performed using the BLAST algorithm (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result, the amino acid sequences of Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2), Peptide 3 (SEQ ID NO: 3), Peptide 4 (SEQ ID NO: 4), Peptide 5 (SEQ ID NO: 5), Peptide 7 (SEQ ID NO: 7), Peptide 9 (SEQ ID NO: 9), Peptide 10 (SEQ ID NO: 10), Peptide 11 (SEQ ID NO: 11), Peptide 12 (SEQ ID NO: 12), Peptide 13 (SEQ ID NO: 13), and Peptide 15 (SEQ ID NO: 15) were not found in human proteins. Therefore, as far as the inventors know, these peptides are derived from SARS-CoV-2 or SARS-CoV or MERS-CoV and are considered to have little potential to cause unintended immune responses against normal human tissues. In conclusion, novel HLA-A * 24:02 or HLA-A * 02:01 restricted epitope peptides were identified and shown to be applicable to peptide vaccines against COVID-19.

Example

[0234] material and method Establishment of CTL clones (limiting dilution method) Cells induced by CTLs in vitro were seeded into 96-well round-bottom microplates (Corning) at a density of 1 cell per well. Two types of human B lymphoblastoid cell lines treated with mitomycin C (1 x 10⁶ each) were then seeded. 4 Cells were cultured with anti-CD3 antibody (final concentration: 30 ng / ml) and IL-2 (final concentration: 150 IU / ml) (culture volume: 150 μl / well). The culture medium used was AIM-V medium containing inactivated type AB serum (SIGMA) (5% ABS / AIM-V medium). After 10 days, 50 μl of 5% ABS / AIM-V medium containing 600 IU / ml of IL-2 was added to the culture (Uchida N et al., Clin Cancer Res 2004, 10(24):8577-8586; Suda T et al., Cancer Sci 2006, 97(5):411-419; Watanabe T et al., Cancer Sci 2005, 96(8):498-506). From day 14 onward, CTLs that showed peptide-specific IFN-γ production in the ELISPOT assay were grown using the previously described method (CTL proliferation procedure). ELISA was performed to re-verify peptide-specific IFN-γ production. The IFN-γ ELISPOT assay and IFN-γ ELISA were performed according to the procedures recommended by the assay kit manufacturer. TISI cells were used as target cells.

[0235] TCR analysis RNA was extracted from SARS-CoV-2 protein-derived peptide-specific CTL clones using the RNeasy mini kit, and cDNA was synthesized. The nucleotide sequences of the TCRα and TCRβ chains were deciphered by Sanger sequencing analysis. After TA cloning, the TCRα chain was sequenced using M13 forward primers (5'-TGTAAAACGACGGCCAGTG-3' (SEQ ID NO: 42) or 5'-CTGGCCGTCGTTTTAC-3' (SEQ ID NO: 43) and M13 reverse primers (5'-CAGGAAACAGCTATGACCAT-3' (SEQ ID NO: 44) or 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 45)). For the TCRβ chain, the forward primer (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3', SEQ ID NO: 46) and reverse primer (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3', SEQ ID NO: 47) were used. We referenced the nucleotide sequences of the TCRα and TCRβ genes registered in 2005, 33 (Database issue): D256-261).

[0236] result Establishment of peptide-specific CTL clones CTL clones that recognize SARS-CoV-2 protein-derived peptides were established using the limiting dilution method. IFN-γ measurements by ELISA revealed that the CTL clones exhibited specific IFN-γ production in response to peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), and 10 (SEQ ID NO: 10) (Figure 5). This confirmed that the CTL clones recognized SARS-CoV-2 protein-derived peptides presented in the HLA.

[0237] Identification of TCRs expressing peptide-specific CTL clones Sanger sequencing analysis identified the CDR3 amino acid sequence of the TCR expressed by SARS-CoV-2 protein-derived peptide-specific CTL clones (Table 3).

[0238] [Table 3] [Examples]

[0239] material and method PBMC A COVID-19 recovered patient (HLA-A) whose RT-PCR test detected SARS-CoV-2-derived DNA (positive) and who subsequently tested negative after the symptomatic period has passed. * PBMCs derived from (24:02 positive) were purchased from Precision For Medicine. SARS-CoV-2 non-infected individuals (HLA-A) were collected before December 2019. * The PBMC derived from the 24:02 positive case was purchased from Cellular Technology Limited.

[0240] PBMC culture PBMCs collected from COVID-19 recovered individuals or SARS-CoV-2 uninfected individuals (5x10) 5 The cells were seeded in 48-well multi-well plates (Corning) at a density of cells per well, and then cultured for 12 days. Complete medium containing inactivated fetal bovine serum (GIBCO) (a mixture of equal volumes of RPMI1640 medium and AIM-V medium) was used. Peptides were added on the first day of culture and 4 days later (final concentration: 10 μg / ml). IL-2 was added 5, 7, and 10 days after the start of culture (final concentration: 120 IU / ml). Post-culture PBMCs were used in tetramer assays.

[0241] Tetramer fabrication QuickSwitch TM Quant HLA-A *Tetramers were prepared for peptides 1 (SEQ ID NO: 1), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), and 13 (SEQ ID NO: 13) using the 24:02 Tetramer Kit-PE (MBL International Corporation) according to the manufacturer's recommended procedure. Exiting peptide bound QuickSwitch TM 50 μl of tetramer and 1 μl of 1 mg / ml peptide solution were mixed. Further addition of 1 μl of peptide exchange factor was performed, and the mixture was allowed to stand at room temperature for more than 4 hours. As a result, a tetramer was obtained in which the exiting peptide was replaced with the target peptide.

[0242] Tetramer assay Tetramer assays were performed on PBMCs derived from COVID-19 recovered individuals or SARS-CoV-2 uninfected individuals. Since peptide-recognizing T cells bind to tetramers via the TCR, PBMCs were treated with 450 nM Dasatinib (Cayman Chemical) (37°C, 30 minutes) to preserve TCR expression on the T cell surface (Lissina A et al., J Immunol Methods 2009, 340(1):11-24). After staining the PBMCs with the tetramer, they were further stained with FITC-labeled anti-CD8 antibody, APC-labeled anti-CD3 antibody, and PE-Cy7-labeled anti-CD4 antibody (all from BD Biosciences). Finally, they were stained with 0.1 μg / ml DAPI solution (BD Biosciences) and analyzed using a flow cytometer (SH800 cell sorter, Sony). Tetramer-positive CD8-positive T cells were identified in the DAPI-negative CD3-positive CD4-negative cell population. PE-labeled HIV tetramer (Medical & Biological Laboratories, Inc.) was used as a negative control.

[0243] result Detection of tetramer-positive CD8-positive T cells CD8-positive T cells recognizing peptides 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), or 13 (SEQ ID NO: 13), derived from SARS-CoV-2 protein, were detected in PBMCs of COVID-19 recovered individuals (Figure 6a). CD8-positive T cells recognizing peptides 1 (SEQ ID NO: 1), 7 (SEQ ID NO: 7), or 13 (SEQ ID NO: 13) were detected in PBMCs of SARS-CoV-2 uninfected individuals (Figure 6b). Peptides 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), and 13 (SEQ ID NO: 13) were suggested to be epitopes that induce peptide-specific CTLs in vivo. It was confirmed that peptides 1 (SEQ ID NO: 1), 7 (SEQ ID NO: 7), and 13 (SEQ ID NO: 13) can be recognized by some CTLs (cross-reactive T cells) that were previously induced by exogenous antigens. These results indicate that peptides 1 (SEQ ID NO: 1), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5), 7 (SEQ ID NO: 7), 9 (SEQ ID NO: 9), 10 (SEQ ID NO: 10), and 13 (SEQ ID NO: 13) can be applied to peptide vaccines against COVID-19 as antigens that stimulate T cells and induce cellular immunity. [Examples]

[0244] material and method PBMC Subjects whose past SARS-CoV-2 infection history is to be investigated (HLA-A * PBMCs are collected from the blood of individuals who tested positive at 24:02. In addition, HLA-A samples are collected from SARS-CoV-2 non-infected individuals (HLA-A) collected before December 2019. * PBMCs derived from the 24:02 positive case will be purchased from Cellular Technology Limited.

[0245] TCR analysis RNA is extracted from PBMCs using the RNeasy mini kit, and then cDNA is synthesized. Alternatively, gDNA (Genomic DNA) is extracted from PBMCs. TCR-α and TCR-β sequences are analyzed using a next-generation sequencer, and a TCR repertoire analysis is performed. The following primers are used to determine the base sequence of CDR3. Forward primer (adapter sequence common to TCR-α and TCR-β, SEQ ID NO: 46): 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3' Reverse primer (for TCR-α, SEQ ID NO: 48): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3' Reverse primer (for TCR-β, SEQ ID NO: 47): 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3'

[0246] result TCR repertoire analysis of subject PBMCs By analyzing the detection frequency of TCR sequences, if TCRs containing CDR3 of TCRα or TCRβ, as shown in Table 3, are detected more frequently than in the control group (PBMCs from non-SARS-CoV-2 infected individuals), it indicates that the subject had been infected with SARS-CoV-2 in the past. [Industrial applicability]

[0247] The present invention provides novel HLA-A24 or HLA-A02-restricted epitope peptides derived from SARS-CoV-2 protein that induce a potent and specific immune response to coronavirus infection and thus have applicability to a wide range of coronavirus infection types. The peptides, compositions, APCs, and CTLs of the present invention can be used as peptide vaccines against coronavirus infections, such as SARS-CoV-2, MERS-CoV, or SARS-CoV infection. Furthermore, the TCR sequences induced by the peptide of the present invention can be used in a method for detecting a history of SARS-CoV-2 infection.

[0248] While the present invention is described in detail in this specification with respect to its particular aspects, it should be understood that the foregoing description is essentially illustrative and descriptive, intended to illustrate the invention and its preferred embodiments. Through conventional experimentation, those skilled in the art will readily recognize that various modifications and alterations can be made therein without departing from the spirit and scope of the invention, whose boundaries and limitations are defined by the appended claims.

