AU2021351255A9 - Sars-cov-2 protein-derived peptide and vaccine containing same - Google Patents

Abstract

The present invention provides a SARS-CoV-2 protein-derived epitope peptide capable of inducing cytotoxic T cells. The present invention also provides: a polynucleotide encoding the peptide; an antigen-presenting cell presenting the peptide; a cytotoxic T cell (CTL) targeting the peptide; and a method for inducing the antigen-presenting cell or the CTL. The present invention further provides a composition or a pharmaceutical composition containing the foregoing as an active ingredient. Furthermore, the present invention provides a method for treating and/or preventing the corona virus infectious disease, and/or a method for suppressing the aggravation of the disease, using the peptide, the polynucleotide, the antigen-presenting cells, the cytotoxic T cells, or the pharmaceutical composition according to the present invention. Also provided is a method for an inducing an immune response to coronavirus infection.

Description

[DESCRIPTION]

[Title of Invention] SARS-COV-2 PROTEIN-DERIVED PEPTIDE AND VACCINE CONTAINING SAME

[Technical Field]

[0001] The present invention relates to the field of biological science, more specifically to the field of prevention of virus infectious diseases. In particular, the present invention relates to novel peptides that are effective as vaccines for preventing infection, methods for either or both of preventing and treating infectious diseases using the peptide(s), and pharmaceutical compositions comprising the peptide(s). The present application claims the benefit of Japanese Patent Application No. 2020 164630 filed on September 30, 2020, the entire contents of which are incorporated by reference herein.

[Background Art]

[0002] The respiratory infectious disease (Coronavirus Disease 2019: COVID-19) caused by infection with the novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2: SARS CoV-2) has spread around the world since it was first reported in Wuhan, China. According to the report from the World Health Organization (WHO), as of September 2020, the number of infected people exceeded 27 million and the number of deaths reached 890 thousand. Moreover, as of April 2021, the number of infected people exceeded 120 million and the number of deaths reached 2.8 million. Effective means for stopping the spread of infection include preventive vaccination; however, there is no vaccine against COVID-19 that has been put to practical use.

[0003] It has been previously known that BCG vaccine has a preventive effect even against infectious diseases other than tuberculosis. A recent study has also reported that BCG vaccination of elderly people aged 65 years and older showed a suppressive effect on infectious diseases (NPL 1: Giamarellos-Bourboulis EJ et al., Cell 2020, Online ahead of print). An association has been suggested between suppression of fatality rates or growth rates of deaths in COVID-19 and BCG vaccination (NPL 2: Toyoshima Y et al., J Hum Genet 2020, Online ahead of print; NPL 3: Berg MK et al., Science Advances 2020, Online ahead of print). In the human defense mechanism against viral infection, innate immune responses led by immune cells (such as macrophages, NK cells, and neutrophils) work first, and then antigen specific immune responses are developed by acquired immunity (humoral immunity and cellular immunity) led by B cells and T cells. After BCG vaccination, the activity of transcribing inflammatory cytokines is enhanced in immune cells, and infection with a virus in this state (trained immunity) triggers rapid release of inflammatory cytokines in the body. This is presumed to result in humoral immune responses by activated B cells and cellular immune responses by T cells suppressing viral proliferation (NPL 4: Netea MG et al., Cell 2020, 181(5): 969-977).

[0004] It is the spike protein that plays a central function in the entry of SARS-CoV-2 into human cells (NPL 5: Hoffmann M et al., Cell 2020, 181(2): 271-280). Most of the vaccines against COVID-19 currently under research and development are primarily aimed at inducing neutralizing antibodies (inducing humoral immunity) against the spike protein of SARS-CoV-2 (NPL 6: Jeyanathan M et al., Nat Rev Immunol 2020, Online ahead of print). However, repeated proliferation of SARS-CoV-2 may cause mutations in its viral genome. When a mutation occurs in the gene encoding the spike protein, vaccine-induced neutralizing antibodies may become ineffective. Moreover, studies conducted on people who had recovered from COVID-19 suggest that neutralizing antibodies induced in the human body may be short-lived (NPL 7: Ibarrondo FJ et al., N Engl J Med 2020, 383(11): 1085-1087; NPL 8: Long QX et al., Nat Med 2020, 26(8): 1200-1204). Accordingly, there are concerns that problems remain in the development of vaccines aimed at inducing humoral immunity by neutralizing antibodies.

[0005] Cellular immunity by T cells is also very important as a defense reaction against infectious diseases. Cytotoxic T lymphocytes (CTLs) are CD8-positive T cells and are induced by presentation of viral antigens (epitope peptides derived from viral proteins) by dendritic cells (DCs). Then, CTLs recognize epitope peptides presented on human leukocyte antigen (HLA) class I molecules expressed on the surface of virus-infected cells and kill these cells. This action destroys the infected cells, where viral particles are replicated, and results in suppression of viral proliferation. Compared to neutralizing antibodies, which mainly inhibit new infection of cells with replicated viral particles, CTLs, which target the replication process itself of viral particles, have a more direct effect in preventing viral proliferation. Accordingly, CTLs are considered to play a role in eliminating viruses from the living body and suppressing aggravation of infectious diseases. The report that the peripheral blood of patients with severe COVID-19 shows a marked decrease in CD8-positive T cells compared to healthy individuals suggests that CTLs are an important factor in suppressing aggravation of COVID-19 (NPL 9: Zheng M et al., Cell Mol Immunol 2020, 17(5): 533-535). Furthermore, part of CTLs are known to remain in the body as memory T cells for a long time. In 2003, the SARS-CoV epidemic occurred and more than 8,000 people were infected. Because of the fact that, 17 years after the epidemic, CTLs against SARS-CoV were still detected in the blood of people who had recovered (NPL 10: Le Bert N et al., Nature 2020, 584(7821): 457-462), CTLs against SARS-CoV-2 may also remain in the body for a long time after being induced. Accordingly, a vaccine aimed at eliciting cellular immunity can be an effective preventive measure against COVID-19. It is considered that CTLs induced by a vaccine comprising an epitope peptide derived from a SARS-CoV-2 protein remain in the body as memory T cells and show cytotoxic activity against virus-infected cells rapidly after SARS-CoV 2 infection, leading to suppression of aggravation of COVID-19. Thus, it is desired to identify an epitope peptide derived from a SARS-CoV-2 protein that can induce CTLs in the human body.

[0006] Candidate epitope peptides determined based on bioinformatics have been reported; however, it has not been verified whether or not they actually bind to an HLA and have the ability to induce CTLs, and no epitope peptide has been identified (NPL 11: Grifoni A et al., Cell Host Microbe 2020, 27(4): 671-680; NPL12: Crooke SN et al., Sci Rep 2020, 10(1): 14179).

[Citation List]

[Non-Patent Literature]

[0007]

[NPL 1] Giamarellos-Bourboulis EJ et al., Cell 2020, Online ahead of print

[NPL 2] Toyoshima Y et al., J Hum Genet 2020, Online ahead of print

[NPL 3] Berg MK et al., Science Advances 2020, Online ahead of print

[NPL 4] Netea MG et al., Cell 2020, 181(5):969-977

[NPL 5] Hoffmann M et al., Cell 2020, 181(2):271-280

[NPL 6] Jeyanathan M et al., Nat Rev Immunol 2020, Online ahead of print

[NPL 7] Ibarrondo FJ et al., N Engl J Med 2020, 383(11):1085-1087

[NPL 8] Long QX et al., Nat Med 2020, 26(8):1200-1204

[NPL 9] Zheng M et al., Cell Mol Immunol 2020, 17(5):533-535

[NPL 10] Le Bert N et al., Nature 2020, 584(7821):457-462

[NPL 11] Grifoni A et al., Cell Host Microbe 2020, 27(4):671-680

[NPL 12] Crooke SN et al., Sci Rep 2020, 10(1):14179

[Summary of Invention]

[0008] The present invention provides epitope peptides that bind to HLA-A*24:02 or HLA A*02:01 and have the ability to induce CTLs, which are selected from peptides derived from four structural proteins and six non-structural proteins that SARS-CoV-2 (reference sequence: GenBank accession number MN908947 (SEQ ID NO: 16)) has. The four structural proteins specifically refer to the following proteins: spike protein (reference sequence: GenBank accession number QHD43416 (SEQ ID NO: 17)); envelope protein (reference sequence: GenBank accession number QHD43418 (SEQ ID NO: 18)); matrix protein (reference sequence: GenBank accession number QHD43419 (SEQ ID NO: 19)); and nucleoprotein (reference sequence: GenBank accession number QHD43423 (SEQ ID NO: 20)). On the other hand, the six non-structural proteins specifically refer to the following proteins: ORFlab (reference sequence: GenBank accession number QHD43415 (SEQ ID NO: 21)); ORF3a (reference sequence: GenBank accession number QHD43417 (SEQ ID NO: 22)); ORF6 (reference sequence: GenBank accession number QHD43420 (SEQ ID NO: 23)); ORF7a (reference sequence: GenBank accession number QHD43421 (SEQ ID NO: 24)); ORF8 (reference sequence: GenBank accession number QHD43422 (SEQ ID NO: 25)); and ORF10 (reference sequence: GenBank accession number QH142199 (SEQ ID NO: 26)). The epitope peptides of the present invention are demonstrated to be able to induce specific and potent immune responses against COVID-19 targeting HLA-A*24:02-positive individuals, who constitute a large population in Asian countries including Japan, or targeting HLA-A*02:01-positive individuals, who constitute a large population in Europe and the United States (Cao K et al., Hum Immunol 2001, 62(9): 1009-1030; Gonzalez-Galarza FF et al., Nucleic Acids Res 2020, 48(D1): D783-D788). Since the amino acid sequences of the epitope peptides provided in the present invention have low similarity to those derived from human proteins, creation of highly safe vaccines that are less likely to cause unexpected side reactions can be expected. On the other hand, since those amino acid sequences are commonly found in SARS-CoV and MERS-CoV, which were epidemic in the past, they may also be found in proteins of new types of coronaviruses that will emerge in the future. Accordingly, vaccines comprising such epitope peptide can be effective against not only the current SARS-CoV-2 but also coronavirus infectious diseases that will be prevalent in the future.

[0009] The present invention also provides compositions comprising one or more types of peptides of the present invention, one or more types of polynucleotides encoding one or more types of peptides of the present invention, APCs of the present invention, exosomes presenting peptides of the present invention, and/or CTLs of the present invention. The compositions of the present invention are preferably pharmaceutical compositions. The pharmaceutical compositions of the present invention can be used for treating and/or preventing coronavirus infectious diseases, as well as suppressing aggravation. They can also be used for inducing an immune response against coronavirus infection. When administered to a subject, a peptide of the present invention is presented on the surface of an APC, and as a result CTLs targeting the peptide are induced. Therefore, another objective of the present invention is to provide compositions inducing CTLs, wherein the compositions comprise one or more types of peptides of the present invention, one or more types of polynucleotides encoding one or more types of peptides of the present invention, APCs of the present invention, and/or exosomes presenting peptides of the present invention.

[0010] A further objective of the present invention is to provide methods of inducing APCs having CTL-inducing ability, wherein the methods comprise a step of contacting one or more types of peptides of the present invention with an APC, or a step of introducing a polynucleotide encoding any one peptide of the present invention into an APC.

[0011] The present invention further provides a method of inducing CTLs, comprising a step of co-culturing a CD8-positive T cell with an APC that presents on its surface a complex of an HLA antigen and a peptide of the present invention, a step of co-culturing a CD8-positive T cell with an exosome that presents on its surface a complex of an HLA antigen and a peptide of the present invention, or a step of introducing into a CD8-positive T cell a vector comprising a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to a peptide of the present invention presented by an HLA antigen on a cell surface.

[0012] A further objective of the present invention is to provide isolated APCs that present on their surface a complex of an HLA antigen and a peptide of the present invention. The present invention further provides isolated CTLs targeting a peptide of the present invention. These APCs and CTLs can be used in immunotherapy for coronavirus infectious diseases.

[0013] Another objective of the present invention is to provide methods of inducing an immune response against coronavirus infection in a subject, wherein the methods comprise a step of administering to the subject a peptide(s) of the present invention, a polynucleotide(s) encoding the peptide(s), an APC(s) of the present invention, an exosome(s) presenting a peptide(s) of the present invention, and/or a CTL(s) of the present invention. Another objective of the present invention is to provide methods of treating and/or preventing coronavirus infectious disease, as well as suppressing aggravation in a subject, wherein the methods comprise a step of administering to the subject a peptide(s) of the present invention, a polynucleotide(s) encoding the peptide(s), an APC(s) of the present invention, an exosome(s) presenting a peptide(s) of the present invention, and/or a CTL(s) of the present invention.

[0014] In addition to the above, other objects and features of the present invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the present invention and the following detailed description are of exemplified embodiments, and not restrictive of the present invention or other alternate embodiments of the present invention. In particular, while the present invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the present invention and is not constructed as limiting of the present invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the present invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.

[Brief Description of Drawings]

[0015] Fig. 1 is composed of images showing the results of IFN-y enzyme-linked immunospot (ELISPOT) assays performed using cells induced with peptides derived from SARS-CoV-2 proteins. In the figure, "(+)" indicates IFN-y production for HLA-A*24:02-expressing target cells (TISI cells) pulsed with a peptide of interest, and "(-)" indicates IFN-y production for TISI cells not pulsed with any peptide (negative control). It can be seen by comparison with the negative control that peptide-specific IFN-y production was observed for 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) (Fig. la). On the other hand, Peptide 6 (SEQ ID NO: 6) is shown as an example of typical negative data for which no peptide-specific IFN- production was observed (Fig. lb).

[0016] Fig. 2 is composed of line graphs showing the results of measuring IFN-y produced by 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), using an enzyme-linked immunosorbent assay (ELISA). These results show that after induction with the peptides, HLA A*24:02-restricted CTL lines were established that produced IFN-y in a peptide-specific manner. In the figure, "(+)" indicates IFN-y production of a CTL line for HLA-A*24:02-expressing target cells (TISI cells) pulsed with a peptide of interest, and "(-)" indicates IFN-y production of a CTL line for TISI cells not pulsed with any peptide. The R/S ratio represents the ratio of the number of CTL line cells, which are responder cells (Responder cells), to the number of target cells (Stimulator cells) that stimulate them.

[0017] Fig. 3 is composed of images showing the results of IFN-y enzyme-linked immunospot (ELISPOT) assays performed using cells induced with peptides derived from SARS-CoV-2 proteins. In the figure, "(+)" indicates IFN-y production for HLA-A*02:01-expressing target cells (T2 cells) pulsed with a peptide of interest, and "(-)" indicates IFN-y production for T2 cells not pulsed with any peptide (negative control). It can be seen by comparison with the negative control that peptide-specific IFN-y production was observed for 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) (Fig. 3a). On the other hand, Peptide 5 (SEQ ID NO: 5) is shown as an example of typical negative data for which no peptide-specific IFN-y production was observed (Fig. 3b).

[0018] Fig. 4 is composed of line graphs showing the results of measuring IFN-y 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 an enzyme-linked immunosorbent assay (ELISA). These results show that after induction with the peptides, HLA-A*02:01-restricted CTL lines were established that produced IFN-y in a peptide-specific manner. In the figure,

"(+)"indicates IFN-y production of a CTL line for HLA-A*02:01-expressing target cells (T2 cells) pulsed with a peptide of interest, and "(-)" indicates IFN-y production of a CTL line for T2 cells not pulsed with any peptide. The R/S ratio represents the ratio of the number of CTL line cells, which are responder cells (Responder cells), to the number of target cells (Stimulator cells) that stimulate them.

[Description of Embodiments]

[0019] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0020] I. Definitions The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated. The terms "isolated" and "purified" used in relation with a substance (for example, peptide, antibody, polynucleotide or such) indicate that the substance does not substantially contain at least one substance that may else be included in a natural source. Thus, an isolated or purified peptide refers to a peptide that does not substantially contain another cellular material, for example, carbohydrate, lipid and other contaminating proteins from the cell or tissue source from which the peptide is derived. When the peptide is chemically synthesized, an isolated or purified peptide refers to a peptide that does not substantially contain a precursor substance or another chemical substance. The phrase "does not substantially contain a cellular material" includes peptide preparations in which the peptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a peptide that does not substantially contain a cellular material encompasses peptide preparations that contain less than about 30%, 20%, 10%, or 5%, 3%, 2% or 1% (dry weight basis) of other cellular materials. When the peptide is recombinantly produced, an isolated or purified peptide does not substantially contain culture medium, which encompasses peptide preparations that contain culture medium less than about 20%, 10%, or 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide preparation. When the peptide is chemically synthesized, an isolated or purified peptide does not substantially contain a precursor substance or other chemical substances, which encompasses peptide preparations that contain a precursor substance or other chemical substances less than about 30%, 20%, 10%, 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide preparation. That a particular peptide preparation is an isolated or purified peptide can be confirmed, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie Brillliant Blue staining or such of the gel. In a preferred embodiment, the peptides and polynucleotides of the present invention are isolated or purified.

[0021] The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably herein, and refer to polymers of amino acid residues. These terms are applied to also non naturally occurring amino acid polymers comprising one or more non-naturally occurring amino acid residues, in addition to naturally occurring amino acid polymers. Non-naturally occurring amino acids include amino acid analogs, amino acid mimetics, and such.

[0022] The term "amino acid" as used herein refers to naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and 0 phosphoserine, etc.). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, and such). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids. Amino acids can be either L amino acids or D-amino acids, and the peptides of the present invention are preferably L-amino acid polymers.

[0023] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein, and refer to a polymer of nucleotides.

[0024] The term "composition" used in the present specification is intended to encompass products that include specified ingredients in specified amounts, and any products generated directly or indirectly from combination of specified ingredients in the specified amounts. When the composition is a pharmaceutical composition, the term "composition" is intended to encompass products including active ingredient(s) and inert ingredient(s), as well as any products generated directly or indirectly from combination, complexation or aggregation of any two or more ingredients, from dissociation of one or more ingredients, or from other types of reactions or interactions of one or more ingredients. Thus, the pharmaceutical compositions of the present invention encompass any compositions made by admixing compounds or cells of the present invention with a pharmaceutically or physiologically acceptable carrier. Without being limited thereto, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" used in the present specification include liquid or solid bulking agents, diluents, excipients, solvents, and encapsulation materials; and mean pharmaceutically or physiologically acceptable materials, compositions, substances or media.

[0025] Unless otherwise specified, the term "virus infectious disease" refers to a coronavirus infectious disease; and examples of coronaviruses include SARS-CoV-2, MERS-CoV, SARS CoV and such, without being limited thereto. In an exemplary embodiment, the "coronavirus infectious disease" is SARS-CoV-2 infectious disease in an HLA-A24- or HLA-A02-positive subject.

[0026] Unless otherwise specified, the terms "cytotoxic T lymphocyte", "cytotoxic T cell" and "CTL" are used interchangeably herein. Unless otherwise specifically indicated, they refer to a sub-group of T lymphocytes that can recognize non-self cells (for example, tumor/cancer cells, virus-infected cells) and induce the death of such cells.

[0027] Unless otherwise specified, the term "HLA-A24" refers to the HLA-A24 type which includes subtypes such as 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 and HLA-A*24:88.

[0028] Unless otherwise specified, the term "HLA-A02 (or HLA-A2)" refers to the HLA-A02 type which includes subtypes such as 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*02:50.

[0029] Unless otherwise specified, the term "coronavirus protein" refers to a protein consisting of the full-length amino acid sequence of each protein encoded by coronavirus genome sequences. Preferred examples include structural and non-structural proteins of SARS-CoV-2. Moreover, examples of coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. The genome sequence (reference sequence) of each of these coronaviruses and the amino acid sequences of coronavirus proteins encoded by it can be obtained with, for example, the following Genbank accession numbers: SARS-CoV-2: NN908947 MERS-CoV: JX869059 SARS-CoV: Tor2: AY274119, BJO1: AY278488, or GZ02: AY390556

[0030] Unless otherwise specified, the term "SARS-CoV-2 protein" refers to a 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 that SARS-CoV-2 has. The four structural sequences and six non-structural proteins are listed in Table 1. In the table, as for the non-structural protein orflab, two ORFs in the reference sequence, 266..13468 and 13468..21555, are linked to form a single ORF by ribosomal frameshifting.

[Table 1] Reference Sequence: Coding Sequence (CDS) Amino Acid Protein GenBank in Genome Sequence Sequence accession (MN908947.3) (SEQ ID NO) number Spike Protein QHD43416 21563..25384 17 Z Envelope Protein QHD43418 26245..26472 18 u- Matrix Protein QHD43419 26523..27191 19 U) Nucleoprotein QHD43423 28274..29533 20 ORFlab QHD43415 266..13468,13468..21555 21 ORF3a QHD43417 25393..26220 22 > Z ORF6 QHD43420 27202..27387 23 ORF7a QHD43421 27394..27759 24 o ORF8 QHD43422 27894..28259 25 ORF10 QH142199 29558..29674 26

[0031] Unless otherwise specified, the terms "coronavirus-infected cells", "virus-infected cells", and "infected cells" are herein used interchangeably, and refer to cells infected with a coronavirus unless otherwise specifically indicated. Examples of coronaviruses include, but are not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0032] As used herein, the term "infection" includes viral entry and/or viral proliferation in cells or body tissues, and disease conditions resulting from viral entry and viral proliferation.

The entry by viral life cycle and the proliferation stage of viral life cycle include, but are not limited to, binding of the viral particle to a cell, introduction of the viral genetic information into the cell, expression of viral proteins, production of new viral particles, and release of the viral particles from the cell.

[0033] In the context of a subject or patient, the phrase "HLA antigen of a subject (or patient) is HLA-A24 or HLA-A02" used herein refers to that a subject or patient has the HLA-A24 or HLA-A02 antigen gene homozygously or heterozygously as the MHC (Major Histocompatibility Complex) Class I molecule, and that the HLA-A24 or HLA-A02 antigen is expressed in the cells of the subject or patient as the HLA antigen.

[0034] As long as the methods and compositions of the present invention are useful in the context of coronavirus infectious disease "treatment", the treatment is considered "efficacious" when it achieves clinical advantages, for example, alleviation of clinical symptoms of coronavirus infectious disease, suppression of aggravation in a subject. Fever, cough, chills, rigors, myalgia and the like are commonly known as main symptoms of coronavirus infectious diseases. It is considered that many infected patients have mild symptoms and recover within about one to two weeks. However, part of patients show symptoms of respiratory distress and may have marked dyspnea, hypoxia, or even acute respiratory distress syndrome (ARDS). According to recent reports, particularly in SARS-CoV-2-infected patients, unique symptoms such as dysosmia and dysgeusia have also been observed in patients with relatively mild symptoms. On the other hand, thrombi and cytokine storms in severely ill patients are known to result in aggravation. When treatment is applied prophylactically, the term "efficacious" means that the treatment retards or prevents development of a coronavirus infectious disease, or prevents or alleviates the clinical symptoms of a coronavirus infectious disease. Effectiveness is determined in relation to any publicly known method for diagnosing or treating a coronavirus infectious disease. For example, when any of the specific symptoms mentioned above is suppressed, the effectiveness of the treatment or prevention is indicated.

[0035] As long as the methods and compositions of the present invention are useful in the context of coronavirus infectious disease "prevention (prophylaxis)", the term "prevention (prophylaxis)" herein includes any work that eases the load of disease-associated mortality or morbidity. Prevention (Prophylaxis) can be carried out at the "primary, secondary and tertiary prevention (prophylaxis) levels". Whereas the primary prevention (prophylaxis) avoids the development of a disease, prevention (prophylaxis) at the secondary and tertiary levels encompasses prevention (prophylaxis) of disease progression and appearance of symptoms, as well as workings intended to reduce adverse effects of the existing disease by restoring functions and reducing disease-associated complications. Alternately, prevention (prophylaxis) can include alleviation of severity of a specific disorder, for example, extensive preventive therapy intended to reduce clinical symptoms such as fever, breathlessness, and upper respiratory infection.

[0036] In the context of the present invention, the treatment and/or prevention (prophylaxis) and/or the suppression of aggravation of coronavirus infectious diseases includes retarding the development of or ameliorating at least one symptom of coronavirus infectious diseases. It also includes inhibition of viral proliferation in coronavirus-infected cells. Effective treatment and/or prevention (prophylaxis) of coronavirus infectious diseases reduces mortality, improves the prognosis of individuals affected with coronavirus infectious diseases, and alleviates symptoms associated with coronavirus infectious diseases. For example, alleviation or improvement of symptoms constitutes effective treatment and/or prevention (prophylaxis), and includes 10%, 20%, 30% or more alleviation or stable conditions of symptoms.

[0037] In the present invention, inhibition of viral proliferation includes inhibition of the viral replication process in infected cells. When each viral protein expressed in the viral replication process is presented as an antigen in virus-infected cells, CTLs induced by the peptide of the present invention injure cellular functions (gene replication and protein translation) of the infected cells by their cytotoxic activity. Alternatively, the CTLs destroy the infected cells themselves. As a result, the replication process of viral particles, which relies on the cellular functions of the infected cells, is inhibited and viral proliferation is inhibited. Furthermore, in the present invention, suppression of aggravation means suppression of the worsening or progression of any symptom associated with coronavirus infections. Specifically, the progression of respiratory distress symptoms that requires interventional treatment such as oxygen inhalation or placement of a ventilator or extracorporeal membrane oxygenation (ECMO) is a typical example of aggravation in coronavirus infectious diseases. Thus, stopping, prevention, or alleviation of the progression (worsening) of those symptoms is included in the suppression of aggravation in the present invention.

[0038] In the context of the present invention, the term "antibody" refers to immunoglobulins and fragments thereof that are specifically reactive to a designated protein or peptide thereof. An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Furthermore, an "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from two or more intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An "antibody" may be antibodies of all classes (e.g., IgA, IgD, IgE, IgG and IgM).

[0039] Unless otherwise specified, the technical terms and scientific terms used herein all have the same meanings as terms commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0040] II. Peptides HLA-A24 is an allele commonly seen in Asians, and HLA-A02 is an allele commonly seen in Caucasians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12; Cao K et al., Hum Immunol 2001, 62(9): 1009-1030; Gonzalez-Galarza FF et al., Nucleic Acids Res 2020, 48(D1): D783-D788). Thus, an effective method of treating coronavirus infectious diseases for a great population of Asians or Caucasians can be provided by providing SARS-CoV-2 protein-derived CTL-inducing peptides restricted to HLA-A24 or HLA-A02. Thus, the present invention provides SARS-CoV-2 protein-derived peptides that are capable of inducing CTLs in an HLA A24- or HLA-A02-restrictive manner.

[0041] The peptides of the present invention are SARS-CoV-2 protein-derived peptides that are capable of inducing CTLs in an HLA-A24- or HLA-A02-restrictive manner. Peptides capable of inducing CTLs in an HLA-A24-restrictive manner include peptides having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. Peptides capable of inducing CTLs in an HLA-A02-restrictive manner include peptides having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13.