[0249] Sequence information SEQUENCE LISTING <110> ONCOTHERAPY SCIENCE, INC. CANCER PRECISION MEDICINE, INC. <120> SARS-CoV-2 PROTEIN-DERIVED PEPTIDES AND VACCINES INCLUDING THE SAME <150> JP 2020-164630 <151> 2020-09-30 <150> US 63 / 236,927 <151> 2021-08-25 <150> PCT / JP2021 / 017159 <151> 2021-04-30 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 1 Ala Tyr Ala Asn Ser Val Phe Asn Ile 1 5 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 2 Thr Ala Tyr Ala Asn Ser Val Phe Asn Ile 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 3 Val Phe Met Ser Glu Ala Lys Cys Trp 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 4 Thr Tyr Ala Ser Ala Leu Trp Glu Ile 1 5 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from surface glycoprotein of SARS-CoV-2 <400> 5 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 6 Tyr Asp Tyr Leu Val Ser Thr Gln Glu Phe 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 7 Tyr Tyr Ser Gln Leu Met Cys Gln Pro Ile 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 8 Ser Tyr Tyr Ser Leu Leu Met Pro Ile 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 9 Arg Tyr Lys Leu Glu Gly Tyr Ala Phe 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 10 Phe Thr Tyr Ala Ser Ala Leu Trp Glu Ile 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 11 Ser Tyr Ser Leu Phe Asp Met Ser Lys Phe 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 12 Arg Tyr Phe Lys Tyr Trp Asp Gln Thr Tyr 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from membrane glycoprotein of SARS-CoV-2 <400> 13 Leu Trp Leu Leu Trp Pro Val Thr Leu 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 14 Thr Tyr Ala Ser Ala Leu Trp Glu Ile Gln 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> an amino acid sequence derived from ORF1ab polyprotein of SARS-CoV-2 <400> 15 Tyr Ala Tyr Leu Arg Lys His Phe Ser Met 1 5 10 <210> 16 <211> 29903 <212> DNA <213> Severe acute respiratory syndrome coronavirus 2 <400> 16 attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc ttgtagatct 60 gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc tgtgcact 120 cacgcagtat attaataac taattactgt cgttgacagg acacgagtaa ctcgtctatc 180 ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt 240 cgtccgggtg tgaccgaaag gtaagatgga gagccttgtc cctggtttca acgagaaaac 300 acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg 360 agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg 420 cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa 480 acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact 540 cgaaggcatt cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600 cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660 tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720 tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga 780 actcatgcgt gagcttaacg gaggggcata cactcgctat gtcgataaca acttctgtgg 840 ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc 900 atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg 960 tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca 1020 gacacctttt gaaattaaat tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa 1080 ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa 1140 gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg 1200 caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca 1260 gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga 1320 aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc 1380 atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg 1440 cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc 1500 ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg 1560 ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga 1620 aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga 1680 gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa 1740 aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac 1800 aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc 1860 tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct 1920 tgaaactgct caaaattctg tgcgtgtttt acagaaggcc gctataacaa tactagatgg 1980 aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040 taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100 gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160 agaaagttt aaagaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220 ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280 ggagagtgtt cagacattct ttaagcttgt aaaataattt ttggctttgt gtgctgactc 2340 tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400 ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460 tctaaaagcc ccaaaagaaa ttatcttctt agaggagaa acacttccca cagaagtgtt 2520 2580 agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640 aatcaaagac agaaaagt actgtgccct tgcacctaat atgatggtaa aaacaatac 2700 cttcacatc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga 2760 2820 acttaatgag aagtgctctg cctatacagt tgaacctcggt acagaagtaa atgagttcgc 2880 ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940 actgggcatt gatttagatg agtggatt ggctacatac tacttatttg atgagtctgg 3000 tgagtttaaa ttggctcac atatgtattg ttctcttac cctccagatg aggatgaaga 3060 agaaggtgat tgtgagaag agagttttga gccatcact caatgagt atggtactga 3120 agatgattac caagtaac ctttggaatt tgtgccact tctgctgctc tcaacctga 3180 frequency frequency ggagatt gggttagatga tgagtcaaactgttg gtxaag 3240 cggcagtgag gatatcaga acaccatt tcaacatt gttgaggttc aaccctcatt 3300 agagatggaa cttacaccag ttgttcagac tattgaagtg atagtttta gtggttattt 3360 aaaacttact gatagtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3420 aaaaccaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc 3480 aggagcctta ataaggcta ctacaatgc catgcaagtt gatctgatg attackagc 3540 tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3600 acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc aacttcttaa 3660 gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg 3720 tatttttggt gctgacccta tacattcttt aagagtttgt gtatagactg ttcgcacaaa 3780 tgtctactta gctgtctttg ataaaaatct ctatgacaaa cttgtttcaa gctttttgga 3840 aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa 3900 gccatttata actgaaagta aaccttcagt tgaacagaga aaacaagatg ataagaaaat 3960 caaagcttgt gttgaagaag ttacaacaac tctggagaaa actaagttcc tcacagaaaa 4020 cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag 4080 tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca 4140 agagggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat 4200 gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca 4260 gggtttaaat ggttacactg tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc 4320 cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc 4380 ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg 4440 tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca 4500 agagggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc 4560 gtcacttatc aacacactta acgatctaaa tgaaactctt gttacaatgc cacttggcta 4620 tgtaacacat ggcttaaatt tggaagaagc tgctcggtat atgagatctc tcaaagtgcc 4680 agctacagtt tctgtttctt cacctgatgc tgttacagcg tataatggtt atcttacttc 4740 ttcttctaaa acacctgaag aacattttat tgaaaccatc tcacttgctg gttcctataa 4800 agattggtcc tattctggac aatctacaca actaggtata gaatttctta agagaggtga 4860 taaaagtgta tattacacta gtaatcctac cacattccac ctagatggtg aagttatcac 4920 ctttgacaat cttaagacac ttctttcttt gagagaagtg aggactatta aggtgtttac 4980 aacagtagac aacattaacc tccacacgca agttgtggac atgtcaatga catatggaca 5040 acagtttggt ccaacttatt tggatggagc tgatgttact aaaataaaac ctcataattc 5100 acatgaaggt aaaacatttt atgttttacc taatgatgac actctacgtg ttgaggcttt 5160 tgagtactac cacacaactg atcctagttt tctgggtagg tacatgtcag cattaaatca 5220 cactaaaaag tggaaatacc cacaagttaa tggtttaact tctattaaat gggcagataa 5280 caactgttat cttgccactg cattgttaac actccaacaa atagagttga agtttaatcc 5340 acctgctcta caagatgctt attacagagc aagggctggt gaagctgcta acttttgtgc 5400 acttatctta gcctactgta ataagacagt aggtgagtta ggtgatgtta gagaaacaat 5460 gagttacttg tttcaacatg ccaatttaga ttcttgcaaa agagtcttga acgtggtgtg 5520 taaaacttgt ggacaacagc agacaaccct taagggtgta gaagctgtta tgtacatggg 5580 cacactttct tatgaacaat ttaagaaagg tgttcagata ccttgtacgt gtggtaaaca 5640 agctacaaaa tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc 5700 tcagtatgaa cttaagcatg gtacatttac tgtgctagt gagtacactg gtaattacca 5760 gtgtggtcac tataaacata taacttctaa agaactttg tattgcatag acggtgcttt 5820 acttacaag tcctcagaat aaaggtcc tattacggat gtttctaca aagaaaacag 5880 ttacacaaca accataaaac cagttactta taaattggat ggtgttgttt gtacagaat 5940 tgaccctaag ttggacaatt attatagaa agacaatct tatttcacag agcaaccaat 6000 tgatcttgta caacacaccaaa cgcaagctc gataatttta agtttgtatg 6060 tgataatatc aaatttgctg atgatttaaa ccagttaact ggttataaga aacctgctc 6120 aagagagctt aaagttacat ttttccctga cttaaatggt gatgtggtgg ctattgatta 6180 taaacaacct acacccttt ttaagaaagg agctaattg ttacataac ctattgttg 6240 gcatgttaac aatgcaacta aaagccac gtataaacca atacctggt gtatacgttg 6300 tctttggagc acaaaaccag ttgaaacatc aaattcgtttt gatgtactga agtcagagga 6360 cgcgcaggga atggataatc ttgcctgcga agatctaaaa ccagtctctg agaagtagt 6420 ggaaaatcct accatacaga aagacgttct tgagtgtaat gtgaaaacta ccgaagttgt aggagacatt attack attack attack tagtttaaaa attack aggttggcca cacagatcta atggctgctt atgtagacaa ttctagtctt actattaga aacctaatga attack gtattaggtt tgaaaaccct tgctactcat ggtttagctg ctgttaatag tgtcccttgg ctaattatag ctaattatgc tagcctttt cttaacaaag ttgttagtac aactactaac atagttacac ggtgtttaa ccgtgtttgt actattata tgccttattt ctttacttta ttgctacaat tgtgtacttt tactagaagt acaaattcta gattaaagc atctatgccg actactatag caaagaatac tgttaagagt gtcggtaaat tttgtctaga ggcttcattt aattattga agtcacctaa tttttctaaa ctgataaata ttataatttg gtttttacta ttagtgttt gcctaggttc tttaatctac tcaaccgctg ctttaggtgt 7020. 7080. tttaatgtct aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa 7140. ctctactaat gtcactattg caacctactg tactggttct ataccttgta gtgtttgtct tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa ttaccatttc 7200 atcttttaaa tgggatttaa ctgcttttgg cttagttgca gagtggtttt tggcatatat 7260 tcttttcact aggtttttct atgtacttgg attggctgca atcatgcaat tgtttttcag 7320 ctatttttgca gtacatttta ttagtaattc ttggcttatg tggttaataa ttaatcttgt 7380 acaaatggcc ccgatttcag ctatggttag aatgtacatc ttctttgcat cattttatta 7440 tgtatggaaa agttatgtgc atgttgtaga cggttgtaat tcatcaactt gtatgatgtg 7500 ttacaaacgt aatagagcaa caagagtcga atgtacaact attgttaatg gtgttagaag 7560 gtccttttat gtctatgcta atggaggtaa aggcttttgc aaactacaca attggaattg 7620 tgttaattgt gatacattct gtgctggtag tacatttatt agtgatgaag ttgcgagaga 7680 7740 tagtgttaca gtgaagaatg gttccatcca tctttacttt gataaagctg gtcaaaagac 7800 ttatgaaga cattctctct ctcattttgt taacttagac aacctgagag ctaataacac 7860 taaaggttca ttgcctatta atgttatagt ttttgatggt aaatcaaaat gtgaagaatc 7920 atctgcaaaa tcagcgtctg tttactacag tcagcttatg tgtcaaccta tactgttact 7980 agatcaggca ttagtgtctg atgttggtga tagtgcggaa gttgcagtta aaatgtttga 8040 tgcttacgtt aatacgtttt catcaacttt taacgtacca atggaaaaac tcaaaacact 8100 agttgcaact gcagaagctg aacttgcaaa gaatgtgtcc ttagacaatg tcttatctac 8160 ttttattca gcagctcggc aagggtttgt tgattcagat gtagaaacta aagatgttgt 8220 tgaatgtctt aaattgtcac atcaatctga catagaagtt actggcgata gttgtaataa 8280 ctatatgctc acctataaca aagttgaaaa catgacaccc cgtgaccttg gtgcttgtat 8340 tgactgtagt gcgcgtcata ttaatgcgca ggtagcaaaa agtcacaaca ttgctttgat 8400 atggaacgtt aaagatttca tgtcattgtc tgaacaacta cgaaaacaaa tacgtagtgc 8460 tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa 8520 tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8580 gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc 8640 tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat 8700 tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 8760 tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 8820 attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac 8880 gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt 8940 tggtaacatc tgttacacac catcaaaact tatagagtac actgactttg caacatcagc 9000 ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc cagtaccata 9060 ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac gccctgacac 9120 acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggttc 9180 tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc 9240 agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag 9300 atctttacca ggagttttct gtggtgtaga tgctgttaaat ttacttacta atatgtttac 9360 accactaatt caacctattg gtgctttgga catatcagca tctagattag ctggtggtat 9420 tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagcttttgg 9480 tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540 ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttatt acttgtactt 9600 gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660 cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720 tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780 tagtacttttt gaaagctg cgctgtgcac cttttgtta aataagaaa tgtatctaaa 9840 gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900 tagtacaag tattttagtg gagcaatgga tacaactagc tacagaag ctgcttgttg 9960 tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc tttaccaacc 10020 accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc 10080 atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg 10140 tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat 10200 gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca 10260 ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct 10320 taaggttgat acagccaatc ctaagacacc tagtataag tttgttcgca ttcaaccagg 10380 agaactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc 10440 tatgaggccc aatttcacta ttaagggttc attccttaat ggttcatgtg gtagtgttgg 10500 ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac 10560 tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca 10620 aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta 10680 cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga 10740 ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagacc atgttgacat 10800 actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa 10860 agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga 10920 tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt 10980 gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcacttt 11040 agttttagtc cagagtactc aatggtcttt gttcttttttt ttgtatgaaa atgcctttt 11100 accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa 11160 gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt atttaatat 11220 ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatac 11280 tagttgtct ggttttaagc taaaagactg tgttatgtat gcatcagctg tagtgttact 11340 aatccttatg acagcaagaa ctgtgtatga tgatggtgct aggagagtgt ggacacttat 11400 gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagatc aagccatttc 11460 catgtgggct cttataatct ctgttacttc taactactca ggtgtagtta caactgtcat 11520 gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac 11580 tggtaataca cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg 11640 ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg gtgtttatga 11700 ttacttagtt tctacacagg agtttagata tatgaattca cagggactac tcccacccaa 11760 gaatagcata gatgccttca aactcaacat taaattgttg ggtgttggtg gcaaaccttg 11820 tatcaaagta gccactgtac agtctaaaat gtcagatgta aagtgcacat cagtagtctt 11880 actctcagtt ttgcaacaac tcagagtaga atcatcatct aaattgtggg ctcaatgtgt 11940 ccagttacac aatgacattc tcttagctaa agatactact gaagcctttg aaaaaatggt 12000 ttcactactt tctgttttgc tttccatgca gggtgctgta gacataaaca agctttgtga 12060 agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta gttcccttcc 12120 atcatatgca gcttttgcta ctgctcaaga agcttatgag caggctgttg ctaatggtga 12180 ttctgaagtt gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga 12240 ccgtgatgca gccatgcaac gtaagttgga aaagatggct gatcaagcta tgacccaaat 12300 gtataaacag gctagatctg aggacaagag ggcaaaagtt actagtgcta tgcagacaat 12360 gcttttcact atgcttagaa agttggataa tgatgcactc aacaacatta tcaacaatgc 12420 aagagatggt tgtgttccct tgaacataat acctcttaca acagcagcca aactaatggt 12480 tgtcatacca gactataaca catataaaaa tacgtgtgat ggtacaacat ttacttatgc 12540 atcagcattg tgggaaatcc aacaggttgt agatgcagat agtaaaattg ttcaacttag 12600 tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa cagctttaag 12660 ggccaattct gctgtcaaat tacagaataa tgagcttagt cctgttgcac tacgacagat 12720 gtcttgtgct gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta 12780 caacacaaca aagggaggta ggtttgtact tgcactgtta tccgatttac aggatttgaa 12840 atgggctaga ttccctaaga gtgatggaac tggtactatc tatacagaac tggaaccacc 12900 ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtattat actttattaa 12960 aggattaaac aacctaaata gaggtatggt acttggtagt ttagctgcca cagtacgtct 13020 acaagctggt aatgcaacag aagtgcctgc caattcaact gtattatctt tctgtgcttt 13080 tgctgtagat gctgctaaag cttacaaaga ttatctagct agtgggggac aaccaatcac 13140 obeygtgtt aagatgttgt gtacacacac tggtactggt caggcaataa cagttacacc 13200 ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt actgccgttg 13260 ccacatagat catccaaatc ctaaaggatt ttgtgactta aaaggtaagt atgtacaaat 13320 acctacaact tgtgctaatg accctgtggg ttttacactt aaaaacacag tctgtaccgt 13380 ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca 13440 gtcagctgat gcacaatcgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca 13500 ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat ctacaatgat 13560 aaagtagctg gttttgctaa attcctaaaa actaattgtt gtcgcttcca agaaaggac 13620 gaagagatgaca atttaattga ttcttacttt gtagttaaga vakacacttt ctctaactac 13680 cacatgaag aacaattta taatttactt aaggattgtc cagctgttgc taacatgac 13740 ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca acgtcttact 13800 aaatacacaa tggcagacct cgtctatgct tgaggcatt tgatgagg taattgtgac 13860 acattaaaag aaatacttgt cacatacaat tgttgtgatg atgattttt cataaaag 13920 gactggtatg attttgtaga aaacccagat atattacgcg tatacgccaa cttaggtgaa 13980 cgtgtacgcc aagctttgtt aaaaacagta cattctgtg atgccatgcg aaatgctggt 14040 attgttggtg tactgacatt agataatcaa gatctcaatg gtactggta tgatttcggt 14100 gatttcatac aaaccacgcc aggtagtgga gttcctgttg tagattctta ttattcattg 14160 ttaatgccta tattaacctt gaccagggct ttaactgcag agtcacatgt tgacactgac 14220 ttaacaaagc cttacattaa gtgggatttg ttaaaatatg acttcacgga agagaggtta 14280 aaactctttg accgttattt taaatattgg gatcagacat accacccaaa ttgtgttaac 14340 tgtttggatg acagatgcat tctgcattgt gcaaacttta atgttttatt ctctacagtg 14400 ttcccaccta caagttttgg accactagtg agaaaaatat ttgttgatgg tgttccattt 14460 gtagtttcaa ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac 14520 ttacatagct ctagacttag ttttaaggaa ttacttgtgt atgctgctga ccctgctatg 14580 cacgctgctt ctggtaatct attactagat aaacgcacta cgtgcttttc agtagctgca 14640 cttactaaca atgttgcttt tcaaactgtc aaacccggta atttaacaa agacttctat 14700 gactttgctg tgtctaaggg tttctttaag gaaggaagtt ctgttgaatt aaaacacttc 14760 ttctttgctc aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta 14820 ccaacaatgt gtgatatcag acaactacta tttgtagttg aagttgttga taagtacttt 14880 gattgttacg atggtggctg tattaatgct aaccaagtca tcgtcaacaa cctagacaaa 14940 tcagctggtt ttccattaa taaatggggt aaggctagac tttattga ttcaatgagt 15000 tatgaggatc aagatgcact ttcgcatat acaaaacgta atgtcatccc tactataact 15060 caaatgaatc ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc 15120 tctatctgta gtactatgac caatagacag tttcatcaaa aattattgaa atcaatagcc 15180 gccactagag gagctactgt agtaattgga acaagcaaat tctatggtgg ttggcacaac 15240 atgttaaaaa ctgtttatag tgatgtagaa aaccctcacc ttatgggttg ggattatcct 15300 aaatgtgata gagccatgcc taacatgctt agaattatgg cctcacttgt tcttgctcgc 15360 aaacatacaa cgtgttgtag cttgtcacac cgtttctata gattagctaa tgagtgtgct 15420 caagtattga gtgaaatggt catgtgtggc ggttcactat atgttaaacc aggtggaacc 15480 tcatcaggag atgccacaac tgcttatgct aatagtgttt ttaacatttg tcaagctgtc 15540 acggccaatg ttaatgcact tttatctact gatggtaaca aaattgccga taagtatgtc 15600 cgcaatttac aacacagact ttatgagtgt cctatagaa atagagatgt tgacacagac 15660 tttgtgaatg agttttacgc atattgcgt aaacatttct caatgatgat actctctgac 15720 gatgctgttg tgtgttcaa tagcacttat gcatctcaag gtctagtggc tagcataaag 15780 aactttaagt cagttcttta tttcaaaac aatgttttta tgtctgaagc aaaatgttgg 15840 actgagactg accttactaa aggacctcat gaattttgct ctcaacatac aatgctagtt 15900 aaacagggtg atgattatgt gtaccttcct tacccagatc catcaagaat cctaggggcc 15960 ggctgttttg tagatgatat cgtaaaaaca gatggtacac ttatgattga acggttcgtg 16020 tctttagcta tagatgctta cccacttact aaacatccta atcaggagta tgctgatgtc 16080 tttcatttgt acttacaata cataagaaag ctacatgatg agttaacagg acacatgtta 16140 gacatgtatt ctgttatgct tactaatgat aacacttcaa ggtattggga acctgagttt 16200 tatgaggcta tgtacacacc gcatacagtc ttacaggctg ttggggcttg tgttctttgc 16260 aattcacaga cttcattaag atgtggtgct tgcatacgta gaccattctt atgttgtaaa tgctgttacg accatgtcat atcaacatca cataaattag tcttgtctgt taatccgtat gtttgcaatg ctccaggttg tgatgtcaca gatgtgactc aactttactt aggaggtatg agctattatt gtaaatcaca taaaccaccc attagttttc cattgtgtgc taatggacaa gtttttggtt fatheraaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca attgcaacat gtgactggac aaatgctggt gattacattt tagctaacac ctgtactgaa agactcaagc tttttgcagc agaaacgctc aaagctactg aggagacatt taaactgtct tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct ttcatgggaa gttggtaac ctagaccacc acttaaccga aattatgtct ttactggtta tcgtgtaact aaaaacagta aagtacaaat aggagagtac acctttgaaa aaggtgacta tggtgatgct gttgtttacc gaggtacaac aacttacaaa ttaaatgttg gtgattattt tgtgctgaca tcacatacag taatgccatt aagtgcacct acactagtgc cacaagagca ctatgttaga 16980 attactggct tatacccaac actcaatatc tcagatgagt tttctagcaa tgttgcaaat 17040 tatcaaaagg ttggtatgca aaagtattct acactccagg gaccacctgg tactggtaag 17100 agtcattttg ctattggcct agctctctac tacccttctg ctcgcatagt gtatacagct 17160 tgctctcatg ccgctgttga tgcactatgt