[0042] CTLs having a cytotoxic activity specific to these peptides can be established by in vitro stimulation of T cells by dendritic cells pulsed with these peptides. The established CTLs show a specific cytotoxic activity against target cells pulsed with each of the peptides.

[0043] CTLs are induced following presentation of coronavirus antigens (epitope peptides derived from coronavirus proteins) and then recognize epitope peptides presented on human leukocyte antigen (HLA) class I molecules expressed on the surface of coronavirus-infected cells to kill these cells. Thus, coronavirus antigens are excellent targets for immunotherapy. Therefore, the peptides of the present invention can be suitably used for immunotherapy of coronavirus infectious diseases. A preferred peptide is a nonapeptide (a peptide consisting of 9 amino acid residues) or a decapeptide (a peptide consisting of 10 amino acid residues), and it is more preferably a peptide consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. For example, a peptide having the amino acid sequence of SEQ ID NO: 1, 2, 4, 7, 10, 12 or 13 is suitable for induction of CTLs that show a specific cytotoxic activity against coronavirus-infected cells having HLA-A24 or HLA-02, and can be suitably used for immunotherapy of coronavirus infectious disease for HLA-A24- or HLA-A02-positive patients. In a more preferred embodiment, the peptide of the present invention is a peptide consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 for HLA-A24-positive patients, and a peptide consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 for HLA-A02-positive patients.

[0044] For the peptides of the present invention, an additional amino acid residue(s) can be made to adjoin the amino acid sequence of the peptide of the present invention, as long as the resultant peptides retain the CTL-inducing ability of the original peptide. The additional amino acid residue(s) may be composed of any types of amino acid(s), 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 the amino acid sequence selected from among 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, in many cases less than about 20 amino acids, and usually less than about 15 amino acids. Therefore, if the original peptide is a nonapeptide, the peptide of the present invention encompasses peptides that are 10 amino-acid long or 11-40 amino-acid long, which are produced by adjoining additional amino acid(s) to the peptide. Furthermore, if the original peptide is a decapeptide, the peptide of the present invention encompasses peptides that are 11-40 amino-acid long. Such a peptide can be, for example, a peptide that is 11-20 amino-acid long or a peptide that is 11-15 amino-acid long. A preferred example of an additional amino acid residue(s) is an amino acid residue(s) 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 a genome sequence of SARS-CoV-2 (for example, SEQ ID NOs: 17 26). Therefore, the peptides of the present invention encompass peptides comprising the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15, and wherein the peptides are peptide fragments of SARS-CoV-2 protein and have CTL-inducing ability.

[0045]

In general, modifications of one, two or more amino acids in a certain peptide do not affect the functions of the peptide, or in some cases even enhance the desired functions of the original peptide. In fact, modified peptides (i.e., peptides composed of the amino acid sequence in which one, two or several amino acid residues are 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). Thus, in one embodiment, the peptides of the present invention can be peptides comprising the amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 and having CTL-inducing ability.

[0046] One skilled in the art can recognize that individual substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids tend to result in the conservation of the properties of the original amino acid side chain(s). Accordingly, those are frequently referred to as "conservative substitutions" or "conservative modifications"; and modification of a protein by "conservative substitution" or "conservative modification" may result in a modified protein that has similar functions as the original protein. Tables of conservative substitutions presenting functionally similar amino acids are well known in the art. Examples of amino acid side chain characteristics that functionally resemble 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 having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). In addition, the following eight groups each contain amino acids that are accepted in the art as conservative substitutions for 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, e.g., Creighton, Proteins 1984).

[0047]

Such conservatively modified peptides are also encompassed in peptides of the present invention. However, peptides of the present invention are not restricted thereto and can include non-conservative modifications, so long as the modified peptide retains the CTL-inducing ability of the original peptide. Furthermore, modified peptides do not exclude CTL inducible peptides derived from polymorphic variants, interspecies homologues, and alleles of SARS-CoV-2 proteins.

[0048] So long as a peptide retains the CTL-inducing ability of an original peptide, one can modify (i.e., substitute, delete, insert and/or add) a small number (for example, 1, 2 or several) or a small percentage of amino acids. Herein, the term "several" means 5 or fewer amino acids, for example, 4 or 3 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 I to 5%.

[0049] When used in the context of immunotherapy, peptides of the present invention should be presented on the surface of a cell or exosome, preferably as a complex with an HLA antigen. Therefore, it is preferable that the peptides of the present invention possess high binding affinity to the HLA antigen. To that end, the peptides can be modified by substitution, deletion, insertion, and/or addition of the amino acid residues to yield a modified peptide having improved binding affinity. Since the regularity of the sequences of peptides displayed by binding to HLA antigens is already 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 peptides of the present invention.

[0050] For example, in peptides having binding affinity for HLA Class I, the second amino acid from the N terminus and the C-terminal amino acid are generally anchor residues involved in the binding to HLA Class I (Rammensee HG, et al., Immunogenetics. 1995; 41(4): 178-228.). For example, peptides having 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 in which the C-terminal amino acid has been substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine tend to have high HLA-A24 binding affinity. Thus, in order to enhance 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 to substitute the C-terminal amino acid with phenylalanine, leucine, isoleucine, tryptophan, or methionine. Accordingly, the present invention encompasses peptides having an amino acid sequence selected from those of 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 has been substituted with phenylalanine, tyrosine, methionine or tryptophan and/or the C terminus has been substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine.

[0051] Similarly, the present invention encompasses peptides comprising an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted, and/or added in the sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, and 15, which peptides have one or both of the features: (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 comprise an amino acid sequence comprising one 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, in the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15.

[0052] Likewise, peptides having high HLA-A02-binding affinity tend to have the second amino acid from the N terminus substituted with leucine or methionine and/or the C-terminal amino acid substituted with valine or leucine. Thus, in order to enhance HLA-A02-binding affinity, it may be desirable to substitute the second amino acid from the N terminus with leucine or methionine and/or to substitute the C-terminal amino acid with valine or leucine. Accordingly, the present invention encompasses peptides having an amino acid sequence selected from those of SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 in which the second amino acid from the N terminus has been substituted with leucine or methionine and/or the C-terminus has been substituted with valine or leucine.

[0053] Similarly, the present invention encompasses peptides comprising an amino acid sequence in which one, two, or several amino acids are substituted, deleted, inserted, and/or added in the sequences of SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13, which peptides have one or both of the features: (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 comprise an amino acid sequence comprising one 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, in the amino acid sequences of SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13.

[0054]

Substitution(s) may be introduced into amino acid(s) not only at the terminus/termini, but also at a position(s) of potential T cell receptor (TCR) recognition site(s) of the peptides. Several research studies have demonstrated that a peptide that has amino acid substitutions, such as CAPI, p5 3 (264-272), Her-2/neu369-377 or gp100(209-217),may have equal to or better activity than that of the original peptide (Zaremba et al. Cancer Res. 57, 4570-4577, 1997; T. K. Hoffmann et al. J Immunol. (2002) Feb 1, 168(3): 1338-47; S. 0. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206; and S. 0. Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-14).

[0055] The present invention also contemplates that one, two or several amino acids can be added to the N terminus and/or C terminus of the peptides of the present invention (for example, peptides consisting of the amino acid sequence selected from among 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, when a peptide in which one, two or several amino acids are added to the N terminus and/or C terminus of a peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 or 15 is contacted with an APC(s), it is incorporated into the APC(s) and processed to become a peptide consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 or 15. It can then induce CTLs through presentation on the cell surface of an APC via the antigen presentation pathway.

[0056] However, when the amino acid sequence of a peptide is identical to a portion of the amino acid sequence of an endogenous or exogenous protein having a different function, side effects such as autoimmune disorders and/or allergic symptoms against specific substances may be induced. Therefore, it is preferable to perform homology searches using available databases to avoid situations in which the amino acid sequence of the peptide matches the amino acid sequence of another protein. When it becomes clear from the homology searches that no peptide exists with as few as 1 or 2 amino acid differences as compared to the objective peptide, the objective peptide can be modified in order to increase its binding affinity with HLA antigens, and/or increase its CTL-inducing ability without danger of such side effects.

[0057] Peptides in which one, two or several amino acids of a peptide of the present invention are modified are predicted to be able to retain CTL-inducing ability of the original peptide; however, it is preferable to verify the CTL-inducing ability of the modified peptides. Herein, the "peptide having CTL-inducing ability (CTL inducibility)" refers to a peptide that induces CTLs through APCs stimulated with the peptide. "CTL induction" includes induction of differentiation into CTLs, induction of CTL activation, induction of CTL proliferation, induction of CTL's cytotoxic activity, induction of CTL-mediated dissolution of target cells, and induction of increase of IFN-gamma production of CTLs.

[0058] The CTL-inducing ability can be confirmed by inducing and stimulating APCs that retain an HLA antigen (for example, B lymphocytes, macrophages, and dendritic cells) with a peptide, and mixing it with CD8-positive T cells; and then measuring IFN-gamma released by CTLs against the target cells. For the APCs, human peripheral blood mononuclear leukocyte derived dendritic cells can be preferably used. As a reaction system, transgenic animals generated to express an HLA antigen can be used. Alternatively, for example, the target cells may be radio-labelled with 5 1Cr or such, and the cytotoxic activity of the peptide-induced CTLs may be calculated from the radioactivity emitted from the target cells. Alternatively, in the presence of peptide-stimulated APCs, it is possible to evaluate the CTL-inducing ability by measuring the IFN-gamma produced and released by CTLs, and visualizing the inhibition zone on the media using anti-IFN-gamma monoclonal antibodies.

[0059] In addition to the modifications above, the peptides of the present invention can be linked to other peptides as long as the resultant linked peptide retains the CTL-inducing ability. An example of an appropriate peptide to be linked with the peptides of the present invention includes other CTL-inducing peptide derived from coronavirus proteins. Further, the peptides of the present invention can also be linked with each other. Suitable linkers for use in linking peptides are known in the art, and for example, linkers such as AAY (P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO: 27) (R. P. M. Sutmuller et al., J Immunol. 2000, 165: 7308-15), or K (S. Ota et al., Can Res. 62, 1471-6, K. S. Kawamura et al., J Immunol. 2002, 168: 5709-15) can be used. Peptides can be linked in various arrangements (for example, catenulate, repeated, etc.), and one can also link three or more peptides.

[0060] The peptides of the present invention can also be linked to other substances as long as the resultant linked peptide retains the CTL-inducing ability. Examples of an appropriate substance to be linked with a peptide of the present invention include, for example, a peptide, a lipid, a sugar or sugar chain, an acetyl group, and a naturally-occurring or synthetic polymer. The peptides of the present invention can be modified by glycosylation, side-chain oxidation, phosphorylation or such, as long as their CTL-inducing ability is not impaired. One can also perform such types of modifications to confer additional functions (for example, targeting function and delivery function) or to stabilize the peptide.

[0061]

For example, to increase the in vivo stability of a peptide, it is known in the art to introduce D-amino acids, amino acid mimetics or non-naturally occurring amino acids, and this concept may also be applied to peptides of the present invention. Peptide stability can be assayed by several methods. For example, stability can be tested by using a peptidase as well as various biological media such as human plasma and serum (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

[0062] Further, as stated above, among the modified peptides in which one, two, or several amino acid residues have been substituted, deleted, inserted and/or added, those having the same or higher activity as compared to original peptides can be screened for or selected. Thus, the present invention also provides methods of screening for or selecting modified peptides that have the same or higher activity than that of the original peptide. Specifically, the present invention provides a method of screening for a peptide having CTL-inducing ability, wherein the method comprises the steps of: (a) generating candidate sequences consisting of an amino acid sequence in which one, two, or several amino acid residues are substituted, deleted, inserted and/or added to the original amino acid sequence consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15; (b) selecting from among the candidate sequences generated in (a), a candidate sequence that does not have a significant homology (sequence identity) with any known human gene product; (c) contacting a peptide consisting of the candidate sequence selected in (b) with APCs; (d) contacting the APCs of (c) with CD8-positive T cells; and (e) selecting a peptide that has an equal to or higher CTL-inducing ability than that of a peptide consisting of the original amino acid sequence. In an embodiment, APCs to be contacted with a peptide are APCs positive for either one of HLA-A02 or HLA-A24, or for both.

[0063] Herein, the peptide of the present invention is also described as a "SARS-CoV-2 peptide(s)" or a "SARS-CoV-2 polypeptide(s)".

[0064] III. Preparation of peptides of the present invention Well known techniques can be used to prepare peptides of the present invention. For example, recombinant DNA technology or chemical synthesis can be used to prepare peptides of the present invention. Peptides of the present invention can be synthesized individually, or as longer polypeptides including two or more peptides. Peptides of the present invention can be isolated from host cells or synthesis reaction products after they are produced in the host cells using recombinant DNA technology or after they are chemically synthesized. That is, peptides of the present invention can be purified or isolated so as not to substantially contain other host cell proteins and fragments thereof, or any other chemical substances.

[0065] The peptides of the present invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation provided such modifications do not destroy the biological activity of the original peptide. Other illustrative modifications include incorporation of D-amino acids or other amino acid mimetics that may be used, for example, to increase the serum half life of the peptides.

[0066] A peptide of the present invention can be obtained through chemical synthesis based on the selected amino acid sequence. Examples of conventional peptide synthesis methods that can be adapted to the synthesis include the methods described in the documents below: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) "Peptide Synthesis" (in Japanese), Maruzen Co., 1975; (iv) "Basics and Experiment of Peptide Synthesis" (in Japanese), Maruzen Co., 1985; (v) "Development of Pharmaceuticals" (in Japanese), Continued Vol. 14 (peptide synthesis), Hirokawa, 1991; (vi) W099/67288; and (vii) Barany G. & Merrifield R.B., Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-18.

[0067] Alternatively, the peptides of the present invention can be obtained adapting any known genetic engineering methods for producing peptides (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 harboring 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. The peptide of the present invention can also be produced in vitro using an in vitro translation system.

[0068] IV. Polynucleotides

The present invention also provides a polynucleotide which encodes any of the peptides of the present invention. These include polynucleotides derived from the naturally occurring SARS-CoV-2 gene (e.g., GenBank Accession No. MN908947 (SEQ ID NO: 16)) as well as those having a conservatively modified nucleotide sequence thereof. Herein, the phrase "conservatively modified nucleotide sequence" refers to sequences which encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a peptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.

[0069] The polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof. A DNA is suitably composed of bases such as A, T, C and G, and T is replaced by U in an RNA.

[0070] The polynucleotide of the present invention can encode multiple peptides of the present invention with or without intervening amino acid sequences in between. For example, the intervening amino acid sequence can provide a cleavage site (e.g., enzyme recognition sequence) of the polynucleotide or the translated peptides. Furthermore, the polynucleotide can include any additional sequences to the coding sequence encoding the peptide of the present invention. For example, the polynucleotide can be a recombinant polynucleotide that includes regulatory sequences required for the expression of the peptide or can be an expression vector (e.g., plasmid) with marker genes and such. In general, such recombinant polynucleotides can be prepared by the manipulation of polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.

[0071] Both recombinant and chemical synthesis techniques can be used to produce the polynucleotides of the present invention. For example, a polynucleotide can be produced by insertion into an appropriate vector, which can be expressed when transfected into a competent cell. Alternatively, a polynucleotide can be amplified using PCR techniques or expression in suitable hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). Alternatively, a polynucleotide can be synthesized using the solid phase techniques, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223 311; Matthes et al., EMBO J 1984, 3: 801-5.

[0072] V. Exosomes The present invention further provides intracellular vesicles, referred to as exosomes, that present complexes formed between the peptides of the present invention and HLA antigens on their surface. Exosomes can be prepared, for example, using the methods detailed in JPH11 510507 and W099/03499, and can be prepared using APCs obtained from patients who are subject to treatment and/or prevention (prophylaxis). The exosomes of the present invention can be inoculated as vaccines, in a fashion similar to the peptides of the present invention.

[0073] The type of the HLA antigens included in the above-described complexes must match that of the subject in need of treatment and/or prevention (prophylaxis). For example, HLA-A24 (for example, HLA-A*24:02) is an allele widely and generally seen in Asian countries including Japan, and HLA-A02 (for example, HLA-A*02:01) is an allele widely and generally seen in Europe and the United States. These HLA antigen types are considered to be suitable for treatment in Asian or Caucasian patients. Typically in clinic, it is possible to select an appropriate peptide that has a high level of binding affinity for a specific HLA antigen or that has CTL-inducing ability by antigen presentation mediated by a specific HLA antigen, by studying in advance the HLA antigen type of the patient in need of treatment.

[0074] The exosomes of the present invention present on their surface a complex of a peptide of the present invention and HLA-A24 or HLA-A02. When the HLA that forms a complex with a peptide of the present invention is HLA-A24, the peptide of the present invention is preferably a peptide having the amino acid sequence selected from among 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 the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof. When the HLA that forms a complex with a peptide of the present invention is HLA A02, the peptide of the present invention is preferably a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof, and more preferably a peptide consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof.

[0075] VI. Antigen-presenting cells (APCs) The present invention further provides APCs that present on their surface complexes formed between HLA antigens and peptides of the present invention. Alternatively, the present invention provides APCs having on their cell surface complexes formed between HLA antigens and peptides of the present invention. The APCs of the present invention can be isolated APCs. When used in the context of cells (APCs, CTLs, etc.), the term "isolated" means that the cells are separated from another type of cells. The APCs of the present invention may be APCs induced from APCs derived from the patient to be subjected to treatment and/or prevention (prophylaxis), and can be administered as a vaccine by themselves or in combination with other drugs including a peptide(s), an exosome(s) or a CTL(s) of the present invention.

[0076] The APCs of the present invention are not limited to specific types of cells, and include cells known to present proteinaceous antigens on their cell surface so as to be recognized by lymphocytes, for example, dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells. Since DC is a representative APC that has the strongest CTL-inducing activity among APCs, DCs can be preferably used as the APCs of the present invention.

[0077] 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 peptides of the present invention. When the peptide of the present invention is administered to a subject, APCs presenting the peptide of the present invention are induced in the body of the subject. Therefore, after the peptides of the present invention are administered to a subject, the APCs of the present invention can be obtained by collecting APCs from the subject. Alternatively, the APCs of the present invention can be obtained by contacting APCs collected from a subject with a peptide of the present invention.

[0078] In order to induce an immune response against coronavirus-infected cells in a subject, the APCs of the present invention can be administered to the subject by themselves or in combination with other drugs including peptide(s), exosome(s) or CTL(s) of the present invention. For example, the ex vivo administration can comprise the following steps of: (a) collecting APCs from a first subject; (b) contacting the APCs of step (a) with a peptide; and

(c) administering the APCs of step (b) to a second subject.

[0079] The first subject and the second subject may be the same individual, or may be different individuals. When the first subject and the second subject are different individuals, it is preferable that the HLAs of the first subject and the second subject are of the same type. The APC obtained in step (b) above can be a vaccine for treatment and/or prevention (prophylaxis) of coronavirus infectious disease.

[0080] The APCs of the present invention obtained by a method such as described above have CTL-inducing ability. The term "CTL-inducing ability (CTL inducibility)" used in the context of an APC(s) refers to the ability of the APC to be able to induce a CTL(s) when placed in contact with a CD8-positive T cell(s). The CTL(s) induced by the APC of the present invention is a CTL(s) specific to SARS-CoV-2 protein and demonstrates a specific cytotoxic activity against SARS-CoV-2 infected cells.

[0081] In addition to the above-described methods, the APCs of the present invention can be prepared by introducing a polynucleotide encoding a peptide of the present invention into APCs in vitro. The polynucleotide to be introduced can be in the form of DNA or RNA. The method of introduction is not particularly limited, and examples thereof include various methods conventionally performed in the art such as lipofection, electroporation and the calcium phosphate method. More specifically, methods described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72, and JP2000-509281 can be used. By introducing a polynucleotide encoding a peptide of the present invention into an APC, the polynucleotide is transcribed and translated in the cell, and then the produced peptide is processed by MHC Class I and proceeds through a presentation pathway to present the peptide of the present invention on the cell surface of the APC.

[0082] In a preferred embodiment, the APC of the present invention is an APC that presents on its cell surface a complex formed between a peptide of the present invention and HLA-A24 (more preferably HLA-A*24:02). When the HLA that forms a complex with a peptide of the present invention is HLA-A24, the peptide of the present invention is preferably a peptide having the amino acid sequence selected from among 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 the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15.

[0083]

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

[0084] The APC(s) of the present invention is preferably an APC(s) induced by a method comprising a step described (a) or (b) below: (a) contacting an APC(s) expressing HLA-A24 (more preferably HLA-A*24:02) or HLA-A02 (more preferably HLA-A*02:01) with a peptide of the present invention; or (b) introducing a polynucleotide encoding a peptide of the present invention into an APC(s) expressing HLA-A24 (more preferably HLA-A*24:02) or HLA-A02 (more preferably HLA A*02:01).

[0085] The peptide of the present invention to be contacted with the HLA-A24-expressing APC(s) is preferably a peptide having the amino acid sequence selected from among 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 the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. The peptide of the present invention to be contacted with the HLA A02-expressing APC(s) is preferably a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof, and more preferably a peptide consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13.

[0086] The present invention provides use of a peptide of the present invention for the manufacture of a pharmaceutical composition that induces an APC(s) having CTL-inducing ability. In addition, the present invention provides a method or process of manufacturing a pharmaceutical composition that induces an APC(s) having CTL-inducing ability. Further, the present invention provides a peptide of the present invention for inducing an APC(s) having CTL-inducing ability.

[0087] VII. Cytotoxic T lymphocytes (CTLs)

The CTL induced by a peptide of the present invention can be used as a vaccine in a similar manner to the peptide of the present invention for enhancing an immune response targeting coronavirus-infected cell in vivo. Thus, the present invention provides CTLs that are induced or activated by a peptide of the present invention. The CTLs of the present invention are CTLs that target a peptide of the present invention, and are capable of binding to a complex of a peptide of the present invention and an HLA antigen. Binding of a CTL to the complex is mediated via a T cell receptor (TCR) present on the cell surface of the CTL. The CTLs of the present invention can be isolated CTLs.

[0088] The CTLs of the present invention can be obtained by (1) administering a peptide of the present invention to a subject, (2) stimulating APCs and CD8-positive T cells, or peripheral blood mononuclear cells (PBMCs) derived from a subject with a peptide of the present invention in vitro, (3) contacting in vitro CD8-positive T cells or PBMCs with APCs or exosomes that present on their surface a complex of an HLA antigen and a peptide of the present invention, or (4) introducing into CD8-positive T cells a vector comprising a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to a peptide of the present invention presented on cell surface via an HLA antigen. The exosomes and APCs used in the method of (2) or (3) above can be prepared by methods described in the "V. Exosomes" and "VI. Antigen presenting cells (APCs)" sections, respectively, and the details of the method of (4) above will be described in the "VIII. T cell receptor (TCR)" section.

[0089] The CTLs of the present invention can be administered by themselves to patients who are subject to treatment and/or prevention (prophylaxis), or in combination with other drugs including peptide(s), APC(s) or exosome(s) of the present invention for the purpose of regulating effects. Further, the CTLs of the present invention can be CTLs induced from CD8-positve T cells derived from the patients who are subject to 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 peptides used to induce the CTLs of the present invention. The target cells may be cells that endogenously express SARS-CoV-2 protein, such as coronavirus-infected cells, or cells transfected with the SARS-CoV-2 gene. Cells that present a peptide of the present invention on their cell surface due to stimulation by the peptide can become a target of attack by the CTLs of the present invention. The cells targeted by the CTLs of the present invention are preferably cells that are positive for HLA-A24 (more preferably HLA-A*24:02) or HLA-A02 (more preferably HLA-A*02:01).

[0090]

In a preferred embodiment, the CTLs of the present invention target specifically cells that express both SARS-CoV-2 protein and HLA-A24 (more preferably HLA-A*24:02) or HLA A02 (more preferably HLA-A*02:01). Herein, that the CTL "targets" cells refers to CTL recognition of cells that present on their cell surface a complex of HLA and a peptide of the present invention and demonstration of a cytotoxic activity against the cells. Further, "specifically target" refers to that the CTLs demonstrate a cytotoxic activity against those cells, but do not show a damaging activity to other cells. The expression "recognize cells" used in the context of CTLs refers to binding to a complex of HLA and a peptide of the present invention presented on cell surface via its TCR, and demonstrating a specific cytotoxic activity against the cell. Therefore, the CTLs of the present invention are preferably CTLs that can bind via TCR to a complex formed between a peptide of the present invention and HLA-A24 (more preferably HLA-A*24:02) or HLA-A02 (more preferably HLA-A*02:01) presented on cell surface. Furthermore, the CTLs of the present invention are preferably CTLs induced by a method comprising a step described in (a) or (b) below: (a) contacting in vitro CD8-positive T cells with APCs or exosomes that present on their surface a complex of a peptide of the present invention and HLA-A24 (more preferably HLA A*24:02) or HLA-A02 (more preferably HLA-A*02:01); or (b) introducing into CD8-positive T cells a polynucleotide encoding each subunit of a TCR capable of binding to a peptide of the present invention presented on cell surface by HLA A24 (more preferably HLA-A*24:02) or HLA-A02 (more preferably HLA-A*02:01).

[0091] VIII. T cell receptors (TCRs) The present invention also provides compositions comprising a polynucleotide encoding each subunit of a TCR capable of binding to a peptide of the present invention presented on cell surface by an HLA antigen, and methods of using the same. The polynucleotide confers CD8 positive T cells with specificity against coronavirus-infected cells through expression of a TCR capable of binding to a peptide of the present invention presented on cell surface by an HLA antigen. Polynucleotides encoding an alpha chain(s) and a beta chain(s) can be identified as the TCR subunit of the CTL induced by a peptide of the present invention by using known methods in the art (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)). For example, PCR methods are preferred for TCR analysis. Without being limited thereto, PCR primers for analysis may be, for example, 5'-R Primer (5'-gtctaccaggcattcgcttcat-3') (SEQ ID NO: 28) as a 5' side primer; and 3-TRa-C Primer (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 29) specific to TCR-alpha-chain C-region, 3-TRb-C1 Primer (5'-tcagaaatcctttctcttgac-3') (SEQ ID NO: 30) specific to TCR-beta-chain Cl-region or 3-TR-beta-C2 Primer (5'-tagcctctggaatctttctctt-3')

(SEQ ID NO: 31) specific to TCR-beta-chain C2-region as 3' side primers. The TCRs formed by introducing the identified polynucleotides into CD8-positive T cells can bind with high binding affinity to the target cells that present a peptide of the present invention, and mediates efficient killing of the target cells presenting a peptide of the present invention in vivo and in vitro.