gagaaggcat taaaatattt gcctatagat 17220 aaatgtagta gaattatacc tgcacgtgct cgtgtagagt gttttgataa attcaaagtg 17280 aattcaacat tagaacagta tgtcttttgt actgtaaatg cattgcctga gacgacagca 17340 gatatagttg tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat 17400 gccagattac gtgctaagca ctatgtgtac attggcgacc ctgctcaatt acctgcacca 17460 cgcacattgc taactaaggg cacactagaa ccagaatatt tcaattcagt gtgtagactt 17520 atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtcc tgctgaaatt 17580 gttgacactg tgagtgcttt ggtttatgat aataagctta aagcacataa agacaaatca 17640 gctcaatgct ttaaaatgtt ttataagggt gttatcacgc atgatgtttc atctgcaatt 17700 aacaggccac aaataggcgt ggtaagagaa ttccttacac gtaaccctgc ttggagaaaa 17760 gctgtcttta tttcacctta taattcacag aatgctgtag cctcaaagat tttgggacta 17820 ccaactcaaa ctgttgattc atcacagggc tcagaatatg actatgtcat attcactcaa 17880 accactgaaa cagctcactc ttgtaatgta aacagattta atgttgctat taccagagca 17940 aaagtaggca tactttgcat aatgtctgat agagaccttt atgacaagtt gcaatttaca 18000 agtcttgaaa ttccacgtag gaatgtggca actttacaag ctgaaaatgt aacaggactc 18060 tttaaagatt gtagtaaggt aatcactggg ttacatccta cacaggcacc tacacacctc 18120 agtgttgaca ctaaattcaa aactgaaggt ttatgtgttg acatacctgg catacctaag 18180 gacatgacct atagaagact catctctatg atgggtttta aaatgaatta tcaagttaat 18240 ggttaccta acatgtttat cacccgcgaa gaagctataa gacatgtacg tgcatggatt 18300 ggcttcgatg tcgaggggtg tcatgctact agagaagctg ttggtaccaa tttaccttta 18360 cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta tgttgataca 18420 cctaataata cagatttttc cagagttagt gctaaaccac cgcctggaga tcaatttaaa 18480 cacctcatac cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta 18540 caaatgttaa gtgacacact taaaaatctc tctgacagag tcgtatttgt cttatgggca 18600 catggctttg agttgacatc tatgaagtat tttgtgaaaa taggacctga gcgcacctgt 18660 tgtctatgtg atagacgtgc cacatgcttt tccactgctt cagacactta tgcctgttgg 18720 catcattcta ttggattga ttacgtctat aatccgttta tgattgatgt tcaacaatgg 18780 ggttttacag gtaacctaca aagcaaccat gatctgtatt gtcaagtcca tggtaatgca 18840 catgtagcta gttgtgatgc aatcatgact aggtgtctag ctgtccacga gtgctttgtt 18900 aagcgtgttg actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg 18960 gcttgtagaa aggttcaaca catggttgtt aaagctgcat tattagcaga caaattccca 19020 gttcttcacg acattggtaa ccctaaagct attaagtgtg tacctcaagc tgatgtagaa 19080 tggaagttct atgatgcaca gccttgtagt gacaaagctt ataaaataga agaattattc 19140 tattcttatg ccacacattc tgacaaattc acagatggtg tatgcctatt ttggaattgc 19200 aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag agtgctatct 19260 aaccttaact tgcctggttg tgatggtggc agtttgtatg taaataaaca tgcattccac 19320 acaccagctt ttgataaaag tgcttttgtt aatttaaaac aattaccatt tttctattac 19380 tctgacagtc catgtgagtc tcatggaaaa caagtagtgt cagatataga ttatgtacca 19440 ctaaagtctg ctacgtgtat aacacgttgc aatttaggtg gtgctgtctg tagacatcat 19500 gctaatgagt acagattgta tctcgatgct tataacatga tgatctcagc tggctttagc 19560 ttgtgggttt acaaacaatt tgatacttat aacctctgga acacttttac aagacttcag 19620 agtttagaaa atgtggcttt taatgttgta aataagggac actttgatgg acaacagggt 19680 gaagtaccag tttctatcat tataacact gtttacacaa aagttgatgg tgttgatgta 19740 gaattgtttg aaaataaaac aacattacct gttaatgtag catttgagct ttgggctaag 19800 cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt ggacattgct 19860 gctaatactg tgatctggga ctacaaaaga gatgctccag cacatatatc tactattggt 19920 gtttgttcta tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact 19980 gtctttttg atggtagagt tgatggtcaa gtagacttat ttagaaatgc ccgtaatggt 20040 gttcttatta cagaaggtag tgttaaaggt ttacaaccat ctgtaggtcc caaacaagct 20100 agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa ttattataag 20160 aaagttgatg gtgttgtcca acaattacct gaaacttact ttactcagag tagaaattta 20220 caagaattta aacccaggag tcaaatggaa attgatttct tagaattagc tatggatgaa 20280 ttcattgaac ggtataaatt agaaggctat gccttcgaac atatcgttta tggagattttt 20340 agtcatagtc agttaggtgg tttacatcta ctgattggac tagctaaacg ttttaaggaa 20400 tcaccttttg aattagaaga ttttattcct atggacagta cagttaaaaa ctattcata acagatgcgc aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat 20520. gattttgttg aaataataaa atcccaagat ttatctgtag tttctaaggt tgtcaaagtg actattgact attack ttcatttatg ctttggtgta aagatggcca tgtagaaaca ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat gcctaatctt 20760. aaaaatgc aaagaatgct attagaaaag tgtgaccttc aaaattatgg tgatagtgca acattaccta aaggcataat gatgaatgtc gcaaaatata ctcaactgtg tcaatattta aacacattaa cattagctgt acccttaat atgagagtta cattttgg tgctggttct 20940. ttgcaccagg ttgcaccagg ttaagacagt ggttgcctac gggtacgctg cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt gattggtgat tgtgcaactg tacatacagc father gatctcatta ttagtgatat gtacgaccct aagactaaaa atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt 21120 gggtttatac aacaaaagct agctcttgga ggttccgtgg ctataaagat aacagaacat 21180 tcttggaatg ctgatcttta taagctcatg ggacacttcg catggtggac agcctttgtt 21240 actaatgtga atgcgtcatc atctgaagca ttttaattg gatgtaatta tcttggcaaa 21300 ccacgcgaac aaatagatgg ttatgtcatg catgcaaatt acatattttg gaggaataca 21360 aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc ccttaaatta 21420 aggggtactg ctgttatgtc tttaaaagaa ggtcaaatca atgatatgat tttatctctt 21480 cttagtaaag gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt 21540 cttgttaaca actaaacgaa caatgtttgt ttttcttgtt ttattgccac tagtctctag 21600 tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac 21660 acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga 21720 cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac 21780 caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc 21840 ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa 21900 gacccagtcc ctacttattg ttaataacgc tactaatgtt gttataaag tctgtgaatt 21960 tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat 22020 ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca 22080 gccttttctt atggaccttg aggaaaaaca gggtaatttc aaaaatctta gggaatttgt 22140 gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt 22200 gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat 22260 taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga 22320 ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag 22380 gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact 22440 tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta 22500 tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac 22560 aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg 22620 gaacaggaag agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc 22680 atttccact tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac 22740 taatgtctat gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg 22800 gcaaactgga aagattgctg attataatta taaattacca gatgatttta caggctgcgt 22860 tatagcttgg aattctaaca atcttgattc taaggttggt ggtaattata attacctgta 22920 tagattgttt aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta 22980 tcaggccggt agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca 23040 atcatatggt ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact 23100 ttcttttgaa cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt 23160 ggttaaaaac aaatgtgtca atttcaactt caatggttta acaggcacag gtgttcttac 23220 tgagtctaac aaaaagtttc tgcctttcca acaatttggc agagacattg ctgacactac 23280 tgatgctgtc cgtgatccac agacacttga gattcttgac attacaccat gttcttttgg 23340 tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg ttctttatca 23400 ggatgttaac tgcacagaag tccctgttgc tattcatgca gatcaactta ctcctacttg 23460 gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc 23520 tgaacatgtc aacaactcat atgagtgtga catacccatt ggtgcaggta tatgcgctag 23580 ttatcagact cagactaatt ctcctcggcg ggcacgtagt gtagctagtc aatccatcat 23640 tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata actctattgc 23700 catacccaca aattttacta ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa 23760 gacatcagta gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt 23820 gttgcaatat ggcagttttt gtacacaatt aaaccgtgct ttaactggaa tagctgttga 23880 acaagacaaa aacacccaag aagtttttgc acaagtcaaa caaatttaca aaacaccacc 23940 attaaagat tttggtggtt ttaatttttc acaaatatta ccagatccat caaaaccaag 24000 caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt 24060 catcaaacaa tatggtgatt gccttggtga tatgctgct agagacctca tttgtgcaca 24120 aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata 24180 cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc 24240 attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca 24300 gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa 24360 aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa 24420 ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat 24480 ttcaagtgtt ttaaatgata tccttcacg tcttgacaaa gttgaggctg aagtgcaaat 24540 tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat 24600 tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt 24660 acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc 24720 tcagtcagca cctcatggtg tagtctctt gcatgtgact tatgtccctg cacaagaaaa 24780 gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg 24840 tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca 24900 aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt 24960 caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga 25020 taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa 25080 tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt 25140 aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc 25200 atggtacatt tggctaggtt ttatagctgg cttgattgcc atagtaatgg tgacaattat gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt gtggatcctg 25320 ctgcaaattt gatgaagacg actctgagcc agtgctcaaa ggagtcaaat tacattacac ataacgaac ttatggattt gtttatgaga atcttcacaa ttggaactgt aactttgaag caaggtgaaa tcaaggatgc tactccttca gattttgttc gcgctactgc aacgataccg atacaagcct cactcccttt cggatggctt attgttggcg ttgcacttct tgctgttttt 25560 cagagcgctt ccaaaatcat aaccctcaaa aagagatggc aactagcact ctccaagggt gttcactttg tttgcaactt gctgttgttg tttgtaacag tttactcaca ccttttgctc gttgctgctg gccttgaagc cccttttctc tatctttatg ctttagtcta cttcttgcag 25740 agtataact ttgtaagaat aataatgagg ctttggcttt gctggaaatg ccgttccaaa aacccattac tttatgatgc caacttttt ctttgctggc attackattg ttacgactat tgtatacctt acaatagtgt aacttcttca attgtcatta cttcaggtga tggcacaaca agtcctattt ctgaacatga ctaccagatt ggtggttata ctgaaaaatg ggaatctgga 25980 gtaaaagact gtgttgtatt acacagttac ttcacttcag actattacca gctgtactca 26040 actcaattga gtacagacac tggtgttgaa catgttacct tcttcatcta caataaaatt 26100 gttgatgagc ctgaagaaca tgtccaaatt cacacaatcg acggttcatc cggagttgtt 26160 aatccagtaa tggaaccaat ttatgatgaa ccgacgacga ctactagcgt gcctttgtaa 26220 gcacaagctg atgagtacga acttatgtac tcattcgttt cggagaagac aggtacgtta 26280 atagttaata gcgtacttct ttttcttgct ttcgtggtat tcttgctagt tacactagcc 26340 atccttactg cgcttcgatt gtgtgcgtac tgctgcaata ttgttaacgt