[0092] A polynucleotide encoding each TCR subunit can be incorporated into an appropriate vector, for example, retrovirus vector. These vectors are well known in the art. The polynucleotide or a vector comprising thereof in an expressible form can be introduced into a CD8-positive T cell, for example, a CD8-positive T cell derived from a patient. The present invention provides off-the-shelf compositions that allow rapid and easy production of modified T cells that have superior coronavirus-infected cell-killing properties by rapid modification of the patient's own T cells (or T cells derived from another subject).

[0093] Herein, a specific TCR is a TCR that can confer a specific cytotoxic activity against target cells by specifically recognizing a complex of a peptide of the present invention and an HLA antigen presented on the surface of the target cell when the TCR is present on the surface of a CD8-positive T cell. Specific recognition of the above-described complex can be confirmed by any known method, and preferable examples thereof include HLA multimer staining analysis using HLA molecules and peptides of the present invention and ELISPOT assay methods. Specific TCR-mediated recognition of target cell by T cell introduced with the above-described polynucleotide and signal transduction in the cell can be confirmed by carrying out an ELISPOT assay. When the above-described TCR is present on the surface of a CD8-positive T cell, whether the TCR can confer a target cell-specific cytotoxic activity against the CD8-positive T cell can also be confirmed by known methods. Preferable methods include, for example, measuring the cytotoxic activity against target cells by a chrome release assay method or such.

[0094] The present invention provides, in the context of HLA-A24, CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each subunit of TCR that binds to, for example, a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15. The present invention provides, in the context of HLA-A02, CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each subunit of TCR that binds to, for example, a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13.

[0095]

The transformed CTLs are capable of homing in vivo and may be propagated by a well known in vitro culturing method (for example, Kawakami et al., J Immunol., 142, 3452-61 (1989)). The CTLs of the present invention can be used to form an immunogenic composition useful for disease treatment or prevention (prophylaxis) in a patient in need of treatment or prevention (prophylaxis) (the contents are incorporated herein for reference W02006/031221).

[0096] IX. Pharmaceutical compositions The present invention further provides compositions or pharmaceutical compositions, comprising at least one active ingredient selected from below: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC of the present invention; (d) an exosome of the present invention; and (e) a CTL of the present invention.

[0097] The pharmaceutical compositions of the present invention can comprise as needed a carrier(s), an excipient(s) or such commonly used in pharmaceuticals without particular limitations, in addition to the active ingredient(s) described above. An example of a carrier that can be used in a pharmaceutical composition of the present invention includes sterilized water, physiological saline, phosphate buffer, culture fluid and such. Therefore, the present invention also provides pharmaceutical compositions, comprising at least one active ingredient selected from (a) to (e) below and a pharmaceutically acceptable carrier: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC of the present invention; (d) an exosome of the present invention; and (e) a CTL of the present invention.

[0098] Further, the pharmaceutical compositions of the present invention can comprise, as needed, stabilizers, suspensions, preservatives, surfactants, solubilizing agents, pH adjusters, aggregation inhibitors and such. The SARS-CoV-2 protein expression significantly up-regulates in coronavirus-infected cells compared with coronavirus non-infected cells. Thus, a peptide of the present invention or a polynucleotide encoding the peptide can be used to any one of purposes selected from treatment, prevention (prophylaxis), and suppression of aggravation of coronavirus infectious disease, and combinations thereof. Therefore, the present invention provides pharmaceutical compositions for any one of purposes selected from treatment, prevention (prophylaxis), and suppression of aggravation of coronavirus infectious disease, or plurality of purposes, the pharmaceutical composition comprising one or more types of peptides or polynucleotides of the present invention as an active ingredient. Alternatively, the peptides of the present invention can be made to be presented on the surface of exosomes or APCs for use as pharmaceutical compositions. In addition, CTLs of the present invention targeting any one of the peptides of the present invention can also be used as an active ingredient of the pharmaceutical compositions of the present invention. The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount or a pharmaceutically effective amount of the above-described active ingredient.

[0099] The pharmaceutical compositions of the present invention may also be used as a vaccine. In the context of the present invention, the term "vaccine" (also called "immunogenic composition") refers to a composition that has a function of inducing an immune response that leads to anti-infective action against coronavirus when inoculated into an animal. Thus, a pharmaceutical composition of the present invention can be used to induce an immune response that leads to anti-invective action against coronavirus. The immune response induced by a peptide, a polynucleotide, an APC, a CTL and a pharmaceutical composition of the present invention is not particularly limited as long as it is an immune response that leads to anti infective action against coronavirus, and examples include induction of coronavirus-infected cell-specific CTLs and induction of coronavirus-infected cell-specific cytotoxic activity. The pharmaceutical compositions of the present invention can be used for any one of treatment, prevention (prophylaxis), and suppression of aggravation of coronavirus infectious diseases, or combination thereof in human subjects or patients. The pharmaceutical compositions of the present invention can be used preferably to a subject positive for HLA-A24 or HLA-A02. Further, the pharmaceutical compositions of the present invention can be used preferably to treat and/or prevent coronavirus infectious diseases, and/or suppress aggravation in a subject having HLA-A24 or HLA-A02.

[0100] In another embodiment, the present invention provides use of an active ingredient selected from below in the manufacture of a pharmaceutical composition for either or both of treating and preventing coronavirus infectious disease: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface;

(d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0101] Alternatively, the present invention further provides an active ingredient selected from below for use in either or both of treating and preventing coronavirus infectious disease: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface; (d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0102] Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for either or both of treating and preventing coronavirus infectious disease, wherein the method or process comprises a step of formulating at least one active ingredient selected from below with a pharmaceutically or physiologically acceptable carrier: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface; (d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0103] In another embodiment, the present invention further provides a method or process for manufacturing a pharmaceutical composition for either or both of treating and preventing coronavirus infectious disease, wherein the method or process comprises a step of mixing an active ingredient selected from below with a pharmaceutically or physiologically acceptable carrier: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface; (d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0104] In another embodiment, the present invention further provides a method for either or both of treating and preventing coronavirus infectious disease, which comprises a step of administering to a subject at least one active ingredient selected from below:

(a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface; (d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0105] In the present invention, peptides having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 are identified as HLA-A24-restricted epitope peptides that can induce a potent and specific immune response. Therefore, pharmaceutical compositions of the present invention comprising at least one peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 are suitable particularly for administration to a subject having HLA-A24 (for example, HLA A*24:02) as an HLA antigen. The same applies to pharmaceutical compositions comprising a polynucleotide encoding any of these peptides (i.e., polynucleotides of the present invention), an APC or exosome that presents these peptides (i.e., APCs or exosomes of the present invention), or a CTL targeting these peptides (i.e., CTLs of the present invention). That is, pharmaceutical compositions comprising an active ingredient in association with a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 are suitable for administration to subjects having HLA-A24 (i.e., HLA-A24-positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition that comprises a peptide having the amino acid sequence of SEQ ID NO: 1, 2, 4, 5, 7, 9, 10 or 13.

[0106] Similarly, in the present invention, peptides having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 are identified as HLA-A02-restricted epitope peptides that can induce a potent and specific immune response. Therefore, pharmaceutical compositions of the present invention comprising at least one peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 are suitable particularly for administration to a subject having HLA-A02 (for example, HLA-A*02:01) as an HLA antigen. The same applies to pharmaceutical compositions comprising a polynucleotide encoding any of these peptides (i.e., polynucleotides of the present invention), an APC or exosome that presents these peptides (i.e., APCs or exosomes of the present invention), or a CTL targeting these peptides (i.e., CTLs of the present invention). That is, pharmaceutical compositions comprising an active ingredient in association with a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 are 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 that comprises a peptide having the amino acid sequence of SEQ ID NO: 1, 2, 10 or 13.

[0107] Coronavirus infectious diseases to be treated and/or prevented by pharmaceutical compositions of the present invention are not particularly limited as long as they are coronavirus infectious diseases, and coronaviruses include SARS-CoV-2, MERS-CoV, SARS-CoV, and such.

[0108] In addition to the active ingredients described above, the pharmaceutical compositions of the present invention can comprise the other peptides that have the ability to induce CTLs against coronavirus-infected cells (for example, the other coronavirus protein-derived CTL inducing peptides), the other polynucleotides encoding the other peptides, the other cells that present the other peptides, or such.

[0109] The pharmaceutical compositions of the present invention may also optionally comprise the other therapeutic substances as an active ingredient, as long as they do not inhibit the anti infective effects against coronavirus of the above-described active ingredients such as peptides of the present invention. For example, the pharmaceutical compositions of the present invention may optionally comprise anti-inflammatory compositions, analgesics, chemotherapeutics and the like. In addition to including the other therapeutic substances to a pharmaceutical composition of the present invention itself, one can also administer the pharmaceutical composition of the present invention sequentially or concurrently with one or more other pharmaceutical compositions. The dose of the pharmaceutical composition of the present invention and the other pharmaceutical compositions depend on, for example, the type of pharmaceutical composition used and the disease being treated, as well as the scheduling and routes of administration.

[0110] It should be understood that in consideration of the formulation type, the pharmaceutical composition of the present invention may include other components conventional in the art, in addition to the ingredients specifically mentioned herein.

[0111] The present invention also provides articles of manufacture or kits that comprise a pharmaceutical composition of the present invention. The articles of manufacture or kits of the present invention can include a container that houses the pharmaceutical composition of the present invention. An example of an appropriate container includes a bottle, a vial or a test tube, but is not limited thereto. The container may be formed of various materials such as glass or plastic. A label may be attached to the container, and the disease or disease state to which the pharmaceutical composition of the present invention should be used may be described in the label. The label may also indicate directions for administration and such.

[0112] The articles of manufacture or kits of the present invention may further comprise a second container that houses pharmaceutically acceptable diluents optionally, in addition to the container that houses the pharmaceutical composition of the present invention. The articles of manufacture or kits of the present invention may further comprise the other materials desirable from a commercial standpoint and the user's perspective, such as the other buffers, diluents, filters, injection needles, syringes, package inserts with instructions for use.

[0113] As needed, the pharmaceutical composition of the present invention can be provided in a pack or dispenser device that can contain one or more units of dosage forms containing active ingredients. The pack can include, for example, a metallic foil or a plastic foil such as a blister pack. Instructions for administration can be attached to the pack or dispenser device.

[0114] (1) Pharmaceutical compositions comprising peptide(s) as an active ingredient The pharmaceutical composition comprising a peptide of the present invention can be formulated by conventional formulation methods as needed. The pharmaceutical compositions of the present invention may comprise as needed in addition to the peptide of the present invention, carriers, excipients and such commonly used in pharmaceuticals without particular limitations. Examples of carriers that can be used in pharmaceutical compositions of the present invention include sterilized water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline, 0.3% glycine, culture fluid, and such. Further, the pharmaceutical compositions of the present invention may comprise as needed stabilizers, suspensions, preservatives, surfactants, solubilizing agents, pH adjusters, aggregation inhibitors, and such. The pharmaceutical compositions of the present invention can induce specific immunity against coronavirus-infected cells, and thus can be applied for the purpose of treatment or prevention (prophylaxis) of coronavirus infectious diseases.

[0115] For example, the pharmaceutical compositions of the present invention can be prepared by dissolving in pharmaceutically or physiologically acceptable water-soluble carriers such as sterilized water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, and Tris buffered saline and adding, as needed, stabilizers, suspensions, preservatives, surfactants, solubilizing agents, pH adjusters, aggregation inhibitors and such, and then sterilizing the peptide solution. The method of sterilizing a peptide solution is not particularly limited, and is preferably carried out by filtration sterilization. Filtration sterilization can be performed using, for example, a filtration sterilization filter of 0.22 micro-m or less in pore diameter. Thefiltration-sterilized peptide solution can be administered to a subject, for example, as an injection, without being limited thereto. The pharmaceutical compositions of the present invention may be prepared as a freeze-dried formulation by freeze drying the above-described peptide solution. The freeze-dried formulation can be prepared by filling the peptide solution prepared as described above into an appropriate container such as an ampule, a vial or a plastic container, followed by freeze drying and encapsulation into the container with a wash-sterilized rubber plug or such after pressure recovery. The freeze-dried formulation can be administered to a subject after it is re-dissolved in pharmaceutically or physiologically acceptable water-soluble carriers such as sterilized water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline and such before administration. Preferred examples of pharmaceutical compositions of the present invention include injections of such a filtration-sterilized peptide solution, and freeze dried formulations that result from freeze-drying the peptide solution. The present invention further encompasses kits comprising such a freeze-dried formulation and re-dissolving solution. The present invention also encompasses kits comprising a container that houses the freeze-dried formulation, which is a pharmaceutical composition of the present invention, and a container that houses a re-dissolving solution thereof.

[0116] The pharmaceutical compositions of the present invention can comprise a combination of two or more types of the peptides of the present invention. The combination of peptides can take a cocktail form of mixed peptides, or can be conjugated with each other using standard techniques. For example, peptides can be chemically linked or expressed as single fusion polypeptide sequences. By administering a peptide of the present invention, the peptide is presented on APCs by an HLA antigen at a high density, and then subsequently CTLs that react specifically to a complex formed between the presented peptide and the HLA antigen are induced. Alternatively, APCs (for example, DCs) are removed from a subject, and subsequently stimulated with peptides of the present invention to obtain APCs that present any of the peptides of the present invention on their cell surface. These APCs are re-administered to a subject to induce CTLs in the subject, and as a result, the aggressiveness towards coronavirus-infected cells can be increased. Neutralizing antibodies may lose their ability to block infection (viral immune escape) due to a mutation in their target epitope. In general, the effect of an antigen mutation is particularly significant in monoclonal antibodies, which depend on a single epitope for antigen binding specificity. On the other hand, when the pharmaceutical composition comprises multiple CTL epitopes (cocktail), even if any of the epitopes is mutated, CTLs recognizing other epitopes are effective. Mixing epitopes derived from multiple proteins is an effective strategy for avoiding a reduction in therapeutic effect due to viral immune escape.

[0117] The pharmaceutical compositions of the present invention may also comprise an adjuvant known for effectively establishing cellular immunity. An adjuvant refers to a compound that enhances the immune response against an antigen that has immunological activity when administered together (or successively) with the antigen. Known adjuvants described in literatures, for example, Clin Microbiol Rev 1994, 7: 277-89, can be used. Examples of a suitable adjuvant include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide and such), alum, cholera toxin, Salmonella toxin, Incomplete Freund's adjuvant (IFA), Complete Freund's adjuvant (CFA), ISCOMatrix, GM CSF and other immunostimulatory cytokines, oligodeoxynucleotide containing the CpG motif (CpG7909 and such), oil-in-water emulsions, Saponin or its derivatives (QS21 and such), lipopolysaccharide such as Lipid A or its derivatives (MPL, RC529, GLA, E6020 and such), lipopeptides, lactoferrin, flagellin, double-stranded RNA or its derivatives (poli IC and such), bacterial DNA, imidazoquinolines (Imiquimod, R848 and such), C-type lectin ligand (trehalose 6,6'-dibehenate (TDB) and such), CDl d ligand (alpha-galactosylceramide and such), squalene emulsions (MF59, ASO3, AF03 and such), PLGA, and such, without being limited thereto. The adjuvant may be contained in another container separate from the pharmaceutical composition comprising a peptide of the present invention in the kits comprising the pharmaceutical composition of the present invention. In this case, the adjuvant and the pharmaceutical composition may be administered to a subject in succession, or mixed together immediately before administration to a subject. Such kits comprising a pharmaceutical composition comprising a peptide of the present invention and an adjuvant are also provided by the present invention. When the pharmaceutical composition of the present invention is a freeze dried formulation, the kit can further comprise a re-dissolving solution. Further, the present invention provides kits comprising a container that houses a pharmaceutical composition of the present invention and a container that stores an adjuvant. The kit can further comprise as needed a container that stores the re-dissolving solution.

[0118] When an oil adjuvant is used as an adjuvant, the pharmaceutical composition of the present invention may be prepared as an emulsion. Emulsions can be prepared, for example, by mixing and stirring the peptide solution prepared as described above and an oil adjuvant. The peptide solution may be one that has been re-dissolved after freeze-drying. The emulsion may be either of the W/O-type emulsion and O/W-type emulsion, and the W/O-type emulsion is preferred for obtaining a high immune response-enhancing effect. IFA can be preferably used as an oil adjuvant, without being limited thereto. Preparation of an emulsion can be carried out immediately before administration to a subject, and in this case, the pharmaceutical composition of the present invention may be provided as a kit comprising the peptide solution of the present invention and an oil adjuvant. When the pharmaceutical composition of the present invention is a freeze-dried formulation, the kit can further comprise a re-dissolving solution.

[0119] Further, the pharmaceutical composition of the present invention may be a liposome formulation within which a peptide of the present invention is encapsulated, a granular formulation in which a peptide is bound to beads with several micrometers in diameter, or a formulation in which a lipid is bound to a peptide.

[0120] In another embodiment of the present invention, the peptide of the present invention may also be administered in the form of a pharmaceutically acceptable salt. Preferred examples of salts include salts with alkali metals (lithium, potassium, sodium and such), salts with alkaline-earth metals (calcium, magnesium and such), salts with other metals (copper, iron, zinc, manganese and such), salts with organic bases, salts with amines, salts with organic acids (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and such), and salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid and such). Therefore, pharmaceutical compositions comprising a pharmaceutically acceptable salt of a peptide of the present invention are also encompassed by the present invention. Further, the "peptide of the present invention" also encompasses, in addition to the free peptide, pharmaceutically acceptable salts thereof.

[0121] In some embodiments, the pharmaceutical compositions of the present invention may further include a component which primes CTLs. Lipids have been identified as substances capable of priming CTLs in vivo against viral antigens. For example, palmitic acid residues can be attached to the epsilon- and alpha-amino groups of a lysine residue and then linked to a peptide of the present invention. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant. As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S glycerylcysteinyl-seryl-serine (P3CSS) can be used to prime CTLs when covalently attached to an appropriate peptide (see, e.g., Deres et al., Nature 1989, 342: 561-4).

[0122]

Examples of methods for administering the peptides or pharmaceutical compositions of the present invention include oral, epidermal, subcutaneous, intramuscular, intraosseous, peritoneal, and intravenous injections, as well as systemic administration or local administration to the vicinity of the targeted sites, but are not limited thereto. A preferred administration method includes subcutaneous injection to the vicinity of lymph nodes such as the armpit or groin. The administration can be performed by single administration or boosted by multiple administrations. The peptides of the present invention can be administered to a subject in a therapeutically or pharmaceutically effective amount for treating coronavirus infectious disease or in a therapeutically or pharmaceutically effective amount for inducing immunity (more specifically CTLs) against coronavirus-infected cells. The dose of the peptides of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age and weight, the method of administration and such. For each of the peptides of the present invention, the dose is usually 0.001 mg - 1000 mg, for example, 0.01 mg - 100 mg, for example, 0.1 mg - 30 mg, for example, 0.1 mg - 10 mg, for example, 0.5 mg - 5 mg. The dosing interval can be once every several days to several months, and for example, the dosing can be done in a once-per-week interval. A skilled artisan can appropriately select a suitable dosage.

[0123] In a preferred embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a peptide of the present invention and a pharmaceutically or physiologically acceptable carrier. In another embodiment, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a peptide of the present invention, a pharmaceutically or physiologically acceptable carrier, and an adjuvant. The pharmaceutical compositions of the present invention can comprise 0.001 mg - 1000 mg, preferably 0.01 mg - 100 mg, more preferably 0.1 mg - 30 mg, even more preferably 0.1 mg - 10 mg, for example, 0.5 mg - 5 mg of a peptide of the present invention. When a pharmaceutical composition of the present invention is an injection, it can comprise a peptide of the present invention at a concentration of 0.001 mg/ml - 1000 mg/ml, preferably 0.01 mg/ml - 100 mg/ml, more preferably 0.1 mg/ml - 30 mg/ml, even more preferably 0.1 mg/ml 10 mg/ml, for example, 0.5 mg/ml - 5 mg/ml. In this case, for example, 0.1 to 5 ml, preferably 0.5 ml to 2 ml of the pharmaceutical composition of the present invention can be administered to a subject by injection. On the other hand, when a pharmacological composition of the present invention comprises an adjuvant, the adjuvant can be contained in an amount that is effective in enhancing the immune response of a subject against the peptide.

[0124] Further, the present invention provides methods of any one of purposes selected from treatment of, prevention (prophylaxis) of and suppression of aggravation of coronavirus infectious disease, or multiple purposes, which comprise administering to a subject a therapeutically effective amount of a peptide of the present invention or a pharmaceutical composition of the present invention. As described above, the peptides of the present invention can be administered to a subject in a single dose of usually 0.001 mg - 1000 mg, for example, 0.01 mg - 100 mg, for example, 0.1 mg - 30 mg, for example, 0.1 mg - 10 mg, or for example, 0.5 mg - 5 mg. In a preferred embodiment, the peptides of the present invention are administered to a subject together with an adjuvant. Further, the dosing interval can be once every several days to several months, preferably once every several days to every month, for example, once every week or once every two weeks. On the other hand, when an adjuvant is administered with a peptide in the method of the present invention, the adjuvant can be administered in an amount that is effective in enhancing the immune response of a subject against the peptide.

[0125] (2) Pharmaceutical compositions containing polynucleotides as the active ingredient The pharmaceutical compositions of the present invention can also contain polynucleotides encoding the peptides of the present invention in an expressible form. Herein, the phrase "in an expressible form" means that the polynucleotide, when introduced into a cell, will be expressed as a peptide of the present invention. In an exemplified embodiment, the sequence of the polynucleotide of the present invention includes regulatory elements necessary for expression of the peptide of the present invention. The polynucleotide(s) of the present invention can be equipped with a sequence necessary to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465 8; US Patent Nos. 5,580,859, 5,589,466, 5,804,566, 5,739,118, 5,736,524, 5,679,647; and W098/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).

[0126] The peptides of the present invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. For example, as a vector to express the peptide of the present invention, vaccinia virus can be used. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351:

456-60. A wide variety of other vectors useful for therapeutic administration or immunization, e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent. See, e.g., 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.

[0127] Delivery of a polynucleotide of the present invention into a patient can be either direct, in which case the patient can be directly exposed to a vector harboring the polynucleotide of the present invention, or indirect, in which case, cells are first transformed with the vector harboring the polynucleotide of the present invention in vitro, then the cells are transplanted into the patient. These two approaches are known, respectively, as in vivo and ex vivo gene therapies.

[0128] For general reviews of the methods of gene therapy, 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 commonly known in the art of recombinant DNA technology which can also be used for 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. Administration may be performed by oral, intradermal, subcutaneous, or intravenous injection, and such. A systemic administration or a local administration to the vicinity of the targeted sites is used. The administration can be performed by single administration or boosted by multiple administrations. The polynucleotides of the present invention can be administered to a subject in a therapeutically or pharmaceutically effective dose for treating coronavirus infectious disease or in a therapeutically or pharmaceutically effective dose for inducing immunity (more specifically CTLs) against coronavirus-infected cells. The dose of a polynucleotide in a suitable carrier or the dose of a polynucleotide in cells transformed with a polynucleotide encoding a peptide of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age and weight, the method of administration and such, and this may be usually 0.001 mg - 1000 mg, for example, 0.01 mg - 100 mg, for example, 0.1 mg - 30 mg, for example, 0.1 mg - 10 mg, or for example, 0.5 mg - 5 mg. The dosing interval can be once every several days to several months, and for example, the dosing can be done in a once-per-week interval. A skilled artisan can appropriately select a suitable dosage.

[0129]

X. Methods of using peptides, exosomes, APCs and CTLs The peptides and polynucleotides of the present invention can be used to induce APCs and CTLs. CTLs can also be induced using the exosomes and APCs of the present invention. The peptides, polynucleotides, exosomes, and APCs can be used in combination with any other compound(s) as long as their CTL-inducing ability is not inhibited. Therefore, CTLs of the present invention can be induced using a pharmaceutical composition comprising any of the peptides, polynucleotides, APCs and exosomes of the present invention. Further, APCs of the present invention can be induced using a pharmaceutical composition comprising a peptide or polynucleotide of the present invention.

[0130] (1) Methods of inducing APCs The present invention provides methods of inducing APCs having CTL-inducing ability, using a peptide(s) or polynucleotide(s) of the present invention.

[0131] The methods of the present invention comprise a step of contacting an APC with a peptide of the present invention in vitro, ex vivo, or in vivo. For example, a method of contacting APCs with the peptide ex vivo may comprise the steps below: (a) collecting APCs from a subject; and (b) contacting the APCs of step (a) with a peptide of the present invention. The above-described APCs are not limited to a particular type of cell, and cells known to present a proteinaceous antigen on their cell surface to be recognized by lymphocytes, for example, DCs, Langerhans cells, macrophages, B cells, and activated T cells can be used. DCs have the most potent CTL-inducing ability among APCs, and thus it is preferable to use DCs. Any peptides of the present invention can be used by themselves or in combination with other peptides of the present invention. Further, peptides of the present invention can be used in combination with other CTL-inducing peptides (for example, other coronavirus protein-derived CTL-inducing peptides).

[0132] Meanwhile, when a peptide of the present invention is administered to a subject, APCs are contacted with the peptide in vivo, and as a result, APCs having a high CTL-inducing ability are induced in the body of the subject. Therefore, the methods of the present invention may comprise a step of administering a peptide of the present invention to a subject. Similarly, when a polynucleotide of the present invention is administered to a subject in an expressible form, a peptide of the present invention is expressed in vivo, the expressed peptide is contacted with APCs in vivo, and as a result APCs having a high CTL-inducing ability are induced in the body of the subject. Therefore, the present invention may also comprise a step of administering a polynucleotide of the present invention to a subject.

[0133] In order to induce APCs having CTL-inducing ability, the present invention may comprise a step of introducing a polynucleotide of the present invention into APCs. For example, the method may comprise the steps below: (a) collecting APCs from a subject; and (b) introducing a polynucleotide encoding a peptide of the present invention into the APCs of step (a). Step (b) can be performed as described in the above "VI. Antigen-presenting cells (APCs)" section.

[0134] Thus, in one embodiment, the present invention provides a method of inducing APCs having CTL-inducing ability, which comprises the step (a) or (b) below: (a) contacting APCs with a peptide of the present invention; or (b) introducing a polynucleotide encoding a peptide of the present invention into APCs.

[0135] Furthermore, the present invention provides a method of preparing APCs having CTL inducing ability, which comprises the step (a) or (b) below: (a) contacting APCs with a peptide of the present invention; or (b) introducing a polynucleotide encoding a peptide of the present invention into APCs.

[0136] The above-described methods can be performed in vitro, ex vivo, or in vivo, and it is preferable to perform them in vitro or ex vivo. APCs used in the above-described methods may be derived from a subject scheduled for administration of the induced APCs, or they may be derived from a different subject. When APCs derived from a subject (donor) different from the subject scheduled for administration are used, the subject of administration and the donor must have the identical HLA type. In the methods of the present invention, when a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof is used as a peptide of the present invention, the HLA type is preferably HLA-A24 (more preferably HLA-A*24:02) in both the subject of administration and the donor. Alternatively, APCs used in the above-described methods are preferably APCs that express HLA-A24 (more preferably HLA-A*24:02). When a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof is used as a peptide of the present invention, the HLA type is preferably HLA-A02 (more preferably HLA-A*02:01) in both the subject of administration and the donor. Alternatively, APCs used in the above-described methods are preferably APCs that express HLA-A02 (more preferably HLA-A*02:01). The APCs can be prepared using known methods from PBMCs after PBMCs are separated from blood collected from a donor by a specific gravity centrifugal method or such.

[0137] In another embodiment, the present invention also provides pharmaceutical compositions that comprise a peptide of the present invention or a polynucleotide encoding the peptide for inducing an APC(s) having CTL-inducing ability.

[0138] Alternatively, the present invention further provides use of a peptide of the present invention or a polynucleotide encoding the peptide in the manufacture of a pharmaceutical composition for inducing an APC(s) having CTL-inducing ability.

[0139] Alternatively, the present invention further provides peptides of the present invention or polynucleotides encoding the peptides for use in the induction of an APC(s) having CTL inducing ability.

[0140] Alternatively, the present invention further provides methods or processes of manufacturing a pharmaceutical composition for inducing an APC(s), wherein the method or process comprises a step of formulating a peptide of the present invention or a polynucleotide encoding the peptide with a pharmaceutically or physiologically acceptable carrier.

[0141] In another embodiment, the present invention further provides methods or processes of manufacturing a pharmaceutical composition for inducing an APC(s) having CTL-inducing ability, wherein the method or process comprises a step of mixing a peptide of the present invention or a polynucleotide encoding the peptide with a pharmaceutically or physiologically acceptable carrier. APCs induced by the methods of the present invention can induce CTLs specific to coronavirus protein (i.e., CTLs of the present invention).

[0142] (2) Methods of inducing CTLs The present invention also provides methods of inducing CTLs using peptides, polynucleotides, exosomes or APCs of the present invention.

[0143]

When a peptide(s), a polynucleotide(s), an exosome(s) or an APC(s) of the present invention is administered to a subject, CTLs are induced in the body of the subject and the strength of the immune response targeting coronavirus-infected cells is enhanced. Therefore, the methods of the present invention may comprise a step of administering a peptide(s), a polynucleotide(s), an APC(s) or an exosome(s) of the present invention to a subject.

[0144] Alternatively, CTLs can be induced by using them in vitro or ex vivo. For example, the methods of the present invention may include the following steps: (a) collecting APCs from a subject; (b) contacting the APCs of step (a) with a peptide of the present invention; and (c) co-culturing the APCs of step (b) with CD8-positive T cells. The induced CTLs may be returned to the subject afterwards.

[0145] The APCs to be co-cultured with the CD8-positive T cells in step (c) above can also be prepared by introducing into APCs a polynucleotide encoding a peptide of the present invention as described above in the "VI. Antigen-presenting cells (APCs)" section. However, the APCs to be used in the methods of the present invention are not limited thereto, and any APCs that present on their surface a complex of an HLA antigen and a peptide of the present invention can be used.

[0146] In the methods of the present invention, instead of such APCs, exosomes that present on their surface a complex of an HLA antigen and a peptide of the present invention can also be used. That is, the methods of the present invention can comprise a step of co-culturing with exosomes that present on their surface a complex of an HLA antigen and a peptide of the present invention. Such exosomes can be prepared by the above-described methods in the "V. Exosomes" section.

[0147] Further, CTLs can also be induced by introducing into a CD8-positive T cell a vector comprising a polynucleotide encoding each subunit of a TCR capable of binding to a peptide of the present invention presented by an HLA antigen on the cell surface. Such transformation can be carried out as described above in the "VIII. T cell receptors (TCRs)" section.

[0148] Accordingly, in one embodiment, the present invention provides methods of inducing CTLs, comprising a step selected from below: (a) co-culturing CD8-positive T cells with APCs that present on their surface a complex of an HLA antigen and a peptide of present invention;

(b) co-culturing CD8-positive T cells with exosomes that present on their surface a complex of an HLA antigen and a peptide of present invention; and (c) introducing into CD8-positive T cells, a vector comprising a polynucleotide encoding each subunit of a TCR capable of binding to a peptide of the present invention presented by an HLA antigen on a cell surface.

[0149] The above-described methods can be performed in vitro, ex vivo, or in vivo, and it is preferable to perform them in vitro or ex vivo. APCs or exosomes and CD8-positive T cells used in the above-described methods may be derived from a subject scheduled for administration of the induced CTLs, or they may be derived from a different subject. When APCs or exosomes and CD8-positive T cells derived from a subject (donor) different from the subject scheduled for administration are used, the subject of administration and the donor must have the identical HLA type. For example, when a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof is used as peptides of the present invention, the HLA type in both the subject of administration and the donor is preferably HLA-A24 (more preferably HLA-A*24:02). Alternatively, APCs or exosomes used in the above-described methods are preferably APCs or exosomes that present on their surface a complex of HLA-A24 (more preferably HLA-A*24:02) and a peptide of the present invention (a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15 or a modified peptide thereof). In this case, the induced CTLs show a specific cytotoxic activity against cells that present a complex of HLA-A24 and a peptide of the present invention (for example, coronavirus-infected HLA-A24-positive cells). When a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof is used as peptides of the present invention, the HLA type in both the subject of administration and the donor is preferably HLA-A02 (more preferably HLA-A*02:01). Alternatively, APCs or exosomes used in the above-described methods are preferably APCs or exosomes that present on their surface a complex of HLA-A02 (more preferably HLA-A*02:01) and a peptide of the present invention (a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13 or a modified peptide thereof). In this case, the induced CTLs show a specific cytotoxic activity against cells that present a complex of HLA-A02 and a peptide of the present invention (for example, coronavirus-infected HLA-A02-positive cells).

[0150] In another embodiment, the present invention also provides compositions or pharmaceutical compositions for inducing CTLs, comprising at least one active ingredient selected from below:

(a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; and (d) an exosome that presents on its surface a peptide of the present invention.

[0151] In another embodiment, the present invention also provides use of an active ingredient selected from below in the manufacture of compositions or pharmaceutical compositions for inducing CTLs: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; and (d) an exosome that presents on its surface a peptide of the present invention.

[0152] Alternatively, the present invention further provides an active ingredient selected from below for use in inducing CTLs: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; and (d) an exosome that presents on its surface a peptide of the present invention.

[0153] Alternatively, the present invention further provides a method or process for manufacturing a composition or pharmaceutical composition for inducing CTLs, which is a method or process that comprises a step of formulating an active ingredient selected from below with a pharmaceutically or physiologically acceptable carrier: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; and (d) an exosome that presents on its surface a peptide of the present invention.

[0154] In another embodiment, the present invention further provides a method or process for manufacturing a composition or pharmaceutical composition for inducing CTLs, which is a method or process that comprises a step of mixing an active ingredient selected from below with a pharmaceutically or physiologically acceptable carrier: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; and

(d) an exosome that presents on its surface a peptide of the present invention.

[0155] XI. Methods of inducing an immune response The present invention further provides methods of inducing an immune response against coronavirus infection. Coronaviruses include SARS-CoV-2, MERS-CoV, SARS-CoV and such, but are not limited thereto. It is preferable that the coronavirus-infected cells express HLA-A24 or HLA-A02.

[0156] The present invention also provides methods of 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 are amino acid sequences commonly found also in SARS-CoV proteins and MERS-CoV proteins. Thus, when an immune response against coronavirus-infected cells is induced, proliferation of viruses in the coronavirus-infected cells is inhibited as a result. Accordingly, the present invention further provides methods of inhibiting proliferation of viruses in coronavirus-infected cells. The methods of the present invention are suitable, in particular, for inhibiting proliferation of coronaviruses in coronavirus-infected cells expressing HLA-A24 or HLA-A02.

[0157] The methods of the present invention may comprise a step of administering a composition comprising any of the peptides of the present invention or a polynucleotide(s) encoding the peptide(s). The methods of the present invention also contemplate administration of APCs or exosomes presenting any of the peptides of the present invention. The details can be referred to the "IX. Pharmaceutical compositions" section, particularly portions describing regarding 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 for inducing an immune response are described in detail in "V. Exosomes", "VI. Antigen-presenting cells (APCs)" and in Items (1) and (2) of "X. Methods of using peptides, exosomes, APCs and CTLs" described above.

[0158] In another embodiment, the present invention provides pharmaceutical compositions or vaccines for inducing an immune response against coronavirus infection, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from below: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention;

(d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0159] Alternatively, the present invention also provides pharmaceutical compositions or vaccines for inducing an immune response against coronavirus-infected cells, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from below: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; (d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0160] Alternatively, the present invention further provides pharmaceutical compositions or vaccines for inhibiting proliferation of coronaviruses in coronavirus-infected cells, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from below: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; (d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0161] In another embodiment, the present invention provides use of an active ingredient selected from below in the manufacture of pharmaceutical compositions or vaccines for inducing an immune response against coronavirus infection: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; (d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0162] Alternatively, the present invention also provides use of an active ingredient selected from below in the manufacture of pharmaceutical compositions or vaccines for inducing an immune response against coronavirus-infected cells: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention;

(d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0163] Alternatively, the present invention further provides use of an active ingredient selected from below in the manufacture of pharmaceutical compositions or vaccines for inhibiting proliferation of coronaviruses in coronavirus-infected cells: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents on its surface a peptide of the present invention; (d) an exosome that presents on its surface a peptide of the present invention; and (e) a CTL of the present invention.

[0164] The present invention further provides methods or processes for manufacturing pharmaceutical compositions that induce an immune response against coronavirus infection, which is a method that may comprise a step of mixing or formulating a peptide of the present invention with a pharmaceutically acceptable carrier.

[0165] Alternatively, the present invention provides methods for inhibiting proliferation of coronaviruses in coronavirus-infected cells or methods of inducing an immune response against coronavirus infection, which comprises a step of administering to a subject vaccines or pharmaceutical compositions comprising an active ingredient selected from below: (a) a peptide of the present invention; (b) a polynucleotide encoding a peptide of the present invention in an expressible form; (c) an APC that presents a peptide of the present invention on its surface; (d) an exosome that presents a peptide of the present invention on its surface; and (e) a CTL of the present invention.

[0166] In the context of the present invention, coronavirus infectious diseases can be treated by administering a peptide, a polynucleotide, an APC, an exosome and/or a CTL of the present invention. Alternatively, an immune response against coronavirus infection can be induced by administering a peptide, a polynucleotide, an APC, an exosome and/or a CTL of the present invention. Examples of such coronaviruses include SARS-CoV-2, MERS-CoV, SARS-CoV and such, but are not limited thereto. Further, an immune response against coronavirus-infected cells can be induced by administering a peptide, a polynucleotide, an APC, an exosome and/or a CTL of the present invention. Therefore, it is preferable to confirm whether the subject to be treated is infected with coronavirus or not before administering a vaccine or pharmaceutical composition comprising an active ingredient described above.

[0167] Thus, in one embodiment, the present invention provides a method of treating a coronavirus infectious disease in a patient in need of the infectious disease treatment, wherein the method comprises the steps below: (i) measuring an expression level of a SARS-CoV-2 gene or a protein encoded thereby in a biological sample collected from a subject infected with coronavirus; (ii) identifying a subject infected with coronavirus based on the expression level of the SARS-CoV-2 gene or the protein encoded thereby measured in (i); and (iii) administering to the subject infected with coronavirus at least one ingredient selected from the group consisting of (a) to (e) above.

[0168] Alternatively, the present invention further provides vaccines and pharmaceutical compositions 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 of identifying or selecting a subject to be treated with at least one active ingredient selected from the group consisting of (a) to (e) above, wherein the method comprises the steps below: (i) measuring an expression level of a SARS-CoV-2 gene or a protein encoded thereby in a biological sample collected from a subject infected with coronavirus; (ii) identifying a subject with coronavirus-infected cells expressing the SARS-CoV-2 gene or the protein encoded thereby based on the expression level of the SARS-CoV-2 gene or the protein encoded thereby measured in (i); and (iii) identifying or selecting the subject identified in (ii) as a subject who may be treated with at least one active ingredient selected from the group consisting of (a) to (e) above.

[0169] Biological samples collected from a subject for measuring the expression level of a SARS-CoV-2 gene or a protein encoded thereby in the above-described methods are not particularly limited, and for example, tissue samples containing coronavirus-infected cells collected by biopsy or such can be preferably used. Alternatively, detecting coronavirus RNAs in throat swab or saliva is generally performed in identifying coronavirus-infected subject. Thus, in the present invention, a coronavirus gene includes genome RNA of coronavirus, or mRNAs transcribed therefrom. The expression level of a SARS-CoV-2 gene or a protein encoded thereby in a biological sample can be measured by known methods, and for example, methods that detect transcription products of the SARS-CoV-2 gene by probes or PCR methods (for example, cDNA microarray method, Northern blot method, RT-PCR method or such), methods that detect translation products of the SARS-CoV-2 gene by antibodies or such (for example, Western blot method, immunostaining method, immunochromatography method or such), and such can be used. Further, biological samples may be blood samples, and in this case, the blood level of an antibody against SARS-CoV-2 protein is measured, and the expression level of SARS-CoV-2 protein may be assessed based on the blood level. The blood level of an antibody against SARS-CoV-2 protein can be measured using known methods, and for example, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and such using the SARS-CoV-2 protein or a peptide of the present invention as an antigen can be used. Alternatively, the expression level of a SARS-CoV-2 protein in a subject may be assessed by detecting CTLs specific to a peptide of the present invention. The level of CTLs specific to a peptide of the present invention can be measured, for example, by isolating PBMCs from the blood collected from a subject and measuring their 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 y released. Complexes of the peptide of the present invention and HLA, mentioned below, can also be used to measure CTL levels. The determination of whether the coronavirus-infected cells that the subject has express a SARS CoV-2 protein may be made by comparison with the measurement results in the same type of biomaterial collected from a subject not infected with the coronavirus. That is, when the level of a measuring object in a biological sample collected from a coronavirus-infected subject is elevated compared to that in the same type of biomaterial collected from a subject not infected with the coronavirus (normal control level), the cells of the coronavirus-infected subject can be judged to express the SARS-CoV-2 protein.

[0170] In a preferred embodiment, it is preferable to confirm the HLA type of the subject before administering at least one active ingredient selected from the group consisting of (a) to (e) above. For example, for the subjects to be administered with an active ingredient in association with a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15, it is preferable to select HLA-A24-positive subjects. For the subjects to be administered with an active ingredient in association with a peptide having the amino acid sequence selected from among SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13, it is preferable to select HLA-A02-positive subjects.

[0171] The present invention further provides complexes of a peptide of the present invention and HLA. The complexes of the present invention described above may be monomers or multimers. When a complex of the present invention is a multimer, the number of polymerization is not particularly limited, and it can be a multimer of any number of polymerization. Examples include a tetramer, pentamer, hexamer and such, but are not limited thereto. The multimers of the present invention also encompass dextramers (W2002/072631) and streptamers (Knabel M et al., Nat Med. 2002 Jun; 8(6): 631-7.). Complexes of a peptide of the present invention and HLA can be prepared according to known methods (for example, Altman JD et al., Science. 1996, 274(5284): 94-6; W02002/072631; W02009/003492; Knabel M et al., Nat Med. 2002 Jun; 8(6): 631-7; and such). The complexes of the present invention, for example, can be used in the quantification of CTLs specific to a peptide of the present invention. For example, a blood sample is collected from a subject administered with a pharmaceutical composition of the present invention, and CD4-negative cells are prepared after separation of PBMCs and contacted with a fluorescent dye-conjugated complex of the present invention. Then, the percentage of CTLs specific to a peptide of the present invention can be measured by flow cytometry analysis. For example, immune response-inducing effects by a pharmaceutical composition of the present invention can be monitored by measuring CTLs specific to a peptide of the present invention before, during and/or after administration of the pharmaceutical composition of the present invention.

[0172] XII. Antibodies The present invention further provides antibodies that bind to the peptide of the present invention. Preferable antibodies bind specifically to a peptide of the present invention, but do not bind (or weakly bind) to one that is not the peptide of the present invention. The binding specificity of an antibody can be confirmed by inhibition assay. That is, if the binding between an antibody to be analyzed and a polypeptide (SEQ ID NOs: 17-26) consisting of an amino acid sequence of each protein encoded by a full-length genome sequence of SARS-CoV-2 is inhibited in the presence of a peptide of the present invention, this antibody is shown to specifically bind to the peptide of the present invention. Antibodies against peptides of the present invention can be used in assays of disease diagnosis and prognosis, as well as subject selection for administration of the pharmaceutical compositions of the present invention and monitoring of the pharmaceutical compositions of the present invention.

[0173] The present invention also provides various immunological assays for detecting and/or quantifying peptides of the present invention or fragments thereof. Such immunological assays include radioimmunoassay, immunochromatography, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA) and such, without being limited thereto, and are performed within the scope of the various immunological assay formats well known in the art.

[0174] The antibodies of the present invention can be used in immunological imaging methods that can detect coronavirus-infected cells, and examples thereof include radioactive scintigraphic imaging using a labelled antibody of the present invention, without being limited thereto. Such assay methods are used clinically in the detection, monitoring, and prognosis of coronavirus infected cells; and examples of such coronaviruses of coronavirus infectious diseases include SARS-CoV-2, MERS-CoV, SARS-CoV and such, without being limited thereto.

[0175] The antibodies of the present invention can be used in any arbitrary form such as monoclonal antibodies or polyclonal antibodies, and may further include anti-sera obtained by immunizing an animal such as a rabbit with a peptide of the present invention, all classes of polyclonal antibodies and monoclonal antibodies, human antibodies, as well as chimeric antibodies and humanized antibodies generated through gene recombination.

[0176] The peptide of the present invention or a fragment thereof used as an antigen for obtaining antibodies can be obtained by chemical synthesis or genetic engineering techniques based on the amino acid sequences disclosed herein.

[0177] The peptide used as an immunizing antigen may be a peptide of the present invention or a fragment of a peptide of the present invention. Further, the peptide may be bound to or conjugated with a carrier for increasing immunogenicity. Keyhole limpet hemocyanin (KLH) is well-known as a carrier. Methods for binding KLH to a peptide are also well known in the art.

[0178] Any mammal can be immunized with an antigen described above, and it is preferable to consider the compatibility with the parent cell used in cell fusion when generating a monoclonal antibody. 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, Catarrhini monkeys (old world monkeys) such as cynomolgus monkey (Macaca fascicularis),rhesus monkeys, hamadryas, and chimpanzee.

[0179] Methods of immunizing animals with an antigen are known in the art. Intraperitoneal injection and subcutaneous injection of an antigen are standard methods for immunizing mammals. More specifically, an antigen is diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, or such. As needed, an antigen suspension solution can be administered to mammals after being mixed with an appropriate amount of a standard adjuvant such as Freund's complete adjuvant and emulsified. Then, it is preferable to administer the antigen mixed with an appropriate amount of a Freund's incomplete adjuvant several times every 4 to 21 days. A suitable carrier may be used for immunization. After the above immunization, the serum can be examined by standard method with respect to increase in the quantity of the desired antibody.

[0180] Polyclonal antibodies against a peptide of the present invention can be prepared by collecting blood from mammals that have been confirmed with an increase in the serum level of the desired antibody after immunization, and separating the serum from blood by any conventional method. A polyclonal antibody may be a polyclonal antibody-containing serum, or a polyclonal antibody-containing fraction may be isolated from the serum. Immunoglobulin G or M can be prepared from fractions that recognize only a peptide of the present invention by, for example, using an affinity column conjugated with the peptide of the present invention, and then further purifying the fractions using a protein A or protein G column.

[0181] In order to prepare monoclonal antibodies, upon confirming an increase in the serum level of the desired antibody after immunization, immune cells are collected from the mammals and subjected to cell fusion. Immune cells used for cell fusion may be preferably obtained from the spleen. For the other parent cells fused with the above immune cells, for example, a mammalian myeloma cell, and more preferably a myeloma cell that has acquired a property for drug selection of fusion cells can be used.

[0182] The above immune cells can be fused with myeloma cells by known methods, for example, the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

[0183] Hybridomas obtained by cell fusion can be selected by culturing them in a standard selection medium such as the HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). Cell culturing is typically continued in the HAT medium for a sufficient period of time (for example, several days to several weeks) to allow death of all other cells (non-fused cells) besides the desired hybridomas. Then, hybridoma cells producing the desired antibody can be screened and cloned by performing a standard limiting dilution.

[0184] In addition to the above methods of immunizing a non-human animal with an antigen for hybridoma preparation, human lymphocytes such as EB virus-infected lymphocytes can be immunized in vitro with a peptide, cells expressing the peptide, or lysates thereof. Then, the immunized lymphocytes can be fused with immortalized human-derived myeloma cells such as U266 to obtain hybridomas producing a desired human antibody capable of binding to the peptide (JPS63-17688).

[0185] Next, the obtained hybridoma is transplanted into the abdominal cavity of a mouse, and the ascites is extracted. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion-exchange chromatography, or affinity column conjugated with the peptide of the present invention.

[0186] Alternatively, antibody-producing immune cells such as the immunized lymphocytes can be immortalized by a cancer gene and used for the preparation of monoclonal antibodies.

[0187] The monoclonal antibodies obtained as such can also be prepared by recombination using genetic engineering techniques (see, e.g., Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies published in United Kingdom by MacMillan Publishers LTD (1990)). For example, an antibody-encoding DNA can be cloned from immune cells such as antibody-producing hybridoma or immunized lymphocytes and inserted into a suitable vector, and then this is introduced into host cells to prepare a recombinant antibody. The present invention also provides recombinant antibodies prepared as described above.

[0188] Further, the antibodies of the present invention may be antibody fragments or modified antibodies, as long as they bind to the peptides of the present invention. For example, the antibody fragments may be Fab, F(ab')2, Fv, or a single chain Fv (scFv) in which Fv fragments derived from an H chain and an L chain are linked with a suitable linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, antibody fragments may be generated by treating an antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, e.g., 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)).

[0189] Antibodies may be modified by conjugation with various molecules such as polyethyleneglycol (PEG). The present invention provides such modified antibodies. Modified antibodies can be obtained by chemically modifying the antibodies. These modification methods are conventional in the art.

[0190] Alternatively, the antibodies of the present invention can be obtained as chimeric antibodies of a non-human antibody-derived variable region and a human antibody-derived constant region, or as humanized antibodies comprising a non-human antibody-derived complementarity determining region (CDR) and a human antibody-derived framework region (FR) and constant region. Such antibodies can be prepared according to known techniques. Humanization can be carried out by substituting a human antibody sequence(s) with a corresponding non-human antibody CDR sequence(s) (see, e.g., Verhoeyen et al., Science 239: 1534-6 (1988)). Thus, such humanized antibodies are chimeric antibodies in which the substantially less than an intact human variable domain has been substituted with a corresponding sequence from a non-human species.

[0191] Intact human antibodies comprising a human variable region in addition to the human framework and constant regions can also be used. Such antibodies can be generated using various techniques known in the art. For example, in vitro methods include use of recombinant libraries of human antibody fragments presented on bacteriophages (for example, Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, human antibodies can also be generated by introducing human immunoglobulin gene loci into transgenic animals, for example, mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in, for example, US Patent Nos. 6,150,584, 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.

[0192] Antibodies obtained as described above may be purified to homogeneity. For example, antibody separation and purification can be performed according to separation methods and purification methods used for general proteins. For example, an antibody can be separated and isolated by appropriately selecting and combining use of column chromatographies such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto. Protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).

[0193] Besides affinity chromatography, exemplary chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography and such (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatography procedures can be carried out by liquid phase chromatography such as HPLC and FPLC.

[0194] The antigen-binding activity of an antibody of the present invention can be measured, for example, by using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence (IF). In the case of ELISA, an antibody of the present invention is immobilized onto a plate, a peptide of the present invention is applied to the plate, and then a sample containing the desired antibody, such as culture supernatant of antibody-producing cells or purified antibodies, is applied. Next, a secondary antibody that recognizes the primary antibody and is labelled with an enzyme such as alkaline phosphatase is applied and the plate is incubated. Then, after washing, an enzyme substrate such as p-nitrophenyl phosphate is applied to the plate, and the antigen-binding activity of the sample is evaluated by measuring absorbance. To assess the binding activity of an antibody, peptide fragments such as C-terminal or N-terminal fragments may be used as an antigen. BlAcore (Pharmacia) may be used to evaluate the activity of an antibody of the present invention.

[0195] It is possible to detect or measure a peptide of the present invention using the above methods, by exposing an antibody of the present invention to a sample assumed to contain the peptide of the present invention, and detecting or measuring an immune complex formed between the antibody and the peptide. For example, an antibody of the present invention can be used to detect a peptide of the present invention present in the blood sample (for example, serum sample) of a subject. Alternatively, an antibody of the present invention present in the blood sample (for example, serum sample) of a subject can also be detected using a peptide of the present invention. The result of measuring a peptide of the present invention or an antibody of the present invention in the blood sample of a subject can be utilized to the subject selection for administration of the pharmaceutical compositions of the present invention or monitoring of the efficacy of the pharmaceutical compositions of the present invention.

[0196] XIII. Vectors and host cells The present invention provides vectors comprising a polynucleotide encoding a peptide of the present invention and host cells introduced with the vectors. A vector of the present invention may be used to keep a polynucleotide of the present invention in a host cell, to express a peptide of the present invention in a host cell, or to administer a polynucleotide of the present invention for gene therapy.

[0197] When E. coli is a host cell and a vector is amplified and produced in a large amount in E. coli (for example, JM109, DH5-alpha, HB101 or XL1-Blue), the vector needs to have a "replication origin" for amplification in E. coli and a marker gene for selection of transformed E. coli (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol). For example, the M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script and such can be used. In addition, pGEM-T, pDIRECT and pT7 can be used for cloning as well as the above vectors. When a vector is used in the production of a peptide of the present invention, an expression vector can be used. For example, an expression vector for expression in E. coli needs to have the above features for amplification in E. coli. When E. coli such as JM109, DH5-alpha, HB101 or XL1-Blue are used as a host cell, the vector needs to have a promoter, for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1989)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like, that can efficiently express the desired gene in E. coli. In that respect, pGEX-5X-1 (Pharmacia), "QlAexpress system" (Qiagen), pEGFP and pET (in this case, the host is preferably BL21 which expresses T7 RNA polymerase), for example, can be used instead of the above vectors. Additionally, the vector may contain a signal sequence for peptide secretion. An exemplary signal sequence that directs the peptide to be secreted to the periplasm of the E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing the vectors into the target host cells include, for example, the calcium chloride method and the electroporation method.