gagtcttgta 26400 aaaccttctt tttacgttta ctctcgtgtt aaaaatctga attcttctag agttcctgat 26460 cttctggtct aaacgaacta aatattatat tagtttttct gtttggaact ttaatttag 26520 ccatggcaga ttccaacggt actattaccg ttgaagagct taaaaagctc cttgaacaat 26580 ggaacctagt aataggtttc ctattcctta catggatttg tcttctacaa tttgcctatg 26640 ccaacaggaa taggttttg tatataatta agttaatttt cctctggctg ttatggccag 26700 taactttagc ttgttttgtg cttgctgctg tttacagaat aaattggatc accggtggaa 26760 ttgctatcgc aatggcttgt cttgtaggct tgatgtggct cagctacttc attgcttct 26820 tcagactgtt tgcgcgtacg cgttccatgt ggtcattcaa tccagaaact aacattcttc 26880 tcaacgtgcc actccatggc actattctga ccagaccgct tctagaaagt gaactcgtaa 26940 tcggagctgt gatccttcgt ggacatcttc gtattgctgg acaccatcta ggacgctgtg 27000 acatcaagga cctgcctaaa gaaatcactg ttgctacatc acgaacgctt tcttattaca 27060 aattgggagc ttcgcagcgt gtagcaggtg actcaggttt tgctgcatac agtcgctaca 27120 ggattggcaa ctataaatta aacacagacc attccagtag cagtgacaat attgctttgc 27180 ttgtacagta agtgacaaca gatgtttcat ctcgttgact ttcaggttac tatagcagag 27240 atattactaa ttattatgag gacttttaaa gtttccattt ggaatcttga ttacatcata 27300 aacctcataa ttaaaaattt atctaagtca ctaactgaga ataaatattc tcaattagat 27360 gaagagcaac caatggagat tgattaaacg aacatgaaaa ttatctttt cttggcactg 27420 ataacactcg ctacttgtga gctttatcac taccaagagt gtgttagagg tacaagaga 27480 cttttaaaag aaccttgctc ttctggaaca tacgagggca attcaccatt tcatcctcta 27540 gctgataaca aatttgcact gacttgcttt agcactcaat ttgcttttgc ttgtcctgac 27600 ggcgtaaaac acgtctatca gttacgtgcc agatcagttt cacctaaact gttcatcaga 27660 caagagaag ttcaagaact ttactctcca atttttctta ttgttgcggc aatagtgttt 27720 ataacacttt gcttcacact caaaagaaag aagaatgat tgaactttca ttaattgact 27780 tctatttgtg ctttttagcc tttctgctat tccttgtttt aattatgctt attatctttt 27840 ggttctcact tgaactgcaa gatcataatg aaacttgtca cgcctaaacg aacatgaaat 27900 ttcttgtttt cttaggaatc atcacaactg tagctgcatt tcaccaagaa tgtagtttac 27960 agtcatgtac tcaacatcaa ccatatgtag ttgatgaccc gtgtcctatt cacttctatt 28020 ctaaatggta tattagagta ggagctagaa aatcagcacc tttaattgaa ttgtgcgtgg 28080 atgaggctgg ttctaaatca cccattcagt acatcgatat cggtaattat acagtttcct 28140 gttaccttt tacaattaat tgccaggaac ctaaattggg tagtcttgta gtgcgttgtt 28200 cgttctatga agacttttta gagtatcatg acgttcgtgt tgttttagat ttcatctaaa 28260 cgaacaaact aaaatgtctg ataatggacc ccaaaatcag cgaaatgcac cccgcattac 28320 gtttggtgga ccctcagatt caactggcag taaccagaat ggagaacgca gtggggcgcg 28380 atcaaaacaa cgtcggcccc aaggtttacc caataatact gcgtcttggt tcaccgctct 28440 cactcaacat ggcaaggaag accttaaatt ccctcgagga caaggcgttc caattaacac 28500 caatagcagt ccagatgacc aaattgcta ctaccgaaga gctaccagac gaattcgtgg 28560 tggtgacggt aaaatgaaag atctcagtcc aagatggtat ttctactacc taggaactgg 28620 gccagaagct ggacttccct atggtgctaa caaagacggc atcatatggg ttgcaactga 28680 gggagccttg aatacaccaa aagatcacat tggcacccgc aatcctgcta aaatgctgc 28740 aatcgtgcta caacttcctc aagcaaac attgccaaaa ggcttctacg cagaagggag 28800 cagaggcggc agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa 28860 ttcaactcca ggcagcagta ggggaacttc tcctgctaga atgggctggca atggcggtga 28920 tgctgctctt gctttgctgc tgcttgacag attgaaccag cttgagagca aaatgtctgg 28980 taaaggccaa caacaaaag gccaaactgt cacaagaaa tctgctgctg aggcttctaa 29040 gaagcctcgg caaaaacgta ctgccaactaa agcatacaat gtaacacaag ctttcggcag 29100 acgtggtcca gaacaaaccc aaggaaattt tgggaccag gaactaatca gacaaggaac 29160 tgattacaaa cattggccgc aaattgcaca atttgcccc agcgcttcag cgttcttcgg 29220 aatgtcgcgc attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc 29280 catcaaattg gatgaaag atccaaattt caaagatcaa gtcattttgc tgaataagca 29340 tattgacgca tacaaaacat tcccaccaac agagcctaaa aaggacaaaa agaagaaggc 29400 tgatgaaact caagccttac cgcagagaca gaagaaacag caaactgtga ctcttcttcc 29460 tgctgcagat ttggatgatt tctccaaaca attgcaacaa tccatgagca gtgctgactc 29520 aactcaggcc taaactcatg cagaccacac aaggcagatg ggctatataa acgttttcgc 29580 ttttccgttt acgatatata gtctactctt gtgcagaatg aattctcgta actacatagc 29640 acaagtagat gtagttaact ttaatctcac atagcaatct ttaatcagtg tgtaacatta 29700 gggaggactt gaaagagcca ccacatttc accgaggcca cgcggagtac gatcgagtgt 29760 acagtgaaca atgctaggga gagctgccta tatggaagag ccctaatgtg taaattaat 29820 tttagtagtg ctatccccat gtgattttaa tagcttctta ggagaatgac aaaaaaaaa 29880 29903 <210> 17 <211> 1273 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 17 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 Will Lew Gly Will Lew His Tyr Thr 1265 1270 <210> 18 <211> 75 <212> PRT <213> Severe acute respiratory syndrome coronavirus <400> 18 Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser 1 5 10 15 Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala 20 25 30 They Have Thr They Have Arg They Have Cys They Have Tyr Cys Asn And They Have Val Asn 35 40 45 Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn 50 55 60 Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val 65 70 75 <210> 19 <211> 222 <212> PRT <213> Severe acute respiratory syndrome coronavirus <400> 19 Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu 1 5 10 15 Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile 20 25 30 Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile 35 40 45 Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys 50 55 60 Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile 65 70 75 80 Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe 85 90 95 Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe 100 105 110 Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile 115 120 125 Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile 130 135 140 Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp 145 150 155 160 Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu 165 170 175 Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly 180 185 190 Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr 195 200 205 Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln 210 215 220 <210> 20 <211> 419 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 20 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 21 <211> 7096 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 21 Met Glu Ser Leu Val Pro Gly Phe Asn Glu Lys Thr His Val Gln Leu 1 5 10 15 Ser Leu Pro Val Leu Gln Val Arg Asp Val Leu Val Arg Gly Phe Gly 20 25 30 Asp Ser Val Glu Glu Val Leu Ser Glu Ala Arg Gln His Leu Lys Asp 35 40 45 Gly Thr Cys Gly Leu Val Glu Val Glu Lys Gly Val Leu Pro Gln Leu 50 55 60 Glu Gln Pro Tyr Val Phe Ile Lys Arg Ser Asp Ala Arg Thr Ala Pro 65 70 75 80 His Gly His Val Met Val Glu Leu Val Ala Glu Leu Glu Gly Ile Gln 85 90 95 Tyr Gly Arg Ser Gly Glu Thr Leu Gly Val Leu Val Pro His Val Gly 100 105 110 Glu Ile Pro Val Ala Tyr Arg Lys Val Leu Leu Arg Lys Asn Gly Asn 115 120 125 Lys Gly Ala Gly Gly His Ser Tyr Gly Ala Asp Leu Lys Ser Phe Asp 130 135 140 Leu Gly Asp Glu Leu Gly Thr Asp Pro Tyr Glu Asp Phe Gln Glu Asn 145 150 155 160 Trp Asn Thr Lys His Ser Ser Gly Val Thr Arg Glu Leu Met Arg Glu 165 170 175 Leu Asn Gly Gly Ala Tyr Thr Arg Tyr Val Asp Asn Asn Phe Cys Gly 180 185 190 Pro Asp Gly Tyr Pro Leu Glu Cys Ile Lys Asp Leu Leu Ala Arg Ala 195 200 205 Gly Lys Ala Ser Cys Thr Leu Ser Glu Gln Leu Asp Phe Ile Asp Thr 210 215 220 Lys Arg Gly Val Tyr Cys Cys Arg Glu His Glu His Glu Ile Ala Trp 225 230 235 240 Tyr Thr Glu Arg Ser Glu Lys Ser Tyr Glu Leu Gln Thr Pro Phe Glu 245 250 255 Ile Lys Leu Ala Lys Lys Phe Asp Thr Phe Asn Gly Glu Cys Pro Asn 260 265 270 Phe Val Phe Pro Leu Asn Ser Ile Ile Lys Thr Ile Gln Pro Arg Val 275 280 285 Glu Lys Lys Lys Leu Asp Gly Phe Met Gly Arg Ile Arg Ser Val Tyr 290 295 300 Pro Val Ala Ser Pro Asn Glu Cys Asn Gln Met Cys Leu Ser Thr Leu 305 310 315 320 Met Lys Cys Asp His Cys Gly Glu Thr Ser Trp Gln Thr Gly Asp Phe 325 330 335 Val Lys Ala Thr Cys Glu Phe Cys Gly Thr Glu Asn Leu Thr Lys Glu 340 345 350 Gly Ala Thr Thr Cys Gly Tyr Leu Pro Gln Asn Ala Val Val Lys Ile 355 360 365 Tyr Cys Pro Ala Cys His Asn Ser Glu Val Gly Pro Glu His Ser Leu 370 375 380 Ala Glu Tyr His Asn Glu Ser Gly Leu Lys Thr Ile Leu Arg Lys Gly 385 390 395 400 Gly Arg Thr Ile Ala Phe Gly Gly Cys Val Phe Ser Tyr Val Gly Cys 405 410 415 His Asn Lys Cys Ala Tyr Trp Val Pro Arg Ala Ser Ala Asn Ile Gly 420 425 430 Cys Asn His Thr Gly Val Val Gly Glu Gly Ser Glu Gly Leu Asn Asp 435 440 445 Asn Leu Leu Glu Ile Leu Gln Lys Glu Lys Val Asn Ile Asn Ile Val 450 455 460 Gly Asp Phe Lys Leu Asn Glu Glu Ile Ala Ile Ile Leu Ala Ser Phe 465 470 475 480 Ser Ala Ser Thr Ser Ala Phe Val Glu Thr Val Lys Gly Leu Asp Tyr 485 490 495 Lys Ala Phe Lys Gln Ile Val Glu Ser Cys Gly Asn Phe Lys Val Thr 500 505 510 Lys Gly Lys Ala Lys Lys Gly Ala Trp Asn Ile Gly Glu Gln Lys Ser 515 520 525 Ile Leu Ser Pro Leu Tyr Ala Phe Ala Ser Glu Ala Ala Arg Val Val 530 535 540 Arg Ser Ile Phe Ser Arg Thr Leu Glu Thr Ala Gln Asn Ser Val Arg 545 550 555 560 Val Leu Gln Lys Ala Ala Ile Thr Ile Leu Asp Gly Ile Ser Gln Tyr 565 570 575 Ser Leu Arg Leu Ile Asp Ala Met Met Phe Thr Ser Asp Leu Ala Thr 580 585 590 Asn Asn Leu Val Val Met Ala Tyr Ile Thr Gly Gly Val Val Gln Leu 595 600 605 Thr Ser Gln Trp Leu Thr Asn Ile Phe Gly Thr Val Tyr Glu Lys Leu 610 615 620 Lys Pro Val Leu Asp Trp Leu Glu Glu Lys Phe Lys Glu Gly Val Glu 625 630 635 640 Phe Leu Arg Asp Gly Trp Glu Ile Val Lys Phe Ile Ser Thr Cys Ala 645 650 655 Cys Glu Ile Val Gly Gly Gln Ile Val Thr Cys Ala Lys Glu Ile Lys 660 665 670 Glu Ser Val Gln Thr Phe Phe Lys Leu Val Asn Lys Phe Leu Ala Leu 675 680 685 Cys Ala Asp Ser Ile Ile Ile Gly Gly Ala Lys Leu Lys Ala Leu Asn 690 695 700 Leu Gly Glu Thr Phe Val Thr His Ser Lys Gly Leu Tyr Arg Lys Cys 705 710 715 720 Val Lys Ser Arg Glu Glu Thr Gly Leu Leu Met Pro Leu Lys Ala Pro 725 730 735 Lys Glu Ile Ile Phe Leu Glu Gly Glu Thr Leu Pro Thr Glu Val Leu 740 745 750 Thr Glu Glu Val Val Leu Lys Thr Gly Asp Leu Gln Pro Leu Glu Gln 755 760 765 Pro Thr Ser Glu Ala Val Glu Ala Pro Leu Val Gly Thr Pro Val Cys 770 775 780 Ile Asn Gly Leu Met Leu Leu Glu Ile Lys Asp Thr Glu Lys Tyr Cys 785 790 795 800 Ala Leu Ala Pro Asn Met Met Val Thr Asn Asn Thr Phe Thr Leu Lys 805 810 815 Gly Gly Ala Pro Thr Lys Val Thr Phe Gly Asp Asp Thr Val Ile Glu 820 825 830 Val Gln Gly Tyr Lys Ser Val Asn Ile Thr Phe Glu Leu Asp Glu Arg 835 840 845 Ile Asp Lys Val Leu Asn Glu Lys Cys Ser Ala Tyr Thr Val Glu Leu 850 855 860 Gly Thr Glu Val Asn Glu Phe Ala Cys Val Val Ala Asp Ala Val Ile 865 870 875 880 Lys Thr Leu Gln Pro Val Ser Glu Leu Leu Thr Pro Leu Gly Ile Asp 885 890 895 Leu Asp Glu Trp Ser Met Ala Thr Tyr Tyr Leu Phe Asp Glu Ser Gly 900 905 910 Glu Phe Lys Leu Ala Ser His Met Tyr Cys Ser Phe Tyr Pro Pro Asp 915 920 925 Glu Asp Glu Glu Glu Gly Asp Cys Glu Glu Glu Glu Phe Glu Pro Ser 930 935 940 Thr Gln Tyr Glu Tyr Gly Thr Glu Asp Asp Tyr Gln Gly Lys Pro Leu 945 950 955 960 Glu Phe Gly Ala Thr Ser