[0198] In addition to E. coli, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac-to-BAC baculovirus expression system" (GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZlpneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-QO1) and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50) can be used for producing the polypeptide of the present invention.

[0199]

In order to express the vector in animal cells such as CHO, COS or NIH3T3 cells, the vector needs to carry a promoter necessary for expression in such cells, for example, the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), the MMLV-LTR promoter, the EF1-alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), the CMV promoter and the like, and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by a drug (e.g., neomycin, G418)). Examples of known vectors with these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

[0200] The embodiments of the present invention are exemplified below based on the above explanation; however, the present invention is not limited to these embodiments.

[1] A peptide of less than 15 amino acids having cytotoxic T cell (CTL)-inducing ability, which comprises the amino acid sequence selected from the group of: (a) the 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;and (b) the amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to the 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] The peptide of [1], which has either or both of features below to the amino acid sequence selected from the group consisting of 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15: (a) the second amino acid from the N terminus is substituted with an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine and tryptophan; and (b) the C-terminal amino acid is substituted with an amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, tryptophan and methionine.

[3] The peptide of [1], which has either or both of features below to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13: (a) the second amino acid from the N terminus is substituted with an amino acid selected from the group consisting of leucine and methionine; and (b) the C-terminal amino acid is substituted with an amino acid selected from the group consisting of valine and leucine.

[4] The peptide of [1], which consists of the 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.

[5] A polynucleotide, which encodes the peptide of 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) below:

(a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[7] The composition of [5], which is a composition for inducing a CTL(s), wherein the active ingredient is at least one ingredient selected from the group consisting of (a) to (d) below: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; and (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen.

[8] The composition of [6], which is a pharmaceutical composition.

[9] The composition of [8], which is a pharmaceutical composition for one or more uses selected from the group consisting of (i) treatment of coronavirus infectious disease, (ii) prevention (prophylaxis) of coronavirus infectious disease and (iii) suppression of aggravation of coronavirus infectious disease.

[10] The composition of [8], which is for inducing an immune response against coronavirus infection.

[11] The composition of [9] or [10], wherein coronavirus of the coronavirus infectious disease is selected from the group consisting of SARS-CoV-2, MERS-CoV and SARS-CoV.

[12] The composition of any one of [6] to [11], which is formulated for administration to a subject positive for HLA-A24 or HLA-A02.

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

[14] A method of inducing a CTL(s), which comprises a step selected from the group consisting of (a) to (c) below:

(a) co-culturing a CD8-positive T cell(s) with an APC(s) that presents on its surface a complex of an HLA antigen and the peptide of any one of [1] to [4]; (b) co-culturing a CD8-positive T cell(s) with an exosome(s) that presents on its surface a complex of an HLA antigen and the peptide of any one of [1] to [4]; and (c) introducing into a CD8-positive T cell(s) a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of any one of [1] to [4] presented by an HLA antigen on a cell surface.

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

[16] The APC of [15], which is induced by the method of [13].

[17] A CTL that targets the peptide of any one of [1] to [4].

[18] The CTL of [17], which is induced by the method of [14].

[19] A method of inducing an immune response against coronavirus infection, which comprises administering to a subject a composition comprising at least one ingredient selected from the group consisting of (a) to (e) below: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[20] A method of any one purpose selected from treatment of, prevention of, and suppression of aggravation of coronavirus infectious disease, or multiple purposes thereof, which comprises administering to a subject at least one ingredient selected from the group consisting of (a) to (e) below: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[21] An antibody that binds to the peptide of any one of [1] to [4].

[22] A method of screening for a peptide having CTL-inducing ability, which comprises the steps of: (a) generating candidate sequences consisting of an amino acid sequence in which one, two or several amino acid residues are substituted, deleted, inserted and/or added to an original amino acid sequence consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15; (b) selecting from among the candidate sequences generated in (a), a candidate sequence that does not have significant homology (sequence identity) with any known human gene product; (c) contacting an APC(s) with a peptide consisting of the candidate sequence selected in (b); (d) contacting the APC(s) of (c) with a CD8-positive T cell(s); and (e) selecting a peptide having an equal to or higher CTL-inducing ability than that of a peptide consisting of the original amino acid sequence.

[23] Use of at least one ingredient selected from the group consisting of (a) to (e) below in the manufacture of a composition for inducing an immune response against coronavirus infection: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [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 one purpose selected from treatment of, prevention of, and suppression of aggravation of coronavirus infectious disease, or multiple purposes thereof: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[25] Use of at least one active ingredient selected from the group consisting of (a) to (e) below for inducing an immune response against coronavirus infection: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[26] Use of at least one active ingredient selected from the group consisting of (a) to (e) below for any one purpose selected from treatment of, prevention of, and suppression of aggravation of coronavirus infectious disease, or multiple purposes thereof: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[27] A method of inducing cytotoxic activity against a coronavirus-infected cell(s), which comprises a step of administering to a subject at least one active ingredient selected from the group consisting of (a) to (e) below: (a) one or more types of peptides of any one of [1] to [4]; (b) one or more types of polynucleotides encoding the peptide(s) of any one of [1] to [4] in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of [1] to [4] and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of [1] to

[4] and an HLA antigen; and (e) a CTL that targets the peptide of any one of [1] to [4].

[28] A freeze-dried formulation comprising one or more types of peptides of any one of [1] to

[4].

[29] A pharmaceutical composition, which is prepared by a method that comprises dissolving one or more types of peptides of any one of [1] to [4] in a water-soluble carrier, and performing filtration sterilization.

[30] A filtration-sterilized aqueous solution, which is an aqueous solution that comprises one or more types of peptides of any one of [1] to [4] and a water-soluble carrier.

[31] An emulsion comprising one or more types of peptides of any one of [1] to [4], a water soluble carrier and an oil adjuvant.

[32] A kit comprising a container that houses the pharmaceutical composition of any one of [8] to [12] and a container that houses an adjuvant.

[33] A kit comprising a container that stores a freeze-dried formulation comprising the peptide of any one of [1] to [4], a container that stores an adjuvant, and a container that stores a re dissolving solution for the freeze-dried formulation.

[34] A kit comprising a container that houses the composition of any one of [6] to [12] and a container that houses an adjuvant.

[0201] The present invention is explained herein in detail with reference to its specific embodiments. However, it should be understood that the above explanation is in fact an illustrative and explanatory explanation, and is intended to explain the present invention and preferred embodiments thereof. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the present invention. Thus, the present invention is not confined to the above explanation, but is intended to be defined by the appended claims and equivalents thereto.

[0202] Hereinbelow, the present invention is described in more detail with reference to the Examples. Nevertheless, while the following materials, method and Examples may serve to assist one of ordinary skill in making and using certain embodiments of the present invention, there are only intended to illustrate aspects of the present invention and thus in no way to limit the scope of the present invention. One of ordinary skill in the art can use methods and materials similar or equivalent to those described herein in the practice or testing of the present invention.

[Examples]

[0203] Materials and Methods Cell lines

TISI cells (HLA-A*24:02/-), human lymphoblastoid cell line, were purchased from the International Histocompatibility Working Group. T2 cells (HLA-A*02:01/-), human lymphoblast cell line, were purchased from ATCC.

[0204] Selection of peptides derived from SARS-CoV-2 SARS-CoV-2 protein-derived 9mer and 1Omer peptides that would be expected to bind to HLA-A*24:02 were determined using "NetMHC4.0" binding prediction server (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). Of these, peptides commonly found in SARS-CoV Tor2 (GenBank accession number AY274119), SARS-CoV BJ1 (GenBank accession number AY278488) and SARS-CoV GZ02 (GenBank accession number AY390556), and also in MERS-CoV (GenBank accession number JX869059) were selected as candidate epitopes (Kiyotani K et al., J Hum Genet 2020, 65(7): 569-575).

[0205] Peptide synthesis The peptides were synthesized by Cosmo Bio Co., Ltd. (Tokyo, Japan) according to solid synthesis method and purified by reversed-phase high-performance liquid chromatography (HPLC). The quality (purity: 90% or higher) of the peptides was guaranteed by HPLC and mass spectrometry analysis. Peptides were dissolved in dimethyl sulfoxide (final concentration: 20 mg/mL) and stored at -80°C.

[0206] In vitro CTL induction Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells to induce cytotoxic T cells (CTLs) specific to a peptide presented on a human leukocyte antigen (HLA). DCs were prepared in vitro as has already been reported in the literature (Nakahara S et al., Cancer Res 2003, 63(14): 4112-4118). Specifically, peripheral blood mononuclear cells (PBMCs) collected from healthy volunteers (HLA-A*24:02- or HLA-A*02:01-positive) were seeded onto a tissue culture dish (Coming) to allow adhesion of monocytes in PBMCs to the dish. The cells were cultured 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) for seven days. AIM-V medium (Invitrogen) containing inactivated Type AB serum (MP Biomedicals) (2% ABS/AIM-V medium) was used as a medium. DCs differentiated from monocytes by cytokine and autologous CD8-positive T cells obtained by using CD8-Positive Isolation Kit (Invitrogen) were mixed at 1:20 ratio (1.5 x 104 DC cells and 3 x 105 CD8-positive T cells) and cultured in a 48-well plate (Coming). Peptides were further added thereto (peptide final concentration: 20 [g/ml). Volume of 2%ABS/AIM-V medium per well was 0.5 ml, and IL-7 (R&D System) and IL-21 (Cell Genix) were added thereto (final concentrations: IL-7, 10 ng/ml; IL-21, 30 ng/ml). Three days after the start of culture, DCs and peptide were again added thereto (peptide final concentration: 20 [g/ml). DCs were prepared at time of use by the same way as described above. Seven days after the start of culture, IL-2 (Novartis), IL-7 and IL-15 (Novoprotein) were added thereto (final concentrations: IL-2, 48 IU/ml; IL7, 5 ng/ml; and IL-15, 5 ng/ml) (Wolfi M et al., Nat Protoc 2014, 9(4): 950-966). On day 9 (after the two DC stimulations) and thereafter, IFN-y production for TISI or T2 cells pulsed with the peptide was confirmed by an enzyme-linked immunospot (ELISPOT) assay.

[0207] CTL proliferation procedure CTLs were proliferated 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). In tissue culture flasks (FALCON), CTLs were cultured in 5% ABS/AIM-V medium (culture medium volume: 25 ml/flask) along with two human B lymphoblastoid cell lines (5 x 106 cells each) treated with mitomycin C and an anti-CD3 antibody (BD biosciences, final concentration: 40 ng/ml). On the day following the start of the culture, IL-2 was added to the culture (final IL-2 concentration: 120 IU/ml). On days 5, 8, and 11, the medium was replaced with 5% ABS/AIM-V medium containing 60 IU/ml IL-2 (final IL-2 concentration: 30 IU/ml) (Yoshimura S et al., PLoS One 2014, 9(1): e85267).

[0208] Confirmation of IFN-y production To confirm peptide-specific IFN-y production of CTLs induced using the peptide, IFN-y ELISPOT assay and IFN-y ELISA were performed. Peptide-pulsed TISI cells or T2 cells were prepared as target cells. IFN-y ELISPOT assay and IFN-y ELISA were performed according to the procedure recommended by the assay kit manufacturer.

[0209] Results Selection of HLA-A*24:02-binding peptides derived from SARS-CoV-2 proteins Tables 2a and 2b show the 9mer and 1Omer peptides derived from SARS-CoV-2 proteins predicted to bind to HLA-A*24:02 by "NetMHC 4.0" in the order of high binding affinity. Peptides that are also commonly found in SARS-CoV and MERS-CoV are shown in Table 2a. Peptides common only to SARS-CoV are listed in Table 2b. A total of 15 peptides were selected as candidate epitope peptides that may have the ability to bind to HLA-A*24:02.

[0210]

[Table 2a] SARS CoV-2 Protein-derived Peptides Predicted to Bind to HLA-A*24:02 (Common to SARS-CoV and MERS-CoV) Peptide Protein Position Length Amino Acid Sequence Binding Affinity (nM) SEQ ID NO 1 ORFIab 5080 9 AYANSVFNI 56 1 2 ORFlab 5079 10 TAYANSVFNI 102 2 3 ORFIab 5184 9 VFMSEAKCW 395 3

The number in "Position" indicates the number of the first amino acid of the peptide counted from the N terminus of the protein. Binding affinity (nM) was calculated using "NetMHC4.0".

[0211]

[Table 2b]

SARS CoV-2 Protein-derived Peptides Predicted to Bind to HLA-A*24:02 (Common Only to SARS-CoV) Peptide Protein Position Length Amino Acid Sequence Binding Affinity (nM) SEQ ID NO 4 ORFlab 4090 9 TYASALWEI 18 4 5 S 897 10 PFAMQMAYRF 39 5 6 ORFlab 3811 10 YDYLVSTQEF 41 6 7 ORFlab 2560 10 YYSQLMCQPI 44 7 8 ORFlab 4628 9 SYYSLLMPI 46 8 9 ORFlab 6676 9 RYKLEGYAF 62 9 10 ORFlab 4089 10 FTYASALWEI 69 10 11 ORFlab 7039 10 SYSLFDMSKF 85 11 12 ORFlab 4677 10 RYFKYWDQTY 90 12 13 M 54 9 LWLLWPVTL 108 13 14 ORFlab 4090 10 TYASALWEIQ 111 14 15 ORFlab 5138 10 YAYLRKHFSM 115 15

The number in "Position" indicates the number of the first amino acid of the peptide counted from the N terminus of the protein. Binding affinity (nM) was calculated using "NetMHC4.0".

[0212] Induction of HLA-A*24:02-restricted CTLs by peptides derived from SARS-CoV-2 proteins Using HLA-A*24:02-positive PBMCs, CTLs specific to peptides derived from SARS CoV-2 proteins were induced according to the protocol described in "Materials and Methods". Peptide-specific IFN-y production by the cells was confirmed by an ELISPOT assay (Fig. 1).

Peptide-specific IFN-y production was observed for 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) (Fig. la). On the other hand, no peptide-specific IFN-y production was observed for the other peptides shown in Tables 2a and 2b. For example, peptide-specific IFN production was not found for Peptide 6 (SEQ ID NO: 6) (Fig. lb). Although all the peptides had the possibility to bind to HLA-A*24:02, as a result, 12 peptides were identified that bind to HLA-A*24:02 and have CTL-inducing ability.

[0213] Establishment of HLA-A*24:02-restricted CTL lines specific to peptides derived from SARS CoV-2 proteins HLA-A*24:02-restricted CTL lines were established by proliferating the cells that showed IFN-y production specific 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 the HLA A*24:02 restricted-IFN-y ELISPOT assay. As a result of measuring IFN-y using ELISA, IFN-y production by CTL lines for HLA-A*24:02-expressing target cells (TISI cells) 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) was observed (Fig. 2). This clearly demonstrated that 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) and Peptide 13 (SEQ ID NO: 13) bind to HLA-A*24:02 and have CTL-inducing ability.

[0214] Induction of HLA-A*02:01-restricted CTLs by peptides derived from SARS-CoV-2 proteins It was verified whether 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), which had been confirmed to have the ability to induce HLA-A*24:02-restricted CTLs, have the ability to induce HLA-A*02:01-restricted CTLs. Using HLA-A*02:01-positive PBMCs, HLA-A*02:01-restricted CTLs were induced according to the protocol described in "Materials and Methods". Peptide-specific IFN-y production by the cells was confirmed by an ELISPOT assay (Fig. 3). Peptide-specific IFN-y production was observed for 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) (Fig. 3a). On the other hand, no peptide-specific IFN-y production was observed for 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). As an example, the result of Peptide 5 (SEQ ID NO: 5) is shown (Fig. 3b). These results revealed that 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), which have the ability to induce HLA-A*24:02-restricted CTLs, also bind to HLA-A*02:01 and have the ability to induce HLA-A*02:01-restricted CTLs.

[0215] Establishment of HLA-A*02:01-restricted CTL lines specific to peptides derived from SARS CoV-2 proteins HLA-A*02:01-restricted CTL lines were established by proliferating the cells that showed IFN-y production specific to 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-y ELISPOT assay. As a result of measuring IFN-y using ELISA, IFN-y production by CTL lines for HLA-A*02:01-expressing target cells (T2 cells) pulsed 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) was observed (Fig. 4). This clearly demonstrated 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) also bind to HLA A*02:01 and have the ability to induce HLA-A*02:01-restricted CTLs.

[0216] Homology analysis of peptides 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 show peptide-specific IFN-y production. Thus, homology analysis was performed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to check 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 amino acid sequences derived from human proteins. 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. Accordingly, as far as the present inventors know, these peptides are derived from SARS-CoV-2, or SARS-CoV or MERS-CoV and are very unlikely to evoke an unintended immune response against human normal tissues. In conclusion, novel HLA A*24:02- or HLA-A*02:01-restricted epitope peptides derived from SARS-CoV-2 proteins were identified and shown to be applicable to peptide vaccines against COVID-19.

[Industrial Applicability]

[0217] The present invention provides novel HLA-A24- or HLA-A02-restricted epitope peptides derived from SARS-CoV-2 proteins that induce a potent and specific immune response against coronavirus infection and thus can have applicability to a wide variety of coronavirus infectious diseases. The peptides, compositions, APCs, and CTLs of the present invention can be used as peptide vaccines against coronavirus infectious diseases, for example SARS-CoV-2, MERS-CoV, or SARS-CoV infectious diseases.

[0218] While the present invention is herein described in detail and with respect to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the present invention, the metes and bounds of which are defined by the appended claims.

SEQUENCE LISTING SEQUENCE LISTING

<110> ONCOTHERAPYSCIENCE, <110> ONCOTHERAPY SCIENCE,INC. INC. CANCER PRECISION CANCER PRECISIONMEDICINE, MEDICINE, INC. INC. KIYOTANI,Kazuma KIYOTANI, Kazuma NAKAMURA,Yusuke NAKAMURA, Yusuke

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Thr Ala Thr Ala Tyr Tyr Ala Ala Asn Asn Ser Ser Val Val Phe Phe Asn Asn Ile Ile 11 5 5 10 10

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<220> <220> <223> aapeptide <223> peptidederived derivedfrom fromORF1ab ORF1abpolyprotein polyproteinofofSARS-CoV-2 SARS-CoV-2

<400> <400> 33

Val Phe Val Phe Met Met Ser Ser Glu Glu Ala Ala Lys Lys Cys Cys Trp Trp 11 5 5

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Thr Tyr Thr Tyr Ala Ala Ser Ser Ala Ala Leu Leu Trp Trp Glu Glu Ile Ile 11 5 5

<210> 55 <210> <211> <211> 10 10 <212> PRT <212> PRT <213> <213> Artificial Sequence Artificial Sequence

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Pro Phe Pro Phe Ala AlaMet MetGln Gln MetMet AlaAla Tyr Tyr Arg Arg Phe Phe

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11 5 5 10 10

<210> <210> 66 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

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Tyr Asp Tyr Asp Tyr TyrLeu LeuVal Val SerSer ThrThr Gln Gln Glu Glu Phe Phe 1 1 5 5 10 10

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Ser Tyr Ser Tyr Tyr TyrSer SerLeu Leu LeuLeu MetMet Pro Pro Ile Ile 1 1 5

4

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Arg Tyr Arg Tyr Lys Lys Leu Leu Glu Glu Gly Gly Tyr Tyr Ala Ala Phe Phe 11 5 5

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Phe Thr Phe Thr Tyr TyrAla AlaSer Ser AlaAla LeuLeu Trp Trp Glu Glu Ile Ile 11 5 5 10 10

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Ser Tyr Ser Tyr Ser SerLeu LeuPhe Phe AspAsp MetMet Ser Ser Lys Lys Phe Phe 1 1 5 5 10

5

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Arg Tyr Arg Tyr Phe PheLys LysTyr Tyr TrpTrp AspAsp Gln Gln Thr Thr Tyr Tyr 1 1 5 5 10 10

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<400> 13 <400> 13

Leu Trp Leu Trp Leu Leu Leu Leu Trp Trp Pro Pro Val Val Thr Thr Leu Leu 11 5 5

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<400> <400> 14 14

Thr Tyr Thr Tyr Ala Ala Ser Ser Ala Ala Leu Leu Trp Trp Glu Glu Ile Ile Gln Gln 11 5 5 10

6

<210> <210> 15 15 <211> <211> 10 10 <212> <212> PRT PRT <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> <223> aapeptide peptidederived derivedfrom fromORF ab polyprotein ORF1ab polyproteinofofSARS-CoV-2 SARS-CoV-2

<400> 15 <400> 15

Tyr Ala Tyr Ala Tyr TyrLeu LeuArg Arg LysLys HisHis Phe Phe Ser Ser Met Met 1 1 5 5 10 10

<210> 16 <210> 16 <211> 29903 <211> 29903 <212> <212> DNA DNA <213> <213> Severe acuterespiratory Severe acute respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 16 16 attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc ttgtagatct attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc ttgtagatct 60 60

gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc ttagtgcact gttctctaaa cgaactttaa aatctgtgtg gctgtcactc ggctgcatgc ttagtgcact 120 120

cacgcagtat aattaataac taattactgt cgttgacagg acacgagtaa ctcgtctatc cacgcagtat aattaataac taattactgt cgttgacagg acacgagtaa ctcgtctatc 180 180

ttctgcaggo tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac atctaggttt 240 240

cgtccgggtgtgaccgaaag cgtccgggtg tgaccgaaag gtaagatgga gtaagatgga gagccttgtc gagccttgtc cctggtttca cctggtttca acgagaaaac acgagaaaac 300 300

acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg acacgtccaa ctcagtttgc ctgttttaca ggttcgcgac gtgctcgtac gtggctttgg 360 360

agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg agactccgtg gaggaggtct tatcagaggc acgtcaacat cttaaagatg gcacttgtgg 420 420

cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa cttagtagaa gttgaaaaag gcgttttgcc tcaacttgaa cagccctatg tgttcatcaa 480 480

acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact acgttcggat gctcgaactg cacctcatgg tcatgttatg gttgagctgg tagcagaact 540 540

cgaaggcatt cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg cgaaggcatt cagtacggtc gtagtggtga gacacttggt gtccttgtcc ctcatgtggg 600 600

cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg cgaaatacca gtggcttacc gcaaggttct tcttcgtaag aacggtaata aaggagctgg 660 660

tggccatagt tacggcgccg atctaaagto atttgactta ggcgacgago ttggcactga tggccatagt tacggcgccg atctaaagtc atttgactta ggcgacgagc ttggcactga 720

7

tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga tccttatgaa gattttcaag aaaactggaa cactaaacat agcagtggtg ttacccgtga 780 780

actcatgcgt gagcttaacg gaggggcata cactcgctat gtcgataaca acttctgtgg actcatgcgt gagcttaacg gaggggcata cactcgctat gtcgataaca acttctgtgg 840 840

ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc ccctgatggc taccctcttg agtgcattaa agaccttcta gcacgtgctg gtaaagcttc 900 900

atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg atgcactttg tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg 960 960

tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca tgaacatgag catgaaattg cttggtacac ggaacgttct gaaaagagct atgaattgca 1020 1020

gacacctttt gaaattaaat tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa gacacctttt gaaattaaat tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa 1080 1080

ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg aaaagaaaaa 1140 1140

gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg gcttgatggc tttatgggta gaattcgatc tgtctatcca gttgcgtcac caaatgaatg 1200 1200

caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca caaccaaatg tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca 1260 1260

gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga gacgggcgat tttgttaaag ccacttgcga attttgtggc actgagaatt tgactaaaga 1320 1320

aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc aggtgccact acttgtggtt acttacccca aaatgctgtt gttaaaattt attgtccagc 1380 1380

atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata atgaatctgg 1440 1440

cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc cttgaaaacc attcttcgta agggtggtcg cactattgcc tttggaggct gtgtgttctc 1500 1500

ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg ttatgttggt tgccataaca agtgtgccta ttgggttcca cgtgctagcg ctaacatagg 1560 1560

ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga ttgtaaccat acaggtgttg ttggagaagg ttccgaaggt cttaatgaca accttcttga 1620 1620

aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac ttaatgaaga 1680 1680

gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa gatcgccatt attttggcat ctttttctgc ttccacaagt gcttttgtgg aaactgtgaa 1740 1740

aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac aggtttggat tataaagcat tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac 1800 1800

aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc aaaaggaaaa gctaaaaaag gtgcctggaa tattggtgaa cagaaatcaa tactgagtcc 1860 1860

tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct tctttatgca tttgcatcag aggctgctcg tgttgtacga tcaattttct cccgcactct 1920 1920

tgaaactgct caaaattctg tgcgtgtttt acagaaggco gctataacaa tactagatgg tgaaactgct caaaattctg tgcgtgtttt acagaaggcc gctataacaa tactagatgg 1980

8

aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac aatttcacag tattcactga gactcattga tgctatgatg ttcacatctg atttggctac 2040 2040

taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg taacaatcta gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg 2100 2100

gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga gctaactaac atctttggca ctgtttatga aaaactcaaa cccgtccttg attggcttga 2160 2160

agagaagttt aaggaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat agagaagttt aaggaaggtg tagagtttct tagagacggt tgggaaattg ttaaatttat 2220 2220

ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa aggaaattaa 2280 2280

ggagagtgtt cagacattct ttaagcttgt aaataaattt ttggctttgt gtgctgactc ggagagtgtt cagacattct ttaagcttgt aaataaattt ttggctttgt gtgctgactc 2340 2340

tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca tatcattatt ggtggagcta aacttaaagc cttgaattta ggtgaaacat ttgtcacgca 2400 2400

ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgco ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa gaaactggcc tactcatgcc 2460 2460

tctaaaagcc ccaaaagaaa ttatcttctt agagggagaa acacttccca cagaagtgtt tctaaaagcc ccaaaagaaa ttatcttctt agagggagaa acacttccca cagaagtgtt 2520 2520

aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac ctactagtga aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac ctactagtga 2580 2580

agctgttgaa gctccattgg ttggtacaco agtttgtatt aacgggctta tgttgctcga agctgttgaa gctccattgg ttggtacacc agtttgtatt aacgggctta tgttgctcga 2640 2640

aatcaaagac acagaaaagt actgtgccct tgcacctaat atgatggtaa caaacaatac aatcaaagac acagaaaagt actgtgccct tgcacctaat atgatggtaa caaacaatac 2700 2700

cttcacactc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga cttcacactc aaaggcggtg caccaacaaa ggttactttt ggtgatgaca ctgtgataga 2760 2760

agtgcaaggt tacaagagtg tgaatatcac ttttgaactt gatgaaagga ttgataaagt agtgcaaggt tacaagagtg tgaatatcac ttttgaactt gatgaaagga ttgataaagt 2820 2820

acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa atgagttcgc acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa atgagttcgc 2880 2880

ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacaco ctgtgttgtg gcagatgctg tcataaaaac tttgcaacca gtatctgaat tacttacacc 2940 2940

actgggcatt gatttagatg agtggagtat ggctacatac tacttatttg atgagtctgg actgggcatt gatttagatg agtggagtat ggctacatac tacttatttg atgagtctgg 3000 3000

tgagtttaaa ttggcttcac atatgtattg ttctttctac cctccagatg aggatgaaga tgagtttaaa ttggcttcac atatgtattg ttctttctac cctccagatg aggatgaaga 3060 3060

agaaggtgat tgtgaagaag aagagtttga gccatcaact caatatgagt atggtactga agaaggtgat tgtgaagaag aagagtttga gccatcaact caatatgagt atggtactga 3120 3120

agatgattac caaggtaaac ctttggaatt tggtgccact tctgctgctc ttcaacctga agatgattac caaggtaaac ctttggaatt tggtgccact tctgctgctc ttcaacctga 3180 3180

agaagagcaa gaagaagatt ggttagatga tgatagtcaa caaactgttg gtcaacaaga agaagagcaa gaagaagatt ggttagatga tgatagtcaa caaactgttg gtcaacaaga 3240 3240

cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggtto aacctcaatt cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggttc aacctcaatt 3300

9

agagatggaa cttacaccag ttgttcagac tattgaagtg aatagtttta gtggttattt agagatggaa cttacaccag ttgttcagac tattgaagtg aatagtttta gtggttattt 3360 3360

aaaacttact gacaatgtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt aaaacttact gacaatgtat acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3420 3420

aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag gaggtgttgc 3480 3480

aggagcctta aataaggcta ctaacaatgc catgcaagtt gaatctgatg attacatago aggagcctta aataaggcta ctaacaatgc catgcaagtt gaatctgatg attacatagc 3540 3540 tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa tactaatgga ccacttaaag tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3600 3600

acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacatto aacttcttaa acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc aacttcttaa 3660 3660

gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg gagtgcttat gaaaatttta atcagcacga agttctactt gcaccattat tatcagctgg 3720 3720 tatttttggt gctgacccta tacattcttt aagagtttgt gtagatactg ttcgcacaaa tatttttggt gctgacccta tacattcttt aagagtttgt gtagatactg ttcgcacaaa 3780 3780

tgtctactta gctgtctttg ataaaaatct ctatgacaaa cttgtttcaa gctttttgga tgtctactta gctgtctttg ataaaaatct ctatgacaaa cttgtttcaa gctttttgga 3840 3840

aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa aatgaagagt gaaaagcaag ttgaacaaaa gatcgctgag attcctaaag aggaagttaa 3900 3900

gccatttata actgaaagta aaccttcagt tgaacagaga aaacaagatg ataagaaaat gccatttata actgaaagta aaccttcagt tgaacagaga aaacaagatg ataagaaaat 3960 3960

caaagcttgt gttgaagaag ttacaacaac tctggaagaa actaagttcc tcacagaaaa caaagcttgt gttgaagaag ttacaacaac tctggaagaa actaagttcc tcacagaaaa 4020 4020 cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca ctcttgttag 4080 4080 tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca tgacattgac atcactttct taaagaaaga tgctccatat atagtgggtg atgttgttca 4140 4140 agagggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat agagggtgtt ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat 4200 4200 gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca gctagcgaaa gctttgagaa aagtgccaac agacaattat ataaccactt acccgggtca 4260 4260 gggtttaaat ggttacactg tagaggaggo aaagacagtg cttaaaaagt gtaaaagtgc gggtttaaat ggttacactg tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc 4320 4320 cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc cttttacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg gaactgtttc 4380 4380 ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg ttggaatttg cgagaaatgc ttgcacatgc agaagaaaca cgcaaattaa tgcctgtctg 4440 4440 tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca tgtggaaact aaagccatag tttcaactat acagcgtaaa tataagggta ttaaaataca 4500 4500 agagggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc agagggtgtg gttgattatg gtgctagatt ttacttttac accagtaaaa caactgtagc 4560

10

gtcacttatcaacacactta gtcacttatc aacacactta acgatctaaa acgatctaaa tgaaactctt tgaaactctt gttacaatgc gttacaatgc cacttggcta cacttggcta 4620 4620

tgtaacacat ggcttaaatt tgtaacacat ggcttaaatt tggaagaagc tggaagaagc tgctcggtat tgctcggtat atgagatctc atgagatctc tcaaagtgcc tcaaagtgcc 4680 4680

agctacagtttctgtttctt agctacagtt tctgtttctt cacctgatgc cacctgatgc tgttacagcg tgttacagcg tataatggtt tataatggtt atcttacttc atcttacttc 4740 4740

ttcttctaaa acacctgaag ttcttctaaa acacctgaag aacattttat aacattttat tgaaaccatc tgaaaccatc tcacttgctg tcacttgctg gttcctataa gttcctataa 4800 4800

agattggtcc tattctggac agattggtcc tattctggac aatctacaca aatctacaca actaggtata actaggtata gaatttctta gaatttctta agagaggtga agagaggtga 4860 4860

taaaagtgtatattacacta taaaagtgta tattacacta gtaatcctac gtaatcctac cacattccac cacattccac ctagatggtg ctagatggtg aagttatcac aagttatcac 4920 4920

ctttgacaatcttaagacac ctttgacaat cttaagacac ttctttcttt ttctttcttt gagagaagtg gagagaagtg aggactatta aggactatta aggtgtttac aggtgtttac 4980 4980

aacagtagac aacattaacc aacagtagac aacattaacc tccacacgca tccacacgca agttgtggac agttgtggac atgtcaatga atgtcaatga catatggaca catatggaca 5040 5040

acagtttggtccaacttatt acagtttggt ccaacttatt tggatggagc tggatggagc tgatgttact tgatgttact aaaataaaac aaaataaaac ctcataattc ctcataattc 5100 5100

acatgaaggtaaaacatttt acatgaaggt aaaacatttt atgttttacc atgttttacc taatgatgac taatgatgac actctacgtg actctacgtg ttgaggcttt ttgaggcttt 5160 5160

tgagtactaccacacaactg tgagtactac cacacaactg atcctagttt atcctagttt tctgggtagg tctgggtagg tacatgtcag tacatgtcag cattaaatca cattaaatca 5220 5220

cactaaaaagtggaaatacc cactaaaaag tggaaatacc cacaagttaa cacaagttaa tggtttaact tggtttaact tctattaaat tctattaaat gggcagataa gggcagataa 5280 5280

caactgttatcttgccactg caactgttat cttgccactg cattgttaac cattgttaac actccaacaa actccaacaa atagagttga atagagttga agtttaatcc agtttaatcc 5340 5340

acctgctctacaagatgctt acctgctcta caagatgctt attacagagc attacagage aagggctggt aagggctggt gaagctgcta gaagctgcta acttttgtgc acttttgtgc 5400 5400

acttatcttagcctactgta acttatctta gcctactgta ataagacagt ataagacagt aggtgagtta aggtgagtta ggtgatgtta ggtgatgtta gagaaacaat gagaaacaat 5460 5460

gagttacttg tttcaacatg gagttacttg tttcaacatg ccaatttaga ccaatttaga ttcttgcaaa ttcttgcaaa agagtcttga agagtcttga acgtggtgtg acgtggtgtg 5520 5520

taaaacttgtggacaacagc taaaacttgt ggacaacagc agacaaccct agacaaccct taagggtgta taagggtgta gaagctgtta gaagctgtta tgtacatggg tgtacatggg 5580 5580

cacactttcttatgaacaat cacactttct tatgaacaat ttaagaaagg ttaagaaagg tgttcagata tgttcagata ccttgtacgt ccttgtacgt gtggtaaaca gtggtaaaca 5640 5640

agctacaaaatatctagtac agctacaaaa tatctagtac aacaggagtc aacaggagtc accttttgtt accttttgtt atgatgtcag atgatgtcag caccacctgc caccacctgc 5700 5700

tcagtatgaacttaagcatg tcagtatgaa cttaagcatg gtacatttac gtacatttac ttgtgctagt ttgtgctagt gagtacactg gagtacactg gtaattacca gtaattacca 5760 5760

gtgtggtcactataaacata gtgtggtcac tataaacata taacttctaa taacttctaa agaaactttg agaaactttg tattgcatag tattgcatag acggtgcttt acggtgcttt 5820 5820

acttacaaagtcctcagaat acttacaaag tcctcagaat acaaaggtcc acaaaggtcc tattacggat tattacggat gttttctaca gttttctaca aagaaaacag aagaaaacag 5880

11

ttacacaacaaccataaaac ttacacaaca accataaaac cagttactta cagttactta taaattggat taaattggat ggtgttgttt ggtgttgttt gtacagaaat gtacagaaat 5940 5940

tgaccctaagttggacaatt tgaccctaag ttggacaatt attataagaa attataagaa agacaattct agacaattct tatttcacag tatttcacag agcaaccaat agcaaccaat 6000 6000

tgatcttgtaccaaaccaac tgatcttgta ccaaaccaac catatccaaa catatccaaa cgcaagcttc cgcaagcttc gataatttta gataatttta agtttgtatg agtttgtatg 6060 6060

tgataatatcaaatttgctg tgataatatc aaatttgctg atgatttaaa atgatttaaa ccagttaact ccagttaact ggttataaga ggttataaga aacctgcttc aacctgcttc 6120 6120

aagagagcttaaagttacat aagagagctt aaagttacat ttttccctga ttttccctga cttaaatggt cttaaatggt gatgtggtgg gatgtggtgg ctattgatta ctattgatta 6180 6180

taaacactacacaccctctt taaacactac acaccctctt ttaagaaagg ttaagaaagg agctaaattg agctaaattg ttacataaac ttacataaac ctattgtttg ctattgtttg 6240 6240

gcatgttaacaatgcaacta gcatgttaac aatgcaacta ataaagccac ataaagccac gtataaacca gtataaacca aatacctggt aatacctggt gtatacgttg gtatacgttg 6300 6300

tctttggagcacaaaaccag tctttggagc acaaaaccag ttgaaacatc ttgaaacatc aaattcgttt aaattcgttt gatgtactga gatgtactga agtcagagga agtcagagga 6360 6360

cgcgcagggaatggataatc cgcgcaggga atggataatc ttgcctgcga ttgcctgcga agatctaaaa agatctaaaa ccagtctctg ccagtctctg aagaagtagt aagaagtagt 6420 6420

ggaaaatcctaccatacaga ggaaaatcct accatacaga aagacgttct aagacgttct tgagtgtaat tgagtgtaat gtgaaaacta gtgaaaacta ccgaagttgt ccgaagttgt 6480 6480

aggagacattatacttaaac aggagacatt atacttaaac cagcaaataa cagcaaataa tagtttaaaa tagtttaaaa attacagaag attacagaag aggttggcca aggttggcca 6540 6540

cacagatcta atggctgctt cacagatcta atggctgctt atgtagacaa atgtagacaa ttctagtctt ttctagtctt actattaaga actattaaga aacctaatga aacctaatga 6600 6600

attatctaga gtattaggtt attatctaga gtattaggtt tgaaaaccct tgaaaaccct tgctactcat tgctactcat ggtttagctg ggtttagctg ctgttaatag ctgttaatag 6660 6660

tgtcccttgg gatactatag tgtcccttgg gatactatag ctaattatgc ctaattatgc taagcctttt taagcctttt cttaacaaag cttaacaaag ttgttagtac ttgttagtac 6720 6720

aactactaac atagttacac aactactaac atagttacac ggtgtttaaa ggtgtttaaa ccgtgtttgt ccgtgtttgt actaattata actaattata tgccttattt tgccttattt 6780 6780

ctttactttattgctacaat ctttacttta ttgctacaat tgtgtacttt tgtgtacttt tactagaagt tactagaagt acaaattcta acaaattcta gaattaaagc gaattaaagc 6840 6840

atctatgccgactactatag atctatgccg actactatag caaagaatac caaagaatac tgttaagagt tgttaagagt gtcggtaaat gtcggtaaat tttgtctaga tttgtctaga 6900 6900

ggcttcatttaattatttga ggcttcattt aattatttga agtcacctaa agtcacctaa tttttctaaa tttttctaaa ctgataaata ctgataaata ttataatttg ttataatttg 6960 6960

gtttttactattaagtgttt gtttttacta ttaagtgttt gcctaggttc gcctaggttc tttaatctac tttaatctac tcaaccgctg tcaaccgctg ctttaggtgt ctttaggtgt 7020 7020

tttaatgtctaatttaggca tttaatgtct aatttaggca tgccttctta tgccttctta ctgtactggt ctgtactggt tacagagaag tacagagaag gctatttgaa gctatttgaa 7080 7080

ctctactaatgtcactattg ctctactaat gtcactattg caacctactg caacctactg tactggttct tactggttct ataccttgta ataccttgta gtgtttgtct gtgtttgtct 7140

12

tagtggtttagattctttag tagtggttta gattctttag acacctatcc acacctatcc ttctttagaa ttctttagaa actatacaaa actatacaaa ttaccatttc ttaccatttc 7200 7200

atcttttaaatgggatttaa atcttttaaa tgggatttaa ctgcttttgg ctgcttttgg cttagttgca cttagttgca gagtggtttt gagtggtttt tggcatatat tggcatatat 7260 7260

tcttttcactaggtttttct tcttttcact aggtttttct atgtacttgg atgtacttgg attggctgca attggctgca atcatgcaat atcatgcaat tgtttttcag tgtttttcag 7320 7320

ctattttgcagtacatttta ctattttgca gtacatttta ttagtaattc ttagtaattc ttggcttatg ttggcttatg tggttaataa tggttaataa ttaatcttgt ttaatcttgt 7380 7380

acaaatggcc ccgatttcag acaaatggcc ccgatttcag ctatggttag ctatggttag aatgtacatc aatgtacatc ttctttgcat ttctttgcat cattttatta cattttatta 7440 7440

tgtatggaaa agttatgtgc tgtatggaaa agttatgtgc atgttgtaga atgttgtaga cggttgtaat cggttgtaat tcatcaactt tcatcaactt gtatgatgtg gtatgatgtg 7500 7500

ttacaaacgt aatagagcaa ttacaaacgt aatagagcaa caagagtcga caagagtcga atgtacaact atgtacaact attgttaatg attgttaatg gtgttagaag gtgttagaag 7560 7560

gtccttttatgtctatgcta gtccttttat gtctatgcta atggaggtaa atggaggtaa aggcttttgc aggcttttgc aaactacaca aaactacaca attggaattg attggaattg 7620 7620

tgttaattgt gatacattct tgttaattgt gatacattct gtgctggtag gtgctggtag tacatttatt tacatttatt agtgatgaag agtgatgaag ttgcgagaga ttgcgagaga 7680 7680

cttgtcactacagtttaaaa cttgtcacta cagtttaaaa gaccaataaa gaccaataaa tcctactgac tcctactgac cagtcttctt cagtcttctt acatcgttga acatcgttga 7740 7740

tagtgttacagtgaagaatg tagtgttaca gtgaagaatg gttccatcca gttccatcca tctttacttt tctttacttt gataaagctg gataaagctg gtcaaaagac gtcaaaagac 7800 7800

ttatgaaaga cattctctct ttatgaaaga cattctctct ctcattttgt ctcattttgt taacttagac taacttagac aacctgagag aacctgagag ctaataacac ctaataacac 7860 7860

taaaggttcattgcctatta taaaggttca ttgcctatta atgttatagt atgttatagt ttttgatggt ttttgatggt aaatcaaaat aaatcaaaat gtgaagaatc gtgaagaatc 7920 7920

atctgcaaaa tcagcgtctg atctgcaaaa tcagcgtctg tttactacag tttactacag tcagcttatg tcagcttatg tgtcaaccta tgtcaaccta tactgttact tactgttact 7980 7980

agatcaggcattagtgtctg agatcaggca ttagtgtctg atgttggtga atgttggtga tagtgcggaa tagtgcggaa gttgcagtta gttgcagtta aaatgtttga aaatgtttga 8040 8040

tgcttacgttaatacgtttt tgcttacgtt aatacgtttt catcaacttt catcaacttt taacgtacca taacgtacca atggaaaaac atggaaaaac tcaaaacact tcaaaacact 8100 8100

agttgcaact gcagaagctg agttgcaact gcagaagctg aacttgcaaa aacttgcaaa gaatgtgtcc gaatgtgtcc ttagacaatg ttagacaatg tcttatctac tcttatctac 8160 8160

ttttatttcagcagctcggc ttttatttca gcagctcggc aagggtttgt aagggtttgt tgattcagat tgattcagat gtagaaacta gtagaaacta aagatgttgt aagatgttgt 8220 8220

tgaatgtcttaaattgtcac tgaatgtctt aaattgtcac atcaatctga atcaatctga catagaagtt catagaagtt actggcgata actggcgata gttgtaataa gttgtaataa 8280 8280

ctatatgctcacctataaca ctatatgctc acctataaca aagttgaaaa aagttgaaaa catgacaccc catgacaccc cgtgaccttg cgtgaccttg gtgcttgtat gtgcttgtat 8340 8340

tgactgtagtgcgcgtcata tgactgtagt gcgcgtcata ttaatgcgca ttaatgcgca ggtagcaaaa ggtagcaaaa agtcacaaca agtcacaaca ttgctttgat ttgctttgat 8400 8400

atggaacgttaaagatttca atggaacgtt aaagatttca tgtcattgtc tgtcattgtc tgaacaacta tgaacaacta cgaaaacaaa cgaaaacaaa tacgtagtgc tacgtagtgc 8460

13

tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa tgctaaaaag aataacttac cttttaagtt gacatgtgca actactagac aagttgttaa 8520 8520

tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca tgttgtaaca acaaagatag cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8580 8580

gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt taataacacc 8640 8640

tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat tgttcatgtc atgtctaaac atactgactt ttcaagtgaa atcataggat acaaggctat 8700 8700

tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgo tgatggtggt gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc 8760 8760

tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc tgattttgac acatggttta gccagcgtgg tggtagttat actaatgaca aagcttgccc 8820 8820

attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac attgattgct gcagtcataa caagagaagt gggttttgtc gtgcctggtt tgcctggcac 8880 8880

gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt gatattacgc acaactaatg gtgacttttt gcatttctta cctagagttt ttagtgcagt 8940 8940

tggtaacatc tgttacacac catcaaaact tatagagtac actgactttg caacatcago tggtaacatc tgttacacac catcaaaact tatagagtac actgactttg caacatcagc 9000 9000

ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaago cagtaccata ttgtgttttg gctgctgaat gtacaatttt taaagatgct tctggtaagc cagtaccata 9060 9060

ttgttatgat accaatgtac tagaaggtto tgttgcttat gaaagtttac gccctgacac ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac gccctgacac 9120 9120

acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggtto acgttatgtg ctcatggatg gctctattat tcaatttcct aacacctacc ttgaaggttc 9180 9180

tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagato tgttagagtg gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc 9240 9240

agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag agaagctggt gtttgtgtat ctactagtgg tagatgggta cttaacaatg attattacag 9300 9300

atctttacca ggagttttct gtggtgtaga tgctgtaaat ttacttacta atatgtttac atctttacca ggagttttct gtggtgtaga tgctgtaaat ttacttacta atatgtttac 9360 9360

accactaatt caacctattg gtgctttgga catatcagca tctatagtag ctggtggtat accactaatt caacctattg gtgctttgga catatcagca tctatagtag ctggtggtat 9420 9420

tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagcttttgg tgtagctatc gtagtaacat gccttgccta ctattttatg aggtttagaa gagcttttgg 9480 9480

tgaatacagt catgtagttg cctttaatac tttactatto cttatgtcat tcactgtact tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat tcactgtact 9540 9540

ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttattt acttgtactt ctgtttaaca ccagtttact cattcttacc tggtgtttat tctgttattt acttgtactt 9600 9600

gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt gacattttat cttactaatg atgtttcttt tttagcacat attcagtgga tggttatgtt 9660 9660

cacaccttta gtacctttct ggataacaat tgcttatato atttgtattt ccacaaagca cacaccttta gtacctttct ggataacaat tgcttatatc atttgtattt ccacaaagca 9720

14

tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt tttctattgg ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9780 9780

tagtactttt gaagaagctg cgctgtgcac ctttttgtta aataaagaaa tgtatctaaa tagtactttt gaagaagctg cgctgtgcac ctttttgtta aataaagaaa tgtatctaaa 9840 9840

gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa gttgcgtagt gatgtgctat tacctcttac gcaatataat agatacttag ctctttataa 9900 9900

taagtacaag tattttagtg gagcaatgga tacaactago tacagagaag ctgcttgttg taagtacaag tattttagtg gagcaatgga tacaactagc tacagagaag ctgcttgttg 9960 9960

tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgtto tttaccaacc tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc tttaccaacc 10020 10020

accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc accacaaacc tctatcacct cagctgtttt gcagagtggt tttagaaaaa tggcattccc 10080 10080

atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaaccg atctggtaaa gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg 10140 10140

tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat tctttggctt gatgacgtag tttactgtcc aagacatgtg atctgcacct ctgaagacat 10200 10200

gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca gcttaaccct aattatgaag atttactcat tcgtaagtct aatcataatt tcttggtaca 10260 10260

ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg tacttaagct 10320 10320

taaggttgat acagccaatc ctaagacacc taagtataag tttgttcgca ttcaaccagg taaggttgat acagccaatc ctaagacacc taagtataag tttgttcgca ttcaaccagg 10380 10380

acagactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc acagactttt tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc 10440 10440

tatgaggccc aatttcacta ttaagggtto attccttaat ggttcatgtg gtagtgttgg tatgaggccc aatttcacta ttaagggttc attccttaat ggttcatgtg gtagtgttgg 10500 10500

ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac ttttaacata gattatgact gtgtctcttt ttgttacatg caccatatgg aattaccaac 10560 10560

tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca tggagttcat gctggcacag acttagaagg taacttttat ggaccttttg ttgacaggca 10620 10620

aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta aacagcacaa gcagctggta cggacacaac tattacagtt aatgttttag cttggttgta 10680 10680

cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga cgctgctgtt ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga 10740 10740

ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagaco atgttgacat ctttaacctt gtggctatga agtacaatta tgaacctcta acacaagacc atgttgacat 10800 10800

actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa actaggacct ctttctgctc aaactggaat tgccgtttta gatatgtgtg cttcattaaa 10860 10860

agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt tattagaaga 10920 10920

tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt tgaatttaca ccttttgatg ttgttagaca atgctcaggt gttactttcc aaagtgcagt 10980 10980

gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcactttt gaaaagaaca atcaagggta cacaccactg gttgttactc acaattttga cttcactttt 11040

15

agttttagto cagagtacto aatggtcttt gttctttttt ttgtatgaaa atgccttttt

agttttagtc cagagtactc aatggtcttt gttctttttt ttgtatgaaa atgccttttt 11100 11100 accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa

accttttgct atgggtatta ttgctatgtc tgcttttgca atgatgtttg tcaaacataa 11160 11160 gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt attttaatat

gcatgcattt ctctgtttgt ttttgttacc ttctcttgcc actgtagctt attttaatat 11220 11220 ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatao

ggtctatatg cctgctagtt gggtgatgcg tattatgaca tggttggata tggttgatac 11280 11280 tagtttgtct ggttttaago taaaagactg tgttatgtat gcatcagctg tagtgttact

tagtttgtct ggttttaagc taaaagactg tgttatgtat gcatcagctg tagtgttact 11340 11340 aatccttatg acagcaagaa ctgtgtatga tgatggtgct aggagagtgt ggacacttat

aatccttatg acagcaagaa ctgtgtatga tgatggtgct aggagagtgt ggacacttat 11400 11400 gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagato aagccattto

gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagatc aagccatttc 11460 11460 catgtgggct cttataatct ctgttactto taactactca ggtgtagtta caactgtcat

catgtgggct cttataatct ctgttacttc taactactca ggtgtagtta caactgtcat 11520 11520 gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac

gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac 11580 11580 tggtaataca cttcagtgta taatgctagt ttattgttto ttaggctatt tttgtacttg

tggtaataca cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg 11640 11640 ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg gtgtttatga

ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg gtgtttatga 11700 11700 ttacttagtt tctacacagg agtttagata tatgaattca cagggactad tcccacccaa

ttacttagtt tctacacagg agtttagata tatgaattca cagggactac tcccacccaa 11760 11760 gaatagcata gatgccttca aactcaacat taaattgttg ggtgttggtg gcaaaccttg

gaatagcata gatgccttca aactcaacat taaattgttg ggtgttggtg gcaaaccttg 11820 11820 tatcaaagta gccactgtad agtctaaaat gtcagatgta aagtgcacat cagtagtctt

tatcaaagta gccactgtac agtctaaaat gtcagatgta aagtgcacat cagtagtctt 11880 11880 actctcagtt ttgcaacaac tcagagtaga atcatcatct aaattgtggg ctcaatgtgt

actctcagtt ttgcaacaac tcagagtaga atcatcatct aaattgtggg ctcaatgtgt 11940 11940 ccagttacac aatgacatto tcttagctaa agatactact gaagcctttg aaaaaatggt

ccagttacac aatgacattc tcttagctaa agatactact gaagcctttg aaaaaatggt 12000 12000 ttcactactt tctgttttgo tttccatgca gggtgctgta gacataaaca agctttgtga

ttcactactt tctgttttgc tttccatgca gggtgctgta gacataaaca agctttgtga 12060 12060 agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta gttcccttco

agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta gttcccttcc 12120 12120 atcatatgca gcttttgcta ctgctcaaga agcttatgag caggctgttg ctaatggtga

atcatatgca gcttttgcta ctgctcaaga agcttatgag caggctgttg ctaatggtga 12180 12180 ttctgaagtt gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga

ttctgaagtt gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga 12240 12240 ccgtgatgca gccatgcaac gtaagttgga aaagatggct gatcaagcta tgacccaaat

ccgtgatgca gccatgcaac gtaagttgga aaagatggct gatcaagcta tgacccaaat 12300

16

gtataaacag gctagatctg aggacaagag ggcaaaagtt actagtgcta tgcagacaat gtataaacag gctagatctg aggacaagag ggcaaaagtt actagtgcta tgcagacaat 12360 12360

gcttttcact atgcttagaa agttggataa tgatgcacto aacaacatta tcaacaatgo gcttttcact atgcttagaa agttggataa tgatgcactc aacaacatta tcaacaatgc 12420 12420

aagagatggt tgtgttccct tgaacataat acctcttaca acagcagcca aactaatggt aagagatggt tgtgttccct tgaacataat acctcttaca acagcagcca aactaatggt 12480 12480

tgtcatacca gactataaca catataaaaa tacgtgtgat ggtacaacat ttacttatgo tgtcatacca gactataaca catataaaaa tacgtgtgat ggtacaacat ttacttatgc 12540 12540

atcagcattg tgggaaatcc aacaggttgt agatgcagat agtaaaattg ttcaacttag atcagcattg tgggaaatcc aacaggttgt agatgcagat agtaaaattg ttcaacttag 12600 12600

tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa cagctttaag tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa cagctttaag 12660 12660

ggccaattct gctgtcaaat tacagaataa tgagcttagt cctgttgcac tacgacagat ggccaattct gctgtcaaat tacagaataa tgagcttagt cctgttgcac tacgacagat 12720 12720

gtcttgtgct gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta gtcttgtgct gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta 12780 12780

caacacaaca aagggaggta ggtttgtact tgcactgtta tccgatttac aggatttgaa caacacaaca aagggaggta ggtttgtact tgcactgtta tccgatttac aggatttgaa 12840 12840

atgggctaga ttccctaaga gtgatggaac tggtactatc tatacagaac tggaaccaco atgggctaga ttccctaaga gtgatggaac tggtactatc tatacagaac tggaaccacc 12900 12900

ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtatttat actttattaa ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtatttat actttattaa 12960 12960

aggattaaac aacctaaata gaggtatggt acttggtagt ttagctgcca cagtacgtct aggattaaac aacctaaata gaggtatggt acttggtagt ttagctgcca cagtacgtct 13020 13020

acaagctggt aatgcaacag aagtgcctgc caattcaact gtattatctt tctgtgcttt acaagctggt aatgcaacag aagtgcctgc caattcaact gtattatctt tctgtgcttt 13080 13080

tgctgtagat gctgctaaag cttacaaaga ttatctagct agtgggggad aaccaatcac tgctgtagat gctgctaaag cttacaaaga ttatctagct agtgggggac aaccaatcac 13140 13140

taattgtgtt aagatgttgt gtacacacac tggtactggt caggcaataa cagttacaco taattgtgtt aagatgttgt gtacacacac tggtactggt caggcaataa cagttacacc 13200 13200

ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt actgccgttg ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt actgccgttg 13260 13260

ccacatagat catccaaatc ctaaaggatt ttgtgactta aaaggtaagt atgtacaaat ccacatagat catccaaatc ctaaaggatt ttgtgactta aaaggtaagt atgtacaaat 13320 13320

acctacaact tgtgctaatg accctgtggg ttttacactt aaaaacacag tctgtaccgt acctacaact tgtgctaatg accctgtggg ttttacactt aaaaacacag tctgtaccgt 13380 13380

ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa ctccgcgaac ccatgcttca 13440 13440

gtcagctgat gcacaatcgt ttttaaacgg gtttgcggtg taagtgcago ccgtcttaca gtcagctgat gcacaatcgt ttttaaacgg gtttgcggtg taagtgcagc ccgtcttaca 13500 13500

ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat ctacaatgat ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat ctacaatgat 13560 13560

aaagtagctg gttttgctaa attcctaaaa actaattgtt gtcgcttcca agaaaaggac aaagtagctg gttttgctaa attcctaaaa actaattgtt gtcgcttcca agaaaaggac 13620

17

gaagatgaca atttaattga ttcttacttt gtagttaaga gacacacttt ctctaactac gaagatgaca atttaattga ttcttacttt gtagttaaga gacacacttt ctctaactac 13680 13680 caacatgaag aaacaattta taatttact+ aaggattgto cagctgttgc taaacatgao caacatgaag aaacaattta taatttactt aaggattgtc cagctgttgc taaacatgac 13740 13740 ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca acgtcttact

ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca acgtcttact 13800 13800 aaatacacaa tggcagacct cgtctatgct ttaaggcatt ttgatgaagg taattgtgac aaatacacaa tggcagacct cgtctatgct ttaaggcatt ttgatgaagg taattgtgac 13860 13860 acattaaaag aaatacttgt cacatacaat tgttgtgatg atgattattt caataaaaag

acattaaaag aaatacttgt cacatacaat tgttgtgatg atgattattt caataaaaag 13920 13920 gactggtatg attttgtaga aaacccagat atattacgcg tatacgccaa cttaggtgaa gactggtatg attttgtaga aaacccagat atattacgcg tatacgccaa cttaggtgaa 13980 13980 cgtgtacgcc aagctttgtt aaaaacagta caattctgtg atgccatgcg aaatgctggt cgtgtacgcc aagctttgtt aaaaacagta caattctgtg atgccatgcg aaatgctggt 14040 14040 attgttggtg tactgacatt agataatcaa gatctcaatg gtaactggta tgatttcggt

attgttggtg tactgacatt agataatcaa gatctcaatg gtaactggta tgatttcggt 14100 14100 gatttcatac aaaccacgcc aggtagtgga gttcctgttg tagattctta ttattcattg gatttcatac aaaccacgcc aggtagtgga gttcctgttg tagattctta ttattcattg 14160 14160 ttaatgccta tattaacctt gaccagggct ttaactgcag agtcacatgt tgacactgad

ttaatgccta tattaacctt gaccagggct ttaactgcag agtcacatgt tgacactgac 14220 14220 ttaacaaagc cttacattaa gtgggatttg ttaaaatatg acttcacgga agagaggtta

ttaacaaagc cttacattaa gtgggatttg ttaaaatatg acttcacgga agagaggtta 14280 14280 aaactctttg accgttattt taaatattgg gatcagacat accacccaaa ttgtgttaac aaactctttg accgttattt taaatattgg gatcagacat accacccaaa ttgtgttaac 14340 14340 tgtttggatg acagatgcat tctgcattgt gcaaacttta atgttttatt ctctacagtg

tgtttggatg acagatgcat tctgcattgt gcaaacttta atgttttatt ctctacagtg 14400 14400 ttcccaccta caagttttgg accactagte agaaaaatat ttgttgatgg tgttccattt

ttcccaccta caagttttgg accactagtg agaaaaatat ttgttgatgg tgttccattt 14460 14460 gtagtttcaa ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac gtagtttcaa ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac 14520 14520 ttacatagct ctagacttag ttttaaggaa ttacttgtgt atgctgctga ccctgctatg ttacatagct ctagacttag ttttaaggaa ttacttgtgt atgctgctga ccctgctatg 14580 14580 cacgctgctt ctggtaatct attactagat aaacgcacta cgtgcttttc agtagctgca cacgctgctt ctggtaatct attactagat aaacgcacta cgtgcttttc agtagctgca 14640 14640 cttactaaca atgttgcttt tcaaactgtc aaacccggta attttaacaa agacttctat

cttactaaca atgttgcttt tcaaactgtc aaacccggta attttaacaa agacttctat 14700 14700 gactttgctg tgtctaaggg tttctttaag gaaggaagtt ctgttgaatt aaaacactto

gactttgctg tgtctaaggg tttctttaag gaaggaagtt ctgttgaatt aaaacacttc 14760 14760 ttctttgctc aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta

ttctttgctc aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta 14820 14820 ccaacaatgt gtgatatcag acaactacta tttgtagttg aagttgttga taagtacttt ccaacaatgt gtgatatcag acaactacta tttgtagttg aagttgttga taagtacttt 14880

18

gattgttacg atggtggctg gattgttacg atggtggctg tattaatgct tattaatgct aaccaagtca aaccaagtca tcgtcaacaa tcgtcaacaa cctagacaaa cctagacaaa 14940 14940

tcagctggtt ttccatttaa tcagctggtt ttccatttaa taaatggggt taaatggggt aaggctagac aaggctagac tttattatga tttattatga ttcaatgagt ttcaatgagt 15000 15000

tatgaggato aagatgcact tttcgcatat acaaaacgta atgtcatccc tactataact tatgaggatc aagatgcact tttcgcatat acaaaacgta atgtcatccc tactataact 15060 15060

caaatgaatc ttaagtatgc caaatgaatc ttaagtatgo cattagtgca cattagtgca aagaatagag aagaatagag ctcgcaccgt ctcgcaccgt agctggtgtc agctggtgtc 15120 15120

tctatctgta gtactatgac caatagacag tttcatcaaa aattattgaa atcaatagcc tctatctgta gtactatgac caatagacag tttcatcaaa aattattgaa atcaatagcc 15180 15180

gccactagaggagctactgt gccactagag gagctactgt agtaattgga agtaattgga acaagcaaat acaagcaaat tctatggtgg tctatggtgg ttggcacaac ttggcacaac 15240 15240

atgttaaaaa ctgtttatag atgttaaaaa ctgtttatag tgatgtagaa tgatgtagaa aaccctcacc aaccctcacc ttatgggttg ttatgggttg ggattatcct ggattatcct 15300 15300

aaatgtgatagagccatgcc aaatgtgata gagccatgcc taacatgctt taacatgctt agaattatgg agaattatgg cctcacttgt cctcacttgt tcttgctcgc tcttgctcgc 15360 15360

aaacatacaa cgtgttgtag aaacatacaa cgtgttgtag cttgtcacac cttgtcacac cgtttctata cgtttctata gattagctaa gattagctaa tgagtgtgct tgagtgtgct 15420 15420

caagtattgagtgaaatggt caagtattga gtgaaatggt catgtgtggc catgtgtggc ggttcactat ggttcactat atgttaaacc atgttaaacc aggtggaacc aggtggaacc 15480 15480

tcatcaggag atgccacaac tcatcaggag atgccacaac tgcttatgct tgcttatgct aatagtgttt aatagtgttt ttaacatttg ttaacatttg tcaagctgtc tcaagctgtc 15540 15540

acggccaatg ttaatgcact acggccaatg ttaatgcact tttatctact tttatctact gatggtaaca gatggtaaca aaattgccga aaattgccga taagtatgtc taagtatgtc 15600 15600

cgcaatttac aacacagact cgcaatttac aacacagact ttatgagtgt ttatgagtgt ctctatagaa ctctatagaa atagagatgt atagagatgt tgacacagac tgacacagac 15660 15660

tttgtgaatg agttttacgc tttgtgaatg agttttacgc atatttgcgt atatttgcgt aaacatttct aaacatttct caatgatgat caatgatgat actctctgac actctctgac 15720 15720

gatgctgttg tgtgtttcaa gatgctgttg tgtgtttcaa tagcacttat tagcacttat gcatctcaag gcatctcaag gtctagtggc gtctagtggc tagcataaag tagcataaag 15780 15780

aactttaagt cagttcttta aactttaagt cagttcttta ttatcaaaac ttatcaaaac aatgttttta aatgttttta tgtctgaagc tgtctgaago aaaatgttgg aaaatgttgg 15840 15840

actgagactg accttactaa actgagactg accttactaa aggacctcat aggacctcat gaattttgct gaattttgct ctcaacatac ctcaacatac aatgctagtt aatgctagtt 15900 15900

aaacagggtg atgattatgt aaacagggtg atgattatgt gtaccttcct gtaccttcct tacccagatc tacccagatc catcaagaat catcaagaat cctaggggcc cctaggggco 15960 15960

ggctgttttg tagatgatat ggctgttttg tagatgatat cgtaaaaaca cgtaaaaaca gatggtacac gatggtacac ttatgattga ttatgattga acggttcgtg acggttcgtg 16020 16020

tctttagcta tagatgctta tctttagcta tagatgctta cccacttact cccacttact aaacatccta aaacatccta atcaggagta atcaggagta tgctgatgtc tgctgatgtc 16080 16080

tttcatttgt acttacaata tttcatttgt acttacaata cataagaaag cataagaaag ctacatgatg ctacatgatg agttaacagg agttaacagg acacatgtta acacatgtta 16140 16140

gacatgtatt ctgttatgct gacatgtatt ctgttatgct tactaatgat tactaatgat aacacttcaa aacacttcaa ggtattggga ggtattggga acctgagttt acctgagttt 16200

19

tatgaggcta tgtacacacc gcatacagto ttacaggctg ttggggcttg tgttctttgc

tatgaggcta tgtacacacc gcatacagtc ttacaggctg ttggggcttg tgttctttgc 16260 16260 aattcacaga cttcattaag atgtggtgct tgcatacgta gaccattctt atgttgtaaa

aattcacaga cttcattaag atgtggtgct tgcatacgta gaccattctt atgttgtaaa 16320 16320 tgctgttacg accatgtcat atcaacatca cataaattag tcttgtctgt taatcogtat

tgctgttacg accatgtcat atcaacatca cataaattag tcttgtctgt taatccgtat 16380 16380 gtttgcaatg ctccaggttg tgatgtcaca gatgtgacto aactttactt aggaggtatg gtttgcaatg ctccaggttg tgatgtcaca gatgtgactc aactttactt aggaggtatg 16440 16440 agctattatt gtaaatcaca taaaccacco attagttttc cattgtgtgc taatggacaa

agctattatt gtaaatcaca taaaccaccc attagttttc cattgtgtgc taatggacaa 16500 16500 gtttttggtt tatataaaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca

gtttttggtt tatataaaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca 16560 16560 attgcaacat gtgactggad aaatgctggt gattacattt tagctaacao ctgtactgaa

attgcaacat gtgactggac aaatgctggt gattacattt tagctaacac ctgtactgaa 16620 16620 agactcaago tttttgcago agaaacgctc aaagctactg aggagacatt taaactgtct

agactcaagc tttttgcagc agaaacgctc aaagctactg aggagacatt taaactgtct 16680 16680 tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct ttcatgggaa

tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct ttcatgggaa 16740 16740 gttggtaaac ctagaccaco acttaaccga aattatgtct ttactggtta tcgtgtaact

gttggtaaac ctagaccacc acttaaccga aattatgtct ttactggtta tcgtgtaact 16800 16800 aaaaacagta aagtacaaat aggagagtac acctttgaaa aaggtgacta tggtgatgct

aaaaacagta aagtacaaat aggagagtac acctttgaaa aaggtgacta tggtgatgct 16860 16860 gttgtttaco gaggtacaac aacttacaaa ttaaatgttg gtgattattt tgtgctgaca

gttgtttacc gaggtacaac aacttacaaa ttaaatgttg gtgattattt tgtgctgaca 16920 16920 tcacatacag taatgccatt aagtgcacct acactagtgo cacaagagca ctatgttaga

tcacatacag taatgccatt aagtgcacct acactagtgc cacaagagca ctatgttaga 16980 16980 attactggct tatacccaac actcaatato tcagatgagt tttctagcaa tgttgcaaat

attactggct tatacccaac actcaatatc tcagatgagt tttctagcaa tgttgcaaat 17040 17040 tatcaaaagg ttggtatgca aaagtattct acactccagg gaccacctgg tactggtaag

tatcaaaagg ttggtatgca aaagtattct acactccagg gaccacctgg tactggtaag 17100 17100 agtcattttg ctattggcct agctctctac tacccttctg ctcgcatagt gtatacagct

agtcattttg ctattggcct agctctctac tacccttctg ctcgcatagt gtatacagct 17160 17160 tgctctcatg ccgctgttga tgcactatgt gagaaggcat taaaatattt gcctatagat tgctctcatg ccgctgttga tgcactatgt gagaaggcat taaaatattt gcctatagat 17220 17220 aaatgtagta gaattataco tgcacgtgct cgtgtagagt gttttgataa attcaaagtg

aaatgtagta gaattatacc tgcacgtgct cgtgtagagt gttttgataa attcaaagtg 17280 17280 aattcaacat tagaacagta tgtcttttgt actgtaaatg cattgcctga gacgacagca

aattcaacat tagaacagta tgtcttttgt actgtaaatg cattgcctga gacgacagca 17340 17340 gatatagttg tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat

gatatagttg tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat 17400 17400 gccagattad gtgctaagca ctatgtgtac attggcgaco ctgctcaatt acctgcacca

gccagattac gtgctaagca ctatgtgtac attggcgacc ctgctcaatt acctgcacca 17460

20

cgcacattgc taactaaggg cacactagaa ccagaatatt tcaattcagt gtgtagactt cgcacattgc taactaaggg cacactagaa ccagaatatt tcaattcagt gtgtagactt 17520 17520

atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtco tgctgaaatt atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtcc tgctgaaatt 17580 17580

gttgacactg tgagtgcttt ggtttatgat aataagctta aagcacataa agacaaatca gttgacactg tgagtgcttt ggtttatgat aataagctta aagcacataa agacaaatca 17640 17640

gctcaatgct ttaaaatgtt ttataagggt gttatcacgc atgatgtttd atctgcaatt gctcaatgct ttaaaatgtt ttataagggt gttatcacgc atgatgtttc atctgcaatt 17700 17700

aacaggccac aaataggcgt ggtaagagaa ttccttacac gtaaccctgc ttggagaaaa aacaggccac aaataggcgt ggtaagagaa ttccttacac gtaaccctgc ttggagaaaa 17760 17760

gctgtcttta tttcacctta taattcacag aatgctgtag cctcaaagat tttgggacta gctgtcttta tttcacctta taattcacag aatgctgtag cctcaaagat tttgggacta 17820 17820

ccaactcaaa ctgttgattc atcacagggo tcagaatatg actatgtcat attcactcaa ccaactcaaa ctgttgattc atcacagggc tcagaatatg actatgtcat attcactcaa 17880 17880

accactgaaa cagctcactc ttgtaatgta aacagattta atgttgctat taccagagca accactgaaa cagctcactc ttgtaatgta aacagattta atgttgctat taccagagca 17940 17940

aaagtaggca tactttgcat aatgtctgat agagaccttt atgacaagtt gcaatttaca aaagtaggca tactttgcat aatgtctgat agagaccttt atgacaagtt gcaatttaca 18000 18000

agtcttgaaa ttccacgtag gaatgtggca actttacaag ctgaaaatgt aacaggacto agtcttgaaa ttccacgtag gaatgtggca actttacaag ctgaaaatgt aacaggactc 18060 18060

tttaaagatt gtagtaaggt aatcactggg ttacatccta cacaggcaco tacacaccto tttaaagatt gtagtaaggt aatcactggg ttacatccta cacaggcacc tacacacctc 18120 18120

agtgttgaca ctaaattcaa aactgaaggt ttatgtgttg acatacctgg catacctaag agtgttgaca ctaaattcaa aactgaaggt ttatgtgttg acatacctgg catacctaag 18180 18180

gacatgacct atagaagact catctctatg atgggtttta aaatgaatta tcaagttaat gacatgacct atagaagact catctctatg atgggtttta aaatgaatta tcaagttaat 18240 18240

ggttacccta acatgtttat cacccgcgaa gaagctataa gacatgtacg tgcatggatt ggttacccta acatgtttat cacccgcgaa gaagctataa gacatgtacg tgcatggatt 18300 18300

ggcttcgatg tcgaggggtg tcatgctact agagaagctg ttggtaccaa tttaccttta ggcttcgatg tcgaggggtg tcatgctact agagaagctg ttggtaccaa tttaccttta 18360 18360

cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta tgttgataca cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta tgttgataca 18420 18420

cctaataata cagatttttc cagagttagt gctaaaccac cgcctggaga tcaatttaaa cctaataata cagatttttc cagagttagt gctaaaccac cgcctggaga tcaatttaaa 18480 18480

cacctcatac cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta cacctcatac cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta 18540 18540

caaatgttaa gtgacacact taaaaatctc tctgacagag tcgtatttgt cttatgggca caaatgttaa gtgacacact taaaaatctc tctgacagag tcgtatttgt cttatgggca 18600 18600

catggctttg agttgacato tatgaagtat tttgtgaaaa taggacctga gcgcacctgt catggctttg agttgacatc tatgaagtat tttgtgaaaa taggacctga gcgcacctgt 18660 18660

tgtctatgtg atagacgtgc cacatgcttt tccactgctt cagacactta tgcctgttgg tgtctatgtg atagacgtgc cacatgcttt tccactgctt cagacactta tgcctgttgg 18720 18720

catcattcta ttggatttga ttacgtctat aatccgttta tgattgatgt tcaacaatgg catcattcta ttggatttga ttacgtctat aatccgttta tgattgatgt tcaacaatgg 18780

21

ggttttacag gtaacctaca aagcaaccat gatctgtatt gtcaagtcca tggtaatgca

ggttttacag gtaacctaca aagcaaccat gatctgtatt gtcaagtcca tggtaatgca 18840 18840 catgtagcta gttgtgatgc aatcatgact aggtgtctag ctgtccacga gtgctttgtt

catgtagcta gttgtgatgc aatcatgact aggtgtctag ctgtccacga gtgctttgtt 18900 18900 aagcgtgttg actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg

aagcgtgttg actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg 18960 18960 gcttgtagaa aggttcaaca catggttgtt aaagctgcat tattagcaga caaattccca

gcttgtagaa aggttcaaca catggttgtt aaagctgcat tattagcaga caaattccca 19020 19020 gttcttcacg acattggtaa ccctaaagct attaagtgtg tacctcaagc tgatgtagaa

gttcttcacg acattggtaa ccctaaagct attaagtgtg tacctcaagc tgatgtagaa 19080 19080 tggaagttct atgatgcaca gccttgtagt gacaaagctt ataaaataga agaattatto

tggaagttct atgatgcaca gccttgtagt gacaaagctt ataaaataga agaattattc 19140 19140 tattcttatg ccacacatto tgacaaatto acagatggtg tatgcctatt ttggaattgo

tattcttatg ccacacattc tgacaaattc acagatggtg tatgcctatt ttggaattgc 19200 19200 aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag agtgctatct

aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag agtgctatct 19260 19260 aaccttaact tgcctggttg tgatggtggc agtttgtatg taaataaaca tgcattccao

aaccttaact tgcctggttg tgatggtggc agtttgtatg taaataaaca tgcattccac 19320 19320 acaccagctt ttgataaaag tgcttttgtt aatttaaaac aattaccatt tttctattac

acaccagctt ttgataaaag tgcttttgtt aatttaaaac aattaccatt tttctattac 19380 19380 tctgacagtc catgtgagto tcatggaaaa caagtagtgt cagatataga ttatgtacca

tctgacagtc catgtgagtc tcatggaaaa caagtagtgt cagatataga ttatgtacca 19440 19440 ctaaagtctg ctacgtgtat aacacgttgc aatttaggtg gtgctgtctg tagacatcat

ctaaagtctg ctacgtgtat aacacgttgc aatttaggtg gtgctgtctg tagacatcat 19500 19500 gctaatgagt acagattgta tctcgatgct tataacatga tgatctcago tggctttago gctaatgagt acagattgta tctcgatgct tataacatga tgatctcagc tggctttagc 19560 19560 ttgtgggttt acaaacaatt tgatacttat aacctctgga acacttttac aagacttcag

ttgtgggttt acaaacaatt tgatacttat aacctctgga acacttttac aagacttcag 19620 19620 agtttagaaa atgtggcttt taatgttgta aataagggad actttgatgg acaacagggt

agtttagaaa atgtggcttt taatgttgta aataagggac actttgatgg acaacagggt 19680 19680 gaagtaccag tttctatcat taataacact gtttacacaa aagttgatgg tgttgatgta

gaagtaccag tttctatcat taataacact gtttacacaa aagttgatgg tgttgatgta 19740 19740 gaattgtttg aaaataaaac aacattacct gttaatgtag catttgagct ttgggctaag

gaattgtttg aaaataaaac aacattacct gttaatgtag catttgagct ttgggctaag 19800 19800 cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt ggacattgct

cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt ggacattgct 19860 19860 gctaatactg tgatctggga ctacaaaaga gatgctccag cacatatato tactattggt

gctaatactg tgatctggga ctacaaaaga gatgctccag cacatatatc tactattggt 19920 19920 gtttgttcta tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact

gtttgttcta tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact 19980 19980 gtcttttttg atggtagagt tgatggtcaa gtagacttat ttagaaatgo ccgtaatggt

gtcttttttg atggtagagt tgatggtcaa gtagacttat ttagaaatgc ccgtaatggt 20040

22

gttcttatta cagaaggtag tgttaaaggt ttacaaccat ctgtaggtcc caaacaagct gttcttatta cagaaggtag tgttaaaggt ttacaaccat ctgtaggtcc caaacaagct 20100 20100

agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa ttattataag agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa ttattataag 20160 20160

aaagttgatg gtgttgtcca acaattacct gaaacttact ttactcagag tagaaattta aaagttgatg gtgttgtcca acaattacct gaaacttact ttactcagag tagaaattta 20220 20220

caagaattta aacccaggag tcaaatggaa attgatttct tagaattago tatggatgaa caagaattta aacccaggag tcaaatggaa attgatttct tagaattagc tatggatgaa 20280 20280

ttcattgaac ggtataaatt agaaggctat gccttcgaac atatcgttta tggagatttt ttcattgaac ggtataaatt agaaggctat gccttcgaac atatcgttta tggagatttt 20340 20340

agtcatagtc agttaggtgg tttacatcta ctgattggad tagctaaacg ttttaaggaa agtcatagtc agttaggtgg tttacatcta ctgattggac tagctaaacg ttttaaggaa 20400 20400

tcaccttttg aattagaaga ttttattcct atggacagta cagttaaaaa ctatttcata tcaccttttg aattagaaga ttttattcct atggacagta cagttaaaaa ctatttcata 20460 20460

acagatgcgc aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat acagatgcgc aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat 20520 20520

gattttgttg aaataataaa atcccaagat ttatctgtag tttctaaggt tgtcaaagtg gattttgttg aaataataaa atcccaagat ttatctgtag tttctaaggt tgtcaaagtg 20580 20580

actattgact atacagaaat ttcatttatg ctttggtgta aagatggcca tgtagaaaca actattgact atacagaaat ttcatttatg ctttggtgta aagatggcca tgtagaaaca 20640 20640

ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat gcctaatctt ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat gcctaatctt 20700 20700

tacaaaatgc aaagaatgct attagaaaag tgtgacctto aaaattatgg tgatagtgca tacaaaatgc aaagaatgct attagaaaag tgtgaccttc aaaattatgg tgatagtgca 20760 20760

acattaccta aaggcataat gatgaatgto gcaaaatata ctcaactgtg tcaatattta acattaccta aaggcataat gatgaatgtc gcaaaatata ctcaactgtg tcaatattta 20820 20820

aacacattaa cattagctgt accctataat atgagagtta tacattttgg tgctggttct aacacattaa cattagctgt accctataat atgagagtta tacattttgg tgctggttct 20880 20880

gataaaggag ttgcaccagg tacagctgtt ttaagacagt ggttgcctac gggtacgctg gataaaggag ttgcaccagg tacagctgtt ttaagacagt ggttgcctac gggtacgctg 20940 20940

cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt gattggtgat cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt gattggtgat 21000 21000

tgtgcaactg tacatacago taataaatgg gatctcatta ttagtgatat gtacgaccct tgtgcaactg tacatacagc taataaatgg gatctcatta ttagtgatat gtacgaccct 21060 21060

aagactaaaa atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt aagactaaaa atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt 21120 21120

gggtttatac aacaaaagct agctcttgga ggttccgtgg ctataaagat aacagaacat gggtttatac aacaaaagct agctcttgga ggttccgtgg ctataaagat aacagaacat 21180 21180

tcttggaatg ctgatcttta taagctcatg ggacacttcg catggtggac agcctttgtt tcttggaatg ctgatcttta taagctcatg ggacacttcg catggtggac agcctttgtt 21240 21240

actaatgtga atgcgtcato atctgaagca tttttaattg gatgtaatta tcttggcaaa actaatgtga atgcgtcatc atctgaagca tttttaattg gatgtaatta tcttggcaaa 21300 21300

ccacgcgaac aaatagatgg ttatgtcatg catgcaaatt acatattttg gaggaataca ccacgcgaac aaatagatgg ttatgtcatg catgcaaatt acatattttg gaggaataca 21360