Ala Ala Leu Gln Pro Glu Glu Glu Gln Glu 965 970 975 Glu Asp Trp Leu Asp Asp Asp Ser Gln Gln Thr Val Gly Gln Gln Asp 980 985 990 Gly Ser Glu Asp Asn Gln Thr Thr Thr Ile Gln Thr Ile Val Glu Val 995 1000 1005 Gln Pro Gln Leu Glu Met Glu Leu Thr Pro Val Val Gln Thr Ile 1010 1015 1020 Glu Val Asn Ser Phe Ser Gly Tyr Leu Lys Leu Thr Asp Asn Val 1025 1030 1035 Tyr Ile Lys Asn Ala Asp Ile Val Glu Glu Ala Lys Lys Val Lys 1040 1045 1050 Pro Thr Val Val Val Asn Ala Ala Asn Val Tyr Leu Lys His Gly 1055 1060 1065 Gly Gly Val Ala Gly Ala Leu Asn Lys Ala Thr Asn Asn Ala Met 1070 1075 1080 Gln Val Glu Ser Asp Asp Tyr Ile Ala Thr Asn Gly Pro Leu Lys 1085 1090 1095 Val Gly Gly Ser Cys Val Leu Ser Gly His Asn Leu Ala Lys His 1100 1105 1110 Cys Leu His Val Val Gly Pro Asn Val Asn Lys Gly Glu Asp Ile 1115 1120 1125 Gln Leu Leu Lys Ser Ala Tyr Glu Asn Phe Asn Gln His Glu Val 1130 1135 1140 Leu Leu Ala Pro Leu Leu Ser Ala Gly Ile Phe Gly Ala Asp Pro 1145 1150 1155 Ile His Ser Leu Arg Val Cys Val Asp Thr Val Arg Thr Asn Val 1160 1165 1170 Tyr Leu Ala Val Phe Asp Lys Asn Leu Tyr Asp Lys Leu Val Ser 1175 1180 1185 Ser Phe Leu Glu Met Lys Ser Glu Lys Gln Val Glu Gln Lys Ile 1190 1195 1200 Ala Glu Ile Pro Lys Glu Glu Val Lys Pro Phe Ile Thr Glu Ser 1205 1210 1215 Lys Pro Ser Val Glu Gln Arg Lys Gln Asp Asp Lys Lys Ile Lys 1220 1225 1230 Ala Cys Val Glu Glu Val Thr Thr Thr Leu Glu Glu Thr Lys Phe 1235 1240 1245 Leu Thr Glu Asn Leu Leu Leu Tyr Ile Asp Ile Asn Gly Asn Leu 1250 1255 1260 His Pro Asp Ser Ala Thr Leu Val Ser Asp Ile Asp Ile Thr Phe 1265 1270 1275 Leu Lys Lys Asp Ala Pro Tyr Ile Val Gly Asp Val Val Gln Glu 1280 1285 1290 Gly Val Leu Thr Ala Val Val Ile Pro Thr Lys Lys Ala Gly Gly 1295 1300 1305 Thr Thr Glu Met Leu Ala Lys Ala Leu Arg Lys Val Pro Thr Asp 1310 1315 1320 Asn Tyr Ile Thr Thr Tyr Pro Gly Gln Gly Leu Asn Gly Tyr Thr 1325 1330 1335 Val Glu Glu Ala Lys Thr Val Leu Lys Lys Cys Lys Ser Ala Phe 1340 1345 1350 Tyr Ile Leu Pro Ser Ile Ile Ser Asn Glu Lys Gln Glu Ile Leu 1355 1360 1365 Gly Thr Val Ser Trp Asn Leu Arg Glu Met Leu Ala His Ala Glu 1370 1375 1380 Glu Thr Arg Lys Leu Met Pro Val Cys Val Glu Thr Lys Ala Ile 1385 1390 1395 Val Ser Thr Ile Gln Arg Lys Tyr Lys Gly Ile Lys Ile Gln Glu 1400 1405 1410 Gly Val Val Asp Tyr Gly Ala Arg Phe Tyr Phe Tyr Thr Ser Lys 1415 1420 1425 Thr Thr Val Ala Ser Leu Ile Asn Thr Leu Asn Asp Leu Asn Glu 1430 1435 1440 Thr Leu Val Thr Met Pro Leu Gly Tyr Val Thr His Gly Leu Asn 1445 1450 1455 Leu Glu Glu Ala Ala Arg Tyr Met Arg Ser Leu Lys Val Pro Ala 1460 1465 1470 Thr Val Ser Val Ser Ser Pro Asp Ala Val Thr Ala Tyr Asn Gly 1475 1480 1485 Tyr Leu Thr Ser Ser Ser Lys Thr Pro Glu Glu His Phe Ile Glu 1490 1495 1500 Thr Ile Ser Leu Ala Gly Ser Tyr Lys Asp Trp Ser Tyr Ser Gly 1505 1510 1515 Gln Ser Thr Gln Leu Gly Ile Glu Phe Leu Lys Arg Gly Asp Lys 1520 1525 1530 Ser Val Tyr Tyr Thr Ser Asn Pro Thr Thr Phe His Leu Asp Gly 1535 1540 1545 Glu Val Ile Thr Phe Asp Asn Leu Lys Thr Leu Leu Ser Leu Arg 1550 1555 1560 Glu Val Arg Thr Ile Lys Val Phe Thr Thr Val Asp Asn Ile Asn 1565 1570 1575 Leu His Thr Gln Val Val Asp Met Ser Met Thr Tyr Gly Gln Gln 1580 1585 1590 Phe Gly Pro Thr Tyr Leu Asp Gly Ala Asp Val Thr Lys Ile Lys 1595 1600 1605 Pro His Asn Ser His Glu Gly Lys Thr Phe Tyr Val Leu Pro Asn 1610 1615 1620 Asp Asp Thr Leu Arg Val Glu Ala Phe Glu Tyr Tyr His Thr Thr 1625 1630 1635 Asp Pro Ser Phe Leu Gly Arg Tyr Met Ser Ala Leu Asn His Thr 1640 1645 1650 Lys Lys Trp Lys Tyr Pro Gln Val Asn Gly Leu Thr Ser Ile Lys 1655 1660 1665 Trp Ala Asp Asn Asn Cys Tyr Leu Ala Thr Ala Leu Leu Thr Leu 1670 1675 1680 Gln Gln Ile Glu Leu Lys Phe Asn Pro Pro Ala Leu Gln Asp Ala 1685 1690 1695 Tyr Tyr Arg Ala Arg Ala Gly Glu Ala Ala Asn Phe Cys Ala Leu 1700 1705 1710 Ile Leu Ala Tyr Cys Asn Lys Thr Val Gly Glu Leu Gly Asp Val 1715 1720 1725 Arg Glu Thr Met Ser Tyr Leu Phe Gln His Ala Asn Leu Asp Ser 1730 1735 1740 Cys Lys Arg Val Leu Asn Val Val Cys Lys Thr Cys Gly Gln Gln 1745 1750 1755 Gln Thr Thr Leu Lys Gly Val Glu Ala Val Met Tyr Met Gly Thr 1760 1765 1770 Leu Ser Tyr Glu Gln Phe Lys Lys Gly Val Gln Ile Pro Cys Thr 1775 1780 1785 Cys Gly Lys Gln Ala Thr Lys Tyr Leu Val Gln Gln Glu Ser Pro 1790 1795 1800 Phe Val Met Met Ser Ala Pro Pro Ala Gln Tyr Glu Leu Lys His 1805 1810 1815 Gly Thr Phe Thr Cys Ala Ser Glu Tyr Thr Gly Asn Tyr Gln Cys 1820 1825 1830 Gly His Tyr Lys His Ile Thr Ser Lys Glu Thr Leu Tyr Cys Ile 1835 1840 1845 Asp Gly Ala Leu Leu Thr Lys Ser Ser Glu Tyr Lys Gly Pro Ile 1850 1855 1860 Thr Asp Val Phe Tyr Lys Glu Asn Ser Tyr Thr Thr Thr Ile Lys 1865 1870 1875 Pro Val Thr Tyr Lys Leu Asp Gly Val Val Cys Thr Glu Ile Asp 1880 1885 1890 Pro Lys Leu Asp Asn Tyr Tyr Lys Lys Asp Asn Ser Tyr Phe Thr 1895 1900 1905 Glu Gln Pro Ile Asp Leu Val Pro Asn Gln Pro Tyr Pro Asn Ala 1910 1915 1920 Ser Phe Asp Asn Phe Lys Phe Val Cys Asp Asn Ile Lys Phe Ala 1925 1930 1935 Asp Asp Leu Asn Gln Leu Thr Gly Tyr Lys Lys Pro Ala Ser Arg 1940 1945 1950 Glu Leu Lys Val Thr Phe Phe Pro Asp Leu Asn Gly Asp Val Val 1955 1960 1965 Ala Ile Asp Tyr Lys His Tyr Thr Pro Ser Phe Lys Lys Gly Ala 1970 1975 1980 Lys Leu Leu His Lys Pro Ile Val Trp His Val Asn Asn Ala Thr 1985 1990 1995 Asn Lys Ala Thr Tyr Lys Pro Asn Thr Trp Cys Ile Arg Cys Leu 2000 2005 2010 Trp Ser Thr Lys Pro Val Glu Thr Ser Asn Ser Phe Asp Val Leu 2015 2020 2025 Lys Ser Glu Asp Ala Gln Gly Met Asp Asn Leu Ala Cys Glu Asp 2030 2035 2040 Leu Lys Pro Val Ser Glu Glu Val Val Glu Asn Pro Thr Ile Gln 2045 2050 2055 Lys Asp Val Leu Glu Cys Asn Val Lys Thr Thr Glu Val Val Gly 2060 2065 2070 Asp Ile Ile Leu Lys Pro Ala Asn Asn Ser Leu Lys Ile Thr Glu 2075 2080 2085 Glu Val Gly His Thr Asp Leu Met Ala Ala Tyr Val Asp Asn Ser 2090 2095 2100 Ser Leu Thr Ile Lys Lys Pro Asn Glu Leu Ser Arg Val Leu Gly 2105 2110 2115 Leu Lys Thr Leu Ala Thr His Gly Leu Ala Ala Val Asn Ser Val 2120 2125 2130 Pro Trp Asp Thr Ile Ala Asn Tyr Ala Lys Pro Phe Leu Asn Lys 2135 2140 2145 Val Val Ser Thr Thr Thr Asn Ile Val Thr Arg Cys Leu Asn Arg 2150 2155 2160 Val Cys Thr Asn Tyr Met Pro Tyr Phe Phe Thr Leu Leu Leu Gln 2165 2170 2175 Leu Cys Thr Phe Thr Arg Ser Thr Asn Ser Arg Ile Lys Ala Ser 2180 2185 2190 Met Pro Thr Thr Ile Ala Lys Asn Thr Val Lys Ser Val Gly Lys 2195 2200 2205 Phe Cys Leu Glu Ala Ser Phe Asn Tyr Leu Lys Ser Pro Asn Phe 2210 2215 2220 Ser Lys Leu Ile Asn Ile Ile Ile Trp Phe Leu Leu Leu Ser Val 2225 2230 2235 Cys Leu Gly Ser Leu Ile Tyr Ser Thr Ala Ala Leu Gly Val Leu 2240 2245 2250 Met Ser Asn Leu Gly Met Pro Ser Tyr Cys Thr Gly Tyr Arg Glu 2255 2260 2265 Gly Tyr Leu Asn Ser Thr Asn Val Thr Ile Ala Thr Tyr Cys Thr 2270 2275 2280 Gly Ser Ile Pro Cys Ser Val Cys Leu Ser Gly Leu Asp Ser Leu 2285 2290 2295 Asp Thr Tyr Pro Ser Leu Glu Thr Ile Gln Ile Thr Ile Ser Ser 2300 2305 2310 Phe Lys Trp Asp Leu Thr Ala Phe Gly Leu Val Ala Glu Trp Phe 2315 2320 2325 Leu Ala Tyr Ile Leu Phe Thr Arg Phe Phe Tyr Val Leu Gly Leu 2330 2335 2340 Ala Ala Ile Met Gln Leu Phe Phe Ser Tyr Phe Ala Val His Phe 2345 2350 2355 Ile Ser Asn Ser Trp Leu Met Trp Leu Ile Ile Asn Leu Val Gln 2360 2365 2370 Met Ala Pro Ile Ser Ala Met Val Arg Met Tyr Ile Phe Phe Ala 2375 2380 2385 Ser Phe Tyr Tyr Val Trp Lys Ser Tyr Val His Val Val Asp Gly 2390 2395 2400 Cys Asn Ser Ser Thr Cys Met Met Cys Tyr Lys Arg Asn Arg Ala 2405 2410 2415 Thr Arg Val Glu Cys Thr Thr Ile Val Asn Gly Val Arg Arg Ser 2420 2425 2430 Phe Tyr Val Tyr Ala Asn Gly Gly Lys Gly Phe Cys Lys Leu His 2435 2440 2445 Asn Trp Asn Cys Val Asn Cys Asp Thr Phe Cys Ala Gly Ser Thr 2450 2455 2460 Phe Ile Ser Asp Glu Val Ala Arg Asp Leu Ser Leu Gln Phe Lys 2465 2470 2475 Arg Pro Ile Asn Pro Thr Asp Gln Ser Ser Tyr Ile Val Asp Ser 2480 2485 2490 Val Thr Val Lys Asn Gly Ser Ile His Leu Tyr Phe Asp Lys Ala 2495 2500 2505 Gly Gln Lys Thr Tyr Glu Arg His Ser Leu Ser His Phe Val Asn 2510 2515 2520 Leu Asp Asn Leu Arg Ala Asn Asn Thr Lys Gly Ser Leu Pro Ile 2525 2530 2535 Asn Val Ile Val Phe Asp Gly Lys Ser Lys Cys Glu Glu Ser Ser 2540 2545 2550 Ala Lys Ser Ala Ser Val Tyr Tyr Ser Gln Leu Met Cys Gln Pro 2555 2560 2565 Ile Leu Leu Leu Asp Gln Ala Leu Val Ser Asp Val Gly Asp Ser 2570 2575 2580 Ala Glu Val Ala Val Lys Met Phe Asp Ala Tyr Val Asn Thr Phe 2585 2590 2595 Ser Ser Thr Phe Asn Val Pro Met Glu Lys Leu Lys Thr Leu Val 2600 2605 2610 Ala Thr Ala Glu Ala Glu Leu Ala Lys Asn Val Ser Leu Asp Asn 2615 2620 2625 Val Leu Ser Thr Phe Ile Ser Ala Ala Arg Gln Gly Phe Val Asp 2630 2635 2640 Ser Asp Val Glu Thr Lys Asp Val Val Glu Cys Leu Lys Leu Ser 2645 2650 2655 His Gln Ser Asp Ile Glu Val Thr Gly Asp Ser Cys Asn Asn Tyr 2660 2665 2670 Met Leu Thr Tyr Asn Lys Val Glu Asn Met Thr Pro Arg Asp Leu 2675 2680 2685 Gly Ala Cys Ile Asp Cys Ser Ala Arg His Ile Asn Ala Gln Val 2690 2695 2700 Ala Lys Ser His Asn Ile Ala Leu Ile Trp Asn Val Lys Asp Phe 2705 2710 2715 Met Ser Leu Ser Glu Gln Leu Arg Lys Gln Ile Arg Ser Ala Ala 2720 2725 2730 Lys Lys Asn Asn Leu Pro Phe Lys Leu Thr Cys Ala Thr Thr Arg 2735 2740 2745 Gln Val Val Asn Val Val Thr Thr Lys Ile Ala Leu Lys Gly Gly 2750 2755 2760 Lys Ile Val Asn Asn Trp Leu Lys Gln Leu Ile Lys Val Thr Leu 2765 2770 2775 Val Phe Leu Phe Val Ala Ala Ile Phe Tyr Leu Ile Thr Pro Val 2780 2785 2790 His Val Met Ser Lys His Thr Asp Phe Ser Ser Glu Ile Ile Gly 2795 2800 2805 Tyr Lys Ala Ile Asp Gly Gly Val Thr Arg Asp Ile Ala Ser Thr 2810 2815 2820 Asp Thr Cys Phe Ala Asn Lys His Ala Asp Phe Asp Thr Trp Phe 2825 2830 2835 Ser Gln Arg Gly Gly Ser Tyr Thr Asn Asp Lys Ala Cys Pro Leu 2840 2845 2850 Ile Ala Ala Val Ile Thr Arg Glu Val Gly Phe Val Val Pro Gly 2855 2860 2865 Leu Pro Gly Thr Ile Leu Arg Thr Thr Asn Gly Asp Phe Leu His 2870 2875 2880 Phe Leu Pro Arg Val Phe Ser Ala Val Gly Asn Ile Cys Tyr Thr 2885 2890 2895 Pro Ser Lys Leu Ile Glu Tyr Thr Asp Phe Ala Thr Ser Ala Cys 2900 2905 2910 Val Leu Ala Ala Glu Cys Thr Ile Phe Lys Asp Ala Ser Gly Lys 2915 2920 2925 Pro Val Pro Tyr Cys Tyr Asp Thr Asn Val Leu Glu Gly Ser Val 2930 2935 2940 Ala Tyr Glu Ser Leu Arg Pro Asp Thr Arg Tyr Val Leu Met Asp 2945 2950 2955 Gly Ser Ile Ile Gln Phe Pro Asn Thr Tyr Leu Glu Gly Ser Val 2960 2965 2970 Arg Val Val Thr Thr Phe Asp Ser Glu Tyr Cys Arg His Gly Thr 2975 2980 2985 Cys Glu Arg Ser Glu Ala Gly Val Cys Val Ser Thr Ser Gly Arg 2990 2995 3000 Trp Val Leu Asn Asn Asp Tyr Tyr Arg Ser Leu Pro Gly Val Phe 3005 3010 3015 Cys Gly Val Asp Ala Val Asn Leu Leu Thr Asn Met Phe Thr Pro 3020 3025 3030 Leu Ile Gln Pro Ile Gly Ala Leu Asp Ile Ser Ala Ser Ile Val 3035 3040 3045 Ala Gly Gly Ile Val Ala Ile Val Val Thr Cys Leu Ala Tyr Tyr 3050 3055 3060 Phe Met Arg Phe Arg Arg Ala Phe Gly Glu Tyr Ser His Val Val 3065 3070 3075 Ala Phe Asn Thr Leu Leu Phe Leu Met Ser Phe Thr Val Leu Cys 3080 3085 3090 Leu Thr Pro Val Tyr Ser Phe Leu Pro Gly Val Tyr Ser Val Ile 3095 3100 3105 Tyr Leu Tyr Leu Thr Phe Tyr Leu Thr Asn Asp Val Ser Phe Leu 3110 3115 3120 Ala His Ile Gln Trp Met Val Met Phe Thr Pro Leu Val Pro Phe 3125 3130 3135 Trp Contains Thr Only Tyr Contains Cys Containing Thr Lys His Phe 3140 3145 3150 Tyr Trp Phe Phe Ser Asn Tyr Leu Lys Arg Arg Val Val Phe Asn 3155 3160 3165 Gly Val Ser Phe Ser Thr Phe Glu Glu Ala Ala Leu Cys Thr Phe 3170 3175 3180 Leu Asn Lys Glu Met Tyr Leu Lys Leu Arg Ser Asp Val Leu 3185 3190 3195 Leu Pro Leu Thr Gln Tyr Asn Arg Tyre Leu Ala Leu Tyr Lys Asn 3200 3205 3210 Tyr Lys Tyr Phe Ser Gly Ala Met Asp Thr Thr Ser Tyr Arg Glu 3215 3220 3225 Ala Ala Cys His Leu Ala Lys Ala Leu Asn Asp Phe Ser Asn 3230 3235 3240 Ser Gly Ser Asp Val Leu Tyr Gln Pro Pro Gln Thr Ser Ile Thr 3245 3250 3255 Ser Ala Val Leu Gln Ser Gly Phe Arg Lys Met Ala Phe Pro Ser 3260 3265 3270 Gly Lys Val Glu Gly Cys Met Val Gln Val Thr Cys Gly Thr Thr 3275 3280 3285 Thr Leu Asn Gly Leu Trp Leu Asp Asp Val Val Tyr Cys Pro Arg 3290 3295 3300 His Val Ile Cys Thr Ser Glu Asp Met Leu Asn Pro Asn Tyr Glu 3305 3310 3315 Asp Leu Leu Ile Arg Lys Ser Asn His Asn Phe Leu Val Gln Ala 3320 3325 3330 Gly Asn Val Gln Leu Arg Val Ile Gly His Ser Met Gln Asn Cys 3335 3340 3345 Val Leu Lys Leu Lys Val Asp Thr Ala Asn Pro Lys Thr Pro Lys 3350 3355 3360 Tyr Lys Phe Val Arg Ile Gln Pro Gly Gln Thr Phe Ser Val Leu 3365 3370 3375 Ala Cys Tyr Asn Gly Ser Pro Ser Gly Val Tyr Gln Cys Ala Met 3380 3385 3390 Arg Pro Asn Phe Thr Ile Lys Gly Ser Phe Leu Asn Gly Ser Cys 3395 3400 3405 Gly Ser Val Gly Phe Asn Ile Asp Tyr Asp Cys Val Ser Phe Cys 3410 3415 3420 Tyr Met His His Met Glu Leu Pro Thr Gly Val His Ala Gly Thr 3425 3430 3435 Asp Leu Glu Gly Asn Phe Tyr Gly Pro Phe Val Asp Arg Gln Thr 3440 3445 3450 Ala Gln Ala Ala Gly Thr Asp Thr Thr Ile Thr Val Asn Val Leu 3455 3460 3465 Ala Trp Leu Tyr Ala Ala Val Ile Asn Gly Asp Arg Trp Phe Leu 3470 3475 3480 Asn Arg Phe Thr Thr Thr Leu Asn Asp Phe Asn Leu Val Ala Met 3485 3490 3495 Lys Tyr Asn Tyr Glu Pro Leu Thr Gln Asp His Val Asp Ile Leu 3500 3505 3510 Gly Pro Leu Ser Ala Gln Thr Gly Ile Ala Val Leu Asp Met Cys 3515 3520 3525 Ala Ser Leu Lys Glu Leu Leu Gln Asn Gly Met Asn Gly Arg Thr 3530 3535 3540 Ile Leu Gly Ser Ala Leu Leu Glu Asp Glu Phe Thr Pro Phe Asp 3545 3550 3555 Val Val Arg Gln Cys Ser Gly Val Thr Phe Gln Ser Ala Val Lys 3560 3565 3570 Arg Thr Ile Lys Gly Thr His His Trp Leu Leu Leu Thr Ile Leu 3575 3580 3585 Thr Ser Leu Leu Val Leu Val Gln Ser Thr Gln Trp Ser Leu Phe 3590 3595 3600 Phe Phe Leu Tyr Glu Asn Ala Phe Leu Pro Phe Ala Met Gly Ile 3605 3610 3615 Ile Ala Met Ser Ala Phe Ala Met Met Phe Val Lys His Lys His 3620 3625 3630 Ala Phe Leu Cys Leu Phe Leu Leu Pro Ser Leu Ala Thr Val Ala 3635 3640 3645 Tyr Phe Asn Met Val Tyr Met Pro Ala Ser Trp Val Met Arg Ile 3650 3655 3660 Met Thr Trp Leu Asp Met Val Asp Thr Ser Leu Ser Gly Phe Lys 3665 3670 3675 Leu Lys Asp Cys Val Met Tyr Ala Ser Ala Val Val Leu Leu Ile 3680 3685 3690 Leu Met Thr Ala Arg Thr Val Tyr Asp Asp Gly Ala Arg Arg Val 3695 3700 3705 Trp Thr Leu Met Asn Val Leu Thr Leu Val Tyr Lys Val Tyr Tyr 3710 3715 3720 Gly Asn Ala Leu Asp Gln Ala Ile Ser Met Trp Ala Leu Ile Ile 3725 3730 3735 Ser Val Thr Ser Asn Tyr Ser Gly Val Val Thr Thr Val Met Phe 3740 3745 3750 Leu Ala Arg Gly Ile Val Phe Met Cys Val Glu Tyr Cys Pro Ile 3755 3760 3765 Phe Phe Ile Thr Gly Asn Thr Leu Gln Cys Ile Met Leu Val Tyr 3770 3775 3780 Cys Phe Leu Gly Tyr Phe Cys Thr Cys Tyr Phe Gly Leu Phe Cys 3785 3790 3795 Leu Leu Asn Arg Tyr Phe Arg Leu Thr Leu Gly Val Tyr Asp Tyr 3800 3805 3810 Leu Val Ser Thr Gln Glu Phe Arg Tyr Met Asn Ser Gln Gly Leu 3815 3820 3825 Leu Pro Pro Lys Asn Ser Ile Asp Ala Phe Lys Leu Asn Ile Lys 3830 3835 3840 Leu Leu Gly Val Gly Gly Lys Pro Cys Ile Lys Val Ala Thr Val 3845 3850 3855 Gln Ser Lys Met Ser Asp Val Lys Cys Thr Ser Val Val Leu Leu 3860 3865 3870 Ser Val Leu Gln Gln Leu Arg Val Glu Ser Ser Ser Lys Leu Trp 3875 3880 3885 Ala Gln Cys Val Gln Leu His Asn Asp Ile Leu Leu Ala Lys Asp 3890 3895 3900 Thr Thr Glu Ala Phe Glu Lys Met Val Ser Leu Leu Ser Val Leu 3905 3910 3915 Leu Ser Met Gln Gly Ala Val Asp Ile Asn Lys Leu Cys Glu Glu 3920 3925 3930 Met Leu Asp Asn Arg Ala Thr Leu Gln Ala Ile Ala Ser Glu Phe 3935 3940 3945 Ser Ser Leu Pro Ser Tyr Ala Ala Phe Ala Thr Ala Gln Glu Ala 3950 3955 3960 Tyr Glu Gln Ala Val Ala Asn Gly Asp Ser Glu Val Val Leu Lys 3965 3970 3975 Lys Leu Lys Lys Ser Leu Asn Val Ala Lys Ser Glu Phe Asp Arg 3980 3985 3990 Asp Ala Ala Met Gln Arg Lys Leu Glu Lys Met Ala Asp Gln Ala 3995 4000 4005 Met Thr Gln Met Tyr Lys Gln Ala Arg Ser Glu Asp Lys Arg Ala 4010 4015 4020 Lys Val Thr Ser Ala Met Gln Thr Met Leu Phe Thr Met Leu Arg 4025 4030 4035 Lys Leu Asp Asn Asp Ala Leu Asn Asn Ile Ile Asn Asn Ala Arg 4040 4045 4050 Asp Gly Cys Val Pro Leu Asn Ile Ile Pro Leu Thr Thr Ala Ala 4055 4060 4065 Lys Leu Met Val Val Ile Pro Asp Tyr Asn Thr Tyr Lys Asn Thr 4070 4075 4080 Cys Asp Gly Thr Thr Phe Thr Tyr Ala Ser Ala Leu Trp Glu Ile 4085 4090 4095 Gln Gln Val Val Asp Ala Asp Ser Lys Ile Val Gln Leu Ser Glu 4100 4105 4110 Ile Ser Met Asp Asn Ser Pro Asn Leu Ala Trp Pro Leu Ile Val 4115 4120 4125 Thr Ala Leu Arg Ala Asn Ser Ala Val Lys Leu Gln Asn Asn Glu 4130 4135 4140 Leu Ser Pro Val Ala Leu Arg Gln Met Ser Cys Ala Ala Gly Thr 4145 4150 4155 Thr Gln Thr Ala Cys Thr Asp Asp Asn Ala Leu Ala Tyr Tyr Asn 4160 4165 4170 Thr Thr Lys Gly Gly Arg Phe Val Leu Ala Leu Leu Ser Asp Leu 4175 4180 4185 Gln Asp Leu Lys Trp Ala Arg Phe Pro Lys Ser Asp Gly Thr Gly 4190 4195 4200 Thr Ile Tyr Thr Glu Leu Glu Pro Pro Cys Arg Phe Val Thr Asp 4205 4210 4215 Thr Pro Lys Gly Pro Lys Val Lys Tyr Leu Tyr Phe Ile Lys Gly 4220 4225 4230 Leu Asn Asn Leu Asn Arg Gly Met Val Leu Gly Ser Leu Ala Ala 4235 4240 4245 Thr Val Arg Leu Gln Ala Gly Asn Ala Thr Glu Val Pro Ala Asn 4250 4255 4260 Ser Thr Val Leu Ser Phe Cys Ala Phe Ala Val Asp Ala Ala Lys 4265 4270 4275 Ala Tyr Lys Asp Tyr Leu Ala Ser Gly Gly Gln Pro Ile Thr Asn 4280 4285 4290 Cys Val Lys Met Leu Cys Thr His Thr Gly Thr Gly Gln Ala Ile 4295 4300 4305 Thr Val Thr Pro Glu Ala Asn Met Asp Gln Glu Ser Phe Gly Gly 4310 4315 4320 Ala Ser Cys Cys Leu Tyr Cys Arg Cys His Ile Asp His Pro Asn 4325 4330 4335 Pro Lys Gly Phe Cys Asp Leu Lys Gly Lys Tyr Val Gln Ile Pro 4340 4345 4350 Thr Thr Cys Ala Asn Asp Pro Val Gly Phe Thr Leu Lys Asn Thr 4355 4360 4365 Val Cys Thr Val Cys Gly Met Trp Lys Gly Tyr Gly Cys Ser Cys 4370 4375 4380 Asp Gln Leu Arg Glu Pro Met Leu Gln Ser Ala Asp Ala Gln Ser 4385 4390 4395 Phe Leu Asn Arg Val Cys Gly Val Ser Ala Ala Arg Leu Thr Pro 4400 4405 4410 Cys Gly Thr Gly Thr Ser Thr Asp Val Val Tyr Arg Ala Phe Asp 4415 4420 4425 Ile Tyr Asn Asp Lys Val Ala Gly Phe Ala Lys Phe Leu Lys Thr 4430 4435 4440 Asn Cys Cys Arg Phe Gln Glu Lys Asp Glu Asp Asp Asn Leu Ile 4445 4450 4455 Asp Ser Tyr Phe Val Val Lys Arg His Thr Phe Ser Asn Tyr Gln 4460 4465 4470 His Glu Glu Thr Ile Tyr Asn Leu Leu Lys Asp Cys Pro Ala Val 4475 4480 4485 Ala Lys His Asp Phe Phe Lys Phe Arg Ile Asp Gly Asp Met Val 4490 4495 4500 Pro His Ile Ser Arg Gln Arg Leu Thr Lys Tyr Thr Met Ala Asp 4505 4510 4515 Leu Val Tyr Ala Leu Arg His Phe Asp Glu Gly Asn Cys Asp Thr 4520 4525 4530 Leu Lys Glu Ile Leu Val Thr Tyr Asn Cys Cys Asp Asp Asp Tyr 4535 4540 4545 Phe Asn Lys Lys Asp Trp Tyr Asp Phe Val Glu Asn Pro Asp Ile 4550 4555 4560 Leu Arg Val Tyr Ala Asn Leu Gly Glu Arg Val Arg Gln Ala Leu 4565 4570 4575 Leu Lys Thr Val Gln Phe Cys Asp Ala Met Arg Asn Ala Gly Ile 4580 4585 4590 Val Gly Val Leu Thr Leu Asp Asn Gln Asp Leu Asn Gly Asn Trp 4595 4600 4605 Tyr Asp Phe Gly Asp Phe Ile Gln Thr Thr Pro Gly Ser Gly Val 4610 4615 4620 Pro Val Val Asp Ser Tyr Tyr Ser Leu Leu Met Pro Ile Leu Thr 4625 4630 4635 Leu Thr Arg Ala Leu Thr Ala Glu Ser His Val Asp Thr Asp Leu 4640 4645 4650 Thr Lys Pro Tyr Ile Lys Trp Asp Leu Leu Lys Tyr Asp Phe Thr 4655 4660 4665 Glu Glu Arg Leu Lys Leu Phe Asp Arg Tyr Phe Lys Tyr Trp Asp 4670 4675 4680 Gln Thr Tyr His Pro Asn Cys Val Asn Cys Leu Asp Asp Arg Cys 4685 4690 4695 Ile Leu His Cys Ala Asn Phe Asn Val Leu Phe Ser Thr Val Phe 4700 4705 4710 Pro Pro Thr Ser Phe Gly Pro Leu Val Arg Lys Ile Phe Val Asp 4715 4720 4725 Gly Val Pro Phe Val Val Ser Thr Gly Tyr His Phe Arg Glu Leu 4730 4735 4740 Gly Val Val His Asn Gln Asp Val Asn Leu His Ser Ser Arg Leu 4745 4750 4755 Ser Phe Lys Glu Leu Leu Val Tyr Ala Ala Asp Pro Ala Met His 4760 4765 4770 Ala Ala Ser Gly Asn Leu Leu Leu Asp Lys Arg Thr Thr Cys Phe 4775 4780 4785 Ser Val Ala Ala Leu Thr Asn Asn Val Ala Phe Gln Thr Val Lys 4790 4795 4800 Pro Gly Asn Phe Asn Lys Asp Phe Tyr Asp Phe Ala Val Ser Lys 4805 4810 4815 Gly Phe Phe Lys Glu Gly Ser Ser Val Glu Leu Lys His Phe Phe 4820 4825 4830 Phe Ala Gln Asp Gly Asn Ala Ala Ile Ser Asp Tyr Asp Tyr Tyr 4835 4840 4845 Arg Tyr Asn Leu Pro Thr Met Cys Asp With Arg Gln Leu Phe 4850 4855 4860 Val Val Glu Val Val Asp Lys Tyr Phe Asp Cys Tyr Asp Gly Gly 4865 4870 4875 How To Do You Have A Leu Asp Lys Ser 4880 4885 4890 Ala Gly Phe Pro Phe Asn Lys Trp Gly Lys Ala Arg Leu Tyr Tyr 4895 4900 4905 Asp Ser Met Ser Tyr Glu Asp Gln Asp Ala Leu Phe Ala Tyr Thr 4910 4915 4920 Lys Arg Asn Val Ile Pro Thr Ile Thr Gln Met Asn Leu Lys Tyr 4925 4930 4935 Ala Ile Ser Ala Lys Asn Arg Ala Arg Thr Val Ala Gly Val Ser 4940 4945 4950 Ile Cys Ser Thr Met Thr Asn Arg Gln Phe His Gln Lys Leu Leu 4955 4960 4965 Lys Ser Ile Ala Ala Thr Arg Gly Ala Thr Val Val Ile Gly Thr 4970 4975 4980 Ser Lys Phe Tyr Gly Gly Trp His Asn Met Leu Lys Thr Val Tyr 4985 4990 4995 Ser Asp Val Glu Asn Pro His Leu Met Gly Trp Asp Tyr Pro Lys 5000 5005 5010 Cys Asp Arg Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Leu 5015 5020 5025 Val Leu Ala Arg Lys His Thr Thr Cys Cys Ser Leu Ser His Arg 5030 5035 5040 Phe Tyr Arg Leu Ala Asn Glu Cys Ala Gln Val Leu Ser Glu Met 5045 5050 5055 Val Met Cys Gly Gly Ser Leu Tyr Val Lys Pro Gly Gly Thr Ser 5060 5065 5070 Ser Gly Asp Ala Thr Thr Ala Tyr Ala Asn Ser Val Phe Asn Ile 5075 5080 5085 Cys Gln Ala Val Thr Ala Asn Val Asn Ala Leu Leu Ser Thr Asp 5090 5095 5100 Gly Asn Lys Ile Ala Asp Lys Tyr Val Arg Asn Leu Gln His Arg 5105 5110 5115 Leu Tyr Glu Cys Leu Tyr Arg Asn Arg Asp Val Asp Thr Asp Phe 5120 5125 5130 Val Asn Glu Phe Tyr Ala Tyr Leu Arg Lys His Phe Ser Met Met 5135 5140 5145 Ile Leu Ser Asp Asp Ala Val Val Cys Phe Asn Ser Thr Tyr Ala 5150 5155 5160 Ser Gln Gly Leu Val Ala Ser Ile Lys Asn Phe Lys Ser Val Leu 5165 5170 5175 Tyr Tyr Gln Asn Asn Val Phe Met Ser Glu Ala Lys Cys Trp Thr 5180 5185 5190 Glu Thr Asp Leu Thr Lys Gly Pro His Glu Phe Cys Ser Gln His 5195 5200 5205 Thr Met Leu Val Lys Gln Gly Asp Asp Tyr Val Tyr Leu Pro Tyr 5210 5215 5220 Pro Asp Pro Ser Arg Ile Leu Gly Ala Gly Cy...

Claims

1. A peptide of less than 15 amino acids having cytotoxic T cell (CTL) inducing ability, comprising an amino acid sequence in which one or two amino acids are substituted for the amino acid sequence of Sequence ID No. 13, wherein the substitution has the following characteristics: (i) A peptide having one or both of the following characteristics relative to the amino acid sequence of SEQ ID NO: 13: (a) The second amino acid from the N-terminus is substituted with an amino acid selected from the group consisting of phenylalanine, tyrosine, and methionine; and (b) The C-terminal amino acid is substituted with an amino acid selected from the group consisting of phenylalanine, isoleucine, tryptophan, or methionine; or (ii) Sequence ID: A peptide having one or both of the following characteristics for the amino acid sequence of 13: (c) The second amino acid from the N-terminus is substituted with an amino acid selected from the group consisting of leucine and methionine; and (d) The C-terminal amino acid is substituted with valine.

2. A peptide having cytotoxic T cell (CTL) inducing ability, comprising an amino acid sequence in which one or two amino acids are substituted in the amino acid sequence of Sequence ID No. 13, wherein the substitution has the following characteristics: (i) A peptide having one or both of the following characteristics relative to the amino acid sequence of SEQ ID NO: 13: (a) The second amino acid from the N-terminus is substituted with an amino acid selected from the group consisting of phenylalanine, tyrosine, and methionine; and (b) The C-terminal amino acid is substituted with an amino acid selected from the group consisting of phenylalanine, isoleucine, tryptophan, or methionine; or (ii) Sequence ID: A peptide having one or both of the following characteristics for the amino acid sequence of 13: (c) The second amino acid from the N-terminus is substituted with an amino acid selected from the group consisting of leucine and methionine; and (d) The C-terminal amino acid is substituted with valine.

3. A polynucleotide encoding the peptide according to claim 1 or 2.

4. A composition comprising a pharmaceutically acceptable carrier and at least one active ingredient selected from the group consisting of (a) to (e) below, for one or more uses selected from the group consisting of (i) treatment of coronavirus infection, (ii) prevention of coronavirus infection, and (iii) suppression of severe coronavirus infection: (a) One or more types of peptides; (b) One or more polynucleotides encoding the peptide of (a) in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide from (a) and the HLA antigen on their cell surface; (d) Exosomes that present a complex of the peptide of (a) and the HLA antigen on their own cell surface; and (e) CTLs that target peptides in (a) A composition wherein the peptide in (a) is a peptide selected from the peptide described in claim 1, a peptide with fewer than 15 amino acids containing the amino acid sequence of SEQ ID NO:

13.

5. A composition comprising a pharmaceutically acceptable carrier and at least one active ingredient selected from the group consisting of (a) to (e) below, for one or more uses selected from the group consisting of (i) treatment of coronavirus infection, (ii) prevention of coronavirus infection, and (iii) suppression of severe coronavirus infection: (a) One or more types of peptides; (b) One or more polynucleotides encoding the peptide of (a) in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide from (a) and the HLA antigen on their cell surface; (d) Exosomes that present a complex of the peptide of (a) and the HLA antigen on their own cell surface; and (e) CTLs that target peptides in (a) A composition wherein the peptide in (a) is a peptide selected from the peptide described in claim 2 and the peptide having the amino acid sequence of SEQ ID NO:

13.

6. A composition for inducing CTLs, comprising a pharmaceutically acceptable carrier and at least one component selected from the group consisting of (a) to (d) below: (a) One or more types of peptides; (b) One or more polynucleotides encoding the peptide of (a) in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide from (a) and the HLA antigen on their cell surface; and (d) Exosomes that present the complex of the peptide from (a) and the HLA antigen on their cell surface. A composition wherein the peptide in (a) is a peptide selected from the peptide described in claim 1, a peptide with fewer than 15 amino acids containing the amino acid sequence of SEQ ID NO:

13.

7. A composition for inducing CTLs, comprising a pharmaceutically acceptable carrier and at least one component selected from the group consisting of (a) to (d) below: (a) One or more types of peptides; (b) One or more polynucleotides encoding the peptide of (a) in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide from (a) and the HLA antigen on their cell surface; and (d) Exosomes that present the complex of the peptide from (a) and the HLA antigen on their cell surface. A composition wherein the peptide in (a) is a peptide selected from the peptide described in claim 2 and the peptide having the amino acid sequence of SEQ ID NO:

13.

8. The composition according to claim 4 or 5, which is a pharmaceutical composition.

9. The composition according to claim 8 for inducing an immune response to coronavirus infection.

10. The composition according to claim 9, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, MERS-CoV, and SARS-CoV.

11. A composition according to any one of claims 4 to 10, formulated for administration to subjects who are HLA-A24 or HLA-A02 positive.

12. A method for inducing CTL-inducing APCs in vitro or ex vivo, comprising steps selected from the following group: (a) The step of contacting the APC with the peptide in vitro or ex vivo, and (b) Step of introducing the polynucleotide encoding the peptide into the APC. A method wherein the peptide is the peptide described in claim 1 or a peptide of less than 15 amino acids comprising the amino acid sequence of SEQ ID NO:

13.

13. A method for inducing CTL-inducing APCs in vitro or ex vivo, comprising steps selected from the following group: (a) The step of contacting the APC with the peptide in vitro or ex vivo, and (b) Step of introducing the polynucleotide encoding the peptide into the APC. A method wherein the peptide is the peptide described in claim 2 or a peptide consisting of the amino acid sequence of SEQ ID NO:

13.

14. A method for inducing CTLs in vitro or ex vivo, including a step selected from the following group: (a) A step in which CD8-positive T cells are co-cultured with APCs that present a complex of HLA antigen and peptide on their surface, (b) A step of co-culturing CD8-positive T cells with exosomes that present a complex of HLA antigen and peptide on their surface, and (c) The step of introducing polynucleotides encoding each subunit of the T cell receptor (TCR) that can bind to peptides presented on the cell surface by HLA antigens into CD8-positive T cells. A method wherein the peptide is the peptide described in claim 1 or a peptide of less than 15 amino acids comprising the amino acid sequence of SEQ ID NO:

13.

15. A method for inducing CTLs in vitro or ex vivo, including a step selected from the following group: (a) A step in which CD8-positive T cells are co-cultured with APCs that present a complex of HLA antigen and peptide on their surface, (b) A step of co-culturing CD8-positive T cells with exosomes that present a complex of HLA antigen and peptide on their surface, and (c) The step of introducing polynucleotides encoding each subunit of the T cell receptor (TCR) that can bind to peptides presented on the cell surface by HLA antigens into CD8-positive T cells. A method wherein the peptide is the peptide described in claim 2 or a peptide consisting of the amino acid sequence of SEQ ID NO:

13.

16. An APC that presents a complex of an HLA antigen and a peptide on its surface, wherein the peptide is selected from the peptide described in claim 1 or 2, a peptide of less than 15 amino acids containing the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO:

13.

17. APC according to claim 16, induced by the method according to claim 12 or 13.

18. A CTL that targets a peptide selected from the peptide described in claim 1 or 2, a peptide with fewer than 15 amino acids containing the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO:

13.

19. The CTL according to claim 18, induced by the method described in claim 14 or 15.

20. An antibody that binds to the peptide according to claim 1 or 2.

21. A method for screening peptides having CTL-inducing ability, comprising the following steps: (a) Sequence ID: A step in creating candidate sequences consisting of amino acid sequences in which one, two, or several amino acid residues are substituted, deleted, inserted, and / or added to an original amino acid sequence consisting of 13 amino acids; (b) The step of selecting candidate sequences from the candidate sequences created in (a) that do not have significant homology (sequence identity) with any known human gene product; (c) The step of contacting the peptide consisting of the candidate sequence selected in (b) with the APC; (d) The step of bringing the APCs in (c) into contact with CD8-positive T cells; and (e) The step of selecting a peptide that has equivalent or higher CTL-inducing ability than the peptide consisting of the original amino acid sequence.

22. An emulsion comprising one or more peptides selected from peptides with fewer than 15 amino acids containing the amino acid sequence of Claim 1 and SEQ ID NO: 13, a water-soluble carrier, and an oily adjuvant.

23. An emulsion comprising one or more peptides selected from the peptide according to claim 2 and the peptide consisting of the amino acid sequence of SEQ ID NO: 13, a water-soluble carrier, and an oily adjuvant.

24. A kit comprising a container containing the composition according to any one of claims 4 to 11, and a container containing an adjuvant.

25. A TCR that recognizes any of the peptides presented on an APC by an HLA antigen, wherein the peptide is the peptide described in claim 1 or a peptide of less than 15 amino acids comprising the amino acid sequence of SEQ ID NO:

13.

26. A TCR that recognizes any of the peptides presented on an APC by an HLA antigen, wherein the peptide is the peptide described in claim 2 or a peptide consisting of the amino acid sequence of SEQ ID NO: 13.

Images