23

aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc ccttaaatta aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc ccttaaatta 21420 21420

aggggtactg ctgttatgtc tttaaaagaa ggtcaaatca atgatatgat tttatctctt aggggtactg ctgttatgtc tttaaaagaa ggtcaaatca atgatatgat tttatctctt 21480 21480

cttagtaaag gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt cttagtaaag gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt 21540 21540

cttgttaaca actaaacgaa caatgtttgt ttttcttgtt ttattgccac tagtctctag cttgttaaca actaaacgaa caatgtttgt ttttcttgtt ttattgccac tagtctctag 21600 21600

tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac tcagtgtgtt aatcttacaa ccagaactca attaccccct gcatacacta attctttcac 21660 21660

acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga acgtggtgtt tattaccctg acaaagtttt cagatcctca gttttacatt caactcagga 21720 21720

cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggad cttgttctta cctttctttt ccaatgttac ttggttccat gctatacatg tctctgggac 21780 21780

caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc caatggtact aagaggtttg ataaccctgt cctaccattt aatgatggtg tttattttgc 21840 21840

ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa ttccactgag aagtctaaca taataagagg ctggattttt ggtactactt tagattcgaa 21900 21900

gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt gacccagtcc ctacttattg ttaataacgc tactaatgtt gttattaaag tctgtgaatt 21960 21960

tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat tcaattttgt aatgatccat ttttgggtgt ttattaccac aaaaacaaca aaagttggat 22020 22020

ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca 22080 22080

gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt 22140 22140

gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt 22200 22200

gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgo caataggtat gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat 22260 22260

taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga 22320 22320

ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag 22380 22380

gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtgcact 22440 22440

tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc actgtagaaa aaggaatcta 22500 22500

tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac tcaaacttct aactttagag tccaaccaac agaatctatt gttagatttc ctaatattac 22560 22560

aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg aaacttgtgc ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg 22620

24

gaacaggaagagaatcagca gaacaggaag agaatcagca actgtgttgc actgtgttgc tgattattct tgattattct gtcctatata gtcctatata attccgcatc attccgcatc 22680 22680

attttccacttttaagtgtt attttccact tttaagtgtt atggagtgtc atggagtgtc tcctactaaa tcctactaaa ttaaatgatc ttaaatgatc tctgctttac tctgctttac 22740 22740

taatgtctatgcagattcat taatgtctat gcagattcat ttgtaattag ttgtaattag aggtgatgaa aggtgatgaa gtcagacaaa gtcagacaaa tcgctccagg tcgctccagg 22800 22800

gcaaactggaaagattgctg gcaaactgga aagattgctg attataatta attataatta taaattacca taaattacca gatgatttta gatgatttta caggctgcgt caggctgcgt 22860 22860

tatagcttgg aattctaaca tatagcttgg aattctaaca atcttgattc atcttgattc taaggttggt taaggttggt ggtaattata ggtaattata attacctgta attacctgta 22920 22920

tagattgttt aggaagtcta tagattgttt aggaagtcta atctcaaacc atctcaaacc ttttgagaga ttttgagaga gatatttcaa gatatttcaa ctgaaatcta ctgaaatcta 22980 22980

tcaggccggt agcacacctt tcaggccggt agcacacctt gtaatggtgt gtaatggtgt tgaaggtttt tgaaggtttt aattgttact aattgttact ttcctttaca ttcctttaca 23040 23040

atcatatggt ttccaaccca atcatatggt ttccaaccca ctaatggtgt ctaatggtgt tggttaccaa tggttaccaa ccatacagag ccatacagag tagtagtact tagtagtact 23100 23100

ttcttttgaa cttctacatg ttcttttgaa cttctacatg caccagcaac caccagcaac tgtttgtgga tgtttgtgga cctaaaaagt cctaaaaagt ctactaattt ctactaattt 23160 23160

ggttaaaaacaaatgtgtca ggttaaaaac aaatgtgtca atttcaactt atttcaactt caatggttta caatggttta acaggcacag acaggcacag gtgttcttac gtgttcttac 23220 23220

tgagtctaacaaaaagtttc tgagtctaac aaaaagtttc tgcctttcca tgcctttcca acaatttggc acaatttggc agagacattg agagacattg ctgacactac ctgacactac 23280 23280

tgatgctgtccgtgatccac tgatgctgtc cgtgatccac agacacttga agacacttga gattcttgac gattcttgac attacaccat attacaccat gttcttttgg gttcttttgg 23340 23340

tggtgtcagt gttataacac tggtgtcagt gttataacac caggaacaaa caggaacaaa tacttctaac tacttctaac caggttgctg caggttgctg ttctttatca ttctttatca 23400 23400

ggatgttaactgcacagaag ggatgttaac tgcacagaag tccctgttgc tccctgttgc tattcatgca tattcatgca gatcaactta gatcaactta ctcctacttg ctcctacttg 23460 23460

gcgtgtttattctacaggtt gcgtgtttat tctacaggtt ctaatgtttt ctaatgtttt tcaaacacgt tcaaacacgt gcaggctgtt gcaggctgtt taataggggc taataggggc 23520 23520

tgaacatgtcaacaactcat tgaacatgtc aacaactcat atgagtgtga atgagtgtga catacccatt catacccatt ggtgcaggta ggtgcaggta tatgcgctag tatgcgctag 23580 23580

ttatcagact cagactaatt ttatcagact cagactaatt ctcctcggcg ctcctcggcg ggcacgtagt ggcacgtagt gtagctagtc gtagctagtc aatccatcat aatccatcat 23640 23640

tgcctacact atgtcacttg tgcctacact atgtcacttg gtgcagaaaa gtgcagaaaa ttcagttgct ttcagttgct tactctaata tactctaata actctattgc actctattgo 23700 23700

catacccacaaattttacta catacccaca aattttacta ttagtgttac ttagtgttac cacagaaatt cacagaaatt ctaccagtgt ctaccagtgt ctatgaccaa ctatgaccaa 23760 23760

gacatcagtagattgtacaa gacatcagta gattgtacaa tgtacatttg tgtacatttg tggtgattca tggtgattca actgaatgca actgaatgca gcaatctttt gcaatctttt 23820 23820

gttgcaatat ggcagttttt gttgcaatat ggcagttttt gtacacaatt gtacacaatt aaaccgtgct aaaccgtgct ttaactggaa ttaactggaa tagctgttga tagctgttga 23880 23880

acaagacaaaaacacccaag acaagacaaa aacacccaag aagtttttgc aagtttttgc acaagtcaaa acaagtcaaa caaatttaca caaatttaca aaacaccacc aaacaccacc 23940

25

aattaaagat tttggtggtt ttaattttto acaaatatta ccagatccat caaaaccaag aattaaagat tttggtggtt ttaatttttc acaaatatta ccagatccat caaaaccaag 24000 24000

caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt caagaggtca tttattgaag atctactttt caacaaagtg acacttgcag atgctggctt 24060 24060

catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca catcaaacaa tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca 24120 24120

aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata aaagtttaac ggccttactg ttttgccacc tttgctcaca gatgaaatga ttgctcaata 24180 24180

cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgo cacttctgca ctgttagcgg gtacaatcac ttctggttgg acctttggtg caggtgctgc 24240 24240

attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg gagttacaca 24300 24300

gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa gaatgttctc tatgagaacc aaaaattgat tgccaaccaa tttaatagtg ctattggcaa 24360 24360

aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa aattcaagac tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa 24420 24420

ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat ccaaaatgca caagctttaa acacgcttgt taaacaactt agctccaatt ttggtgcaat 24480 24480

ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat 24540 24540

tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgacto aacaattaat tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc aacaattaat 24600 24600

tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt tagagctgca gaaatcagag cttctgctaa tcttgctgct actaaaatgt cagagtgtgt 24660 24660

acttggacaa tcaaaaagag ttgatttttg tggaaagggo tatcatctta tgtccttccc acttggacaa tcaaaaagag ttgatttttg tggaaagggc tatcatctta tgtccttccc 24720 24720

tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa tcagtcagca cctcatggtg tagtcttctt gcatgtgact tatgtccctg cacaagaaaa 24780 24780

gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacacttto ctcgtgaagg gaacttcaca actgctcctg ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg 24840 24840

tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca tgtctttgtt tcaaatggca cacactggtt tgtaacacaa aggaattttt atgaaccaca 24900 24900

aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt aatcattact acagacaaca catttgtgtc tggtaactgt gatgttgtaa taggaattgt 24960 24960

caacaacaca gtttatgato ctttgcaaco tgaattagac tcattcaagg aggagttaga caacaacaca gtttatgatc ctttgcaacc tgaattagac tcattcaagg aggagttaga 25020 25020

taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa taaatatttt aagaatcata catcaccaga tgttgattta ggtgacatct ctggcattaa 25080 25080

tgcttcagtt gtaaacatto aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt 25140 25140

aaatgaatct ctcatcgato tccaagaact tggaaagtat gagcagtata taaaatggcc aaatgaatct ctcatcgatc tccaagaact tggaaagtat gagcagtata taaaatggcc 25200

26

atggtacatt tggctaggtt atggtacatt tggctaggtt ttatagctgg ttatagctgg cttgattgcc cttgattgcc atagtaatgg atagtaatgg tgacaattat tgacaattat 25260 25260

gctttgctgt atgaccagtt gctttgctgt atgaccagtt gctgtagttg gctgtagttg tctcaagggc tctcaagggc tgttgttctt tgttgttctt gtggatcctg gtggatcctg 25320 25320

ctgcaaatttgatgaagacg ctgcaaattt gatgaagacg actctgagcc actctgagcc agtgctcaaa agtgctcaaa ggagtcaaat ggagtcaaat tacattacac tacattacac 25380 25380

ataaacgaacttatggattt ataaacgaac ttatggattt gtttatgaga gtttatgaga atcttcacaa atcttcacaa ttggaactgt ttggaactgt aactttgaag aactttgaag 25440 25440

caaggtgaaatcaaggatgc caaggtgaaa tcaaggatgc tactccttca tactccttca gattttgttc gattttgttc gcgctactgc gcgctactgc aacgataccg aacgataccg 25500 25500

atacaagcct cactcccttt atacaagcct cactcccttt cggatggctt cggatggctt attgttggcg attgttggcg ttgcacttct ttgcacttct tgctgttttt tgctgttttt 25560 25560

cagagcgctt ccaaaatcat cagagcgctt ccaaaatcat aaccctcaaa aaccctcaaa aagagatggc aagagatggc aactagcact aactagcact ctccaagggt ctccaagggt 25620 25620

gttcactttgtttgcaactt gttcactttg tttgcaactt gctgttgttg gctgttgttg tttgtaacag tttgtaacag tttactcaca tttactcaca ccttttgctc ccttttgctc 25680 25680

gttgctgctg gccttgaagc gttgctgctg gccttgaagc cccttttctc cccttttctc tatctttatg tatctttatg ctttagtcta ctttagtcta cttcttgcag cttcttgcag 25740 25740

agtataaactttgtaagaat agtataaact ttgtaagaat aataatgagg aataatgagg ctttggcttt ctttggcttt gctggaaatg gctggaaatg ccgttccaaa ccgttccaaa 25800 25800

aacccattac tttatgatgc aacccattac tttatgatgc caactatttt caactatttt ctttgctggc ctttgctggc atactaattg atactaattg ttacgactat ttacgactat 25860 25860

tgtataccttacaatagtgt tgtatacctt acaatagtgt aacttcttca aacttcttca attgtcatta attgtcatta cttcaggtga cttcaggtga tggcacaaca tggcacaaca 25920 25920

agtcctattt ctgaacatga agtcctattt ctgaacatga ctaccagatt ctaccagatt ggtggttata ggtggttata ctgaaaaatg ctgaaaaatg ggaatctgga ggaatctgga 25980 25980

gtaaaagact gtgttgtatt gtaaaagact gtgttgtatt acacagttac acacagttac ttcacttcag ttcacttcag actattacca actattacca gctgtactca gctgtactca 26040 26040

actcaattga gtacagacac actcaattga gtacagacac tggtgttgaa tggtgttgaa catgttacct catgttacct tcttcatcta tcttcatcta caataaaatt caataaaatt 26100 26100

gttgatgagcctgaagaaca gttgatgagc ctgaagaaca tgtccaaatt tgtccaaatt cacacaatcg cacacaatcg acggttcatc acggttcatc cggagttgtt cggagttgtt 26160 26160

aatccagtaatggaaccaat aatccagtaa tggaaccaat ttatgatgaa ttatgatgaa ccgacgacga ccgacgacga ctactagcgt ctactagcgt gcctttgtaa gcctttgtaa 26220 26220

gcacaagctgatgagtacga gcacaagctg atgagtacga acttatgtac acttatgtac tcattcgttt tcattcgttt cggaagagac cggaagagac aggtacgtta aggtacgtta 26280 26280

atagttaatagcgtacttct atagttaata gcgtacttct ttttcttgct ttttcttgct ttcgtggtat ttcgtggtat tcttgctagt tcttgctagt tacactagcc tacactagcc 26340 26340

atccttactgcgcttcgatt atccttactg cgcttcgatt gtgtgcgtac gtgtgcgtac tgctgcaata tgctgcaata ttgttaacgt ttgttaacgt gagtcttgta gagtcttgta 26400 26400

aaaccttctttttacgttta aaaccttctt tttacgttta ctctcgtgtt ctctcgtgtt aaaaatctga aaaaatctga attcttctag attcttctag agttcctgat agttcctgat 26460 26460

cttctggtctaaacgaacta cttctggtct aaacgaacta aatattatat aatattatat tagtttttct tagtttttct gtttggaact gtttggaact ttaattttag ttaattttag 26520

27

ccatggcagattccaacggt ccatggcaga ttccaacggt actattaccg actattaccg ttgaagagct ttgaagagct taaaaagctc taaaaagctc cttgaacaat cttgaacaat 26580 26580

ggaacctagt aataggtttc ggaacctagt aataggtttc ctattcctta ctattcctta catggatttg catggatttg tcttctacaa tcttctacaa tttgcctatg tttgcctatg 26640 26640

ccaacaggaataggtttttg ccaacaggaa taggtttttg tatataatta tatataatta agttaatttt agttaatttt cctctggctg cctctggctg ttatggccag ttatggccag 26700 26700

taactttagcttgttttgtg taactttagc ttgttttgtg cttgctgctg cttgctgctg tttacagaat tttacagaat aaattggatc aaattggatc accggtggaa accggtggaa 26760 26760

ttgctatcgcaatggcttgt ttgctatcgc aatggcttgt cttgtaggct cttgtaggct tgatgtggct tgatgtggct cagctacttc cagctactto attgcttctt attgcttctt 26820 26820

tcagactgtttgcgcgtacg tcagactgtt tgcgcgtacg cgttccatgt cgttccatgt ggtcattcaa ggtcattcaa tccagaaact tccagaaact aacattcttc aacattcttc 26880 26880

tcaacgtgccactccatggc tcaacgtgcc actccatggc actattctga actattctga ccagaccgct ccagaccgct tctagaaagt tctagaaagt gaactcgtaa gaactcgtaa 26940 26940

tcggagctgtgatccttcgt tcggagctgt gatccttcgt ggacatcttc ggacatcttc gtattgctgg gtattgctgg acaccatcta acaccatcta ggacgctgtg ggacgctgtg 27000 27000

acatcaaggacctgcctaaa acatcaagga cctgcctaaa gaaatcactg gaaatcactg ttgctacatc ttgctacatc acgaacgctt acgaacgctt tcttattaca tcttattaca 27060 27060

aattgggagcttcgcagcgt aattgggagc ttcgcagcgt gtagcaggtg gtagcaggtg actcaggttt actcaggttt tgctgcatac tgctgcatac agtcgctaca agtcgctaca 27120 27120

ggattggcaactataaatta ggattggcaa ctataaatta aacacagacc aacacagacc attccagtag attccagtag cagtgacaat cagtgacaat attgctttgc attgctttgc 27180 27180

ttgtacagtaagtgacaaca ttgtacagta agtgacaaca gatgtttcat gatgtttcat ctcgttgact ctcgttgact ttcaggttac ttcaggttac tatagcagag tatagcagag 27240 27240

atattactaattattatgag atattactaa ttattatgag gacttttaaa gacttttaaa gtttccattt gtttccattt ggaatcttga ggaatcttga ttacatcata ttacatcata 27300 27300

aacctcataattaaaaattt aacctcataa ttaaaaattt atctaagtca atctaagtca ctaactgaga ctaactgaga ataaatattc ataaatattc tcaattagat tcaattagat 27360 27360

gaagagcaaccaatggagat gaagagcaac caatggagat tgattaaacg tgattaaacg aacatgaaaa aacatgaaaa ttattctttt ttattctttt cttggcactg cttggcactg 27420 27420

ataacactcgctacttgtga ataacactcg ctacttgtga gctttatcac gctttatcac taccaagagt taccaagagt gtgttagagg gtgttagagg tacaacagta tacaacagta 27480 27480

cttttaaaagaaccttgctc cttttaaaag aaccttgctc ttctggaaca ttctggaaca tacgagggca tacgagggca attcaccatt attcaccatt tcatcctcta tcatcctcta 27540 27540

gctgataacaaatttgcact gctgataaca aatttgcact gacttgcttt gacttgcttt agcactcaat agcactcaat ttgcttttgc ttgcttttgc ttgtcctgac ttgtcctgac 27600 27600

ggcgtaaaac acgtctatca ggcgtaaaac acgtctatca gttacgtgcc gttacgtgcc agatcagttt agatcagttt cacctaaact cacctaaact gttcatcaga gttcatcaga 27660 27660

caagaggaagttcaagaact caagaggaag ttcaagaact ttactctcca ttactctcca atttttctta atttttctta ttgttgcggc ttgttgcggc aatagtgttt aatagtgttt 27720 27720

ataacactttgcttcacact ataacacttt gcttcacact caaaagaaag caaaagaaag acagaatgat acagaatgat tgaactttca tgaactttca ttaattgact ttaattgact 27780

28

tctatttgtgctttttagcc tctatttgtg ctttttagcc tttctgctat tttctgctat tccttgtttt tccttgtttt aattatgctt aattatgctt attatctttt attatctttt 27840 27840

ggttctcact tgaactgcaa ggttctcact tgaactgcaa gatcataatg gatcataatg aaacttgtca aaacttgtca cgcctaaacg cgcctaaacg aacatgaaat aacatgaaat 27900 27900

ttcttgtttt cttaggaatc ttcttgtttt cttaggaatc atcacaactg atcacaactg tagctgcatt tagctgcatt tcaccaagaa tcaccaagaa tgtagtttac tgtagtttac 27960 27960

agtcatgtactcaacatcaa agtcatgtac tcaacatcaa ccatatgtag ccatatgtag ttgatgaccc ttgatgaccc gtgtcctatt gtgtcctatt cacttctatt cacttctatt 28020 28020

ctaaatggtatattagagta ctaaatggta tattagagta ggagctagaa ggagctagaa aatcagcacc aatcagcacc tttaattgaa tttaattgaa ttgtgcgtgg ttgtgcgtgg 28080 28080

atgaggctgg ttctaaatca atgaggctgg ttctaaatca cccattcagt cccattcagt acatcgatat acatcgatat cggtaattat cggtaattat acagtttcct acagtttcct 28140 28140

gtttaccttt tacaattaat gtttaccttt tacaattaat tgccaggaac tgccaggaac ctaaattggg ctaaattggg tagtcttgta tagtcttgta gtgcgttgtt gtgcgttgtt 28200 28200

cgttctatgaagacttttta cgttctatga agacttttta gagtatcatg gagtatcatg acgttcgtgt acgttcgtgt tgttttagat tgttttagat ttcatctaaa ttcatctaaa 28260 28260

cgaacaaact aaaatgtctg cgaacaaact aaaatgtctg ataatggacc ataatggacc ccaaaatcag ccaaaatcag cgaaatgcac cgaaatgcac cccgcattac cccgcattac 28320 28320

gtttggtggaccctcagatt gtttggtgga ccctcagatt caactggcag caactggcag taaccagaat taaccagaat ggagaacgca ggagaacgca gtggggcgcg gtggggcgcg 28380 28380

atcaaaacaacgtcggcccc atcaaaacaa cgtcggcccc aaggtttacc aaggtttacc caataatact caataatact gcgtcttggt gcgtcttggt tcaccgctct tcaccgctct 28440 28440

cactcaacatggcaaggaag cactcaacat ggcaaggaag accttaaatt accttaaatt ccctcgagga ccctcgagga caaggcgttc caaggcgttc caattaacac caattaacac 28500 28500

caatagcagtccagatgacc caatagcagt ccagatgacc aaattggcta aaattggcta ctaccgaaga ctaccgaaga gctaccagac gctaccagac gaattcgtgg gaattcgtgg 28560 28560

tggtgacggt aaaatgaaag tggtgacggt aaaatgaaag atctcagtcc atctcagtcc aagatggtat aagatggtat ttctactacc ttctactacc taggaactgg taggaactgg 28620 28620

gccagaagct ggacttccct gccagaagct ggacttccct atggtgctaa atggtgctaa caaagacggc caaagacggc atcatatggg atcatatggg ttgcaactga ttgcaactga 28680 28680

gggagccttg aatacaccaa gggagccttg aatacaccaa aagatcacat aagatcacat tggcacccgc tggcacccgc aatcctgcta aatcctgcta acaatgctgc acaatgctgc 28740 28740

aatcgtgcta caacttcctc aatcgtgcta caacttcctc aaggaacaac aaggaacaac attgccaaaa attgccaaaa ggcttctacg ggcttctacg cagaagggag cagaagggag 28800 28800

cagaggcggcagtcaagcct cagaggcggc agtcaagcct cttctcgttc cttctcgttc ctcatcacgt ctcatcacgt agtcgcaaca agtcgcaaca gttcaagaaa gttcaagaaa 28860 28860

ttcaactcca ggcagcagta ttcaactcca ggcagcagta ggggaacttc ggggaacttc tcctgctaga tcctgctaga atggctggca atggctggca atggcggtga atggcggtga 28920 28920

tgctgctctt gctttgctgc tgctgctctt gctttgctgc tgcttgacag tgcttgacag attgaaccag attgaaccag cttgagagca cttgagagca aaatgtctgg aaatgtctgg 28980 28980

taaaggccaacaacaacaag taaaggccaa caacaacaag gccaaactgt gccaaactgt cactaagaaa cactaagaaa tctgctgctg tctgctgctg aggcttctaa aggcttctaa 29040 29040

gaagcctcgg caaaaacgta gaagcctcgg caaaaacgta ctgccactaa ctgccactaa agcatacaat agcatacaat gtaacacaag gtaacacaag ctttcggcag ctttcggcag 29100

29

acgtggtccagaacaaaccc acgtggtcca gaacaaaccc aaggaaattt aaggaaattt tggggaccag tggggaccag gaactaatca gaactaatca gacaaggaac gacaaggaac 29160 29160

tgattacaaa cattggccgc tgattacaaa cattggccgc aaattgcaca aaattgcaca atttgccccc atttgccccc agcgcttcag agcgcttcag cgttcttcgg cgttcttcgg 29220 29220

aatgtcgcgcattggcatgg aatgtcgcgc attggcatgg aagtcacacc aagtcacacc ttcgggaacg ttcgggaacg tggttgacct tggttgacct acacaggtgc acacaggtgc 29280 29280

catcaaattggatgacaaag catcaaattg gatgacaaag atccaaattt atccaaattt caaagatcaa caaagatcaa gtcattttgc gtcattttgc tgaataagca tgaataagca 29340 29340

tattgacgcatacaaaacat tattgacgca tacaaaacat tcccaccaac tcccaccaac agagcctaaa agagcctaaa aaggacaaaa aaggacaaaa agaagaaggc agaagaaggo 29400 29400

tgatgaaactcaagccttac tgatgaaact caagccttac cgcagagaca cgcagagaca gaagaaacag gaagaaacag caaactgtga caaactgtga ctcttcttcc ctcttcttcc 29460 29460

tgctgcagat ttggatgatt tgctgcagat ttggatgatt tctccaaaca tctccaaaca attgcaacaa attgcaacaa tccatgagca tccatgagca gtgctgactc gtgctgactc 29520 29520

aactcaggcc taaactcatg aactcaggcc taaactcatg cagaccacac cagaccacac aaggcagatg aaggcagatg ggctatataa ggctatataa acgttttcgc acgttttcgc 29580 29580

ttttccgttt acgatatata ttttccgttt acgatatata gtctactctt gtctactctt gtgcagaatg gtgcagaatg aattctcgta aattctcgta actacatagc actacatage 29640 29640

acaagtagatgtagttaact acaagtagat gtagttaact ttaatctcac ttaatctcac atagcaatct atagcaatct ttaatcagtg ttaatcagtg tgtaacatta tgtaacatta 29700 29700

gggaggacttgaaagagcca gggaggactt gaaagagcca ccacattttc ccacattttc accgaggcca accgaggcca cgcggagtac cgcggagtac gatcgagtgt gatcgagtgt 29760 29760

acagtgaaca atgctaggga acagtgaaca atgctaggga gagctgccta gagctgccta tatggaagag tatggaagag ccctaatgtg ccctaatgtg taaaattaat taaaattaat 29820 29820

tttagtagtgctatccccat tttagtagtg ctatccccat gtgattttaa gtgattttaa tagcttctta tagcttctta ggagaatgac ggagaatgac aaaaaaaaaa aaaaaaaaaa 29880 29880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 29903 29903

<210> <210> 17 17 <211> <211> 1273 1273 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 17 17

Met Phe Met Phe Val ValPhe PheLeu Leu ValVal LeuLeu Leu Leu Pro Pro Leu Ser Leu Val Val Ser SerGln SerCys Gln ValCys Val 1 1 5 5 10 10 15 15

Asn Leu Asn Leu Thr Thr Thr Thr Arg Arg Thr Thr Gln Gln Leu Leu Pro Pro Pro Pro Ala Ala Tyr Tyr Thr Thr Asn Asn Ser Ser Phe Phe 20 20 25 25 30

30

Thr Arg Thr Arg Gly GlyVal ValTyr Tyr TyrTyr ProPro Asp Asp Lys Lys Val Arg Val Phe Phe Ser ArgSer SerVal Ser LeuVal Leu 35 35 40 40 45 45

His Ser His Ser Thr ThrGln GlnAsp Asp LeuLeu PhePhe Leu Leu Pro Pro Phe Ser Phe Phe Phe Asn SerVal AsnThr Val TrpThr Trp 50 50 55 55 60 60

Phe His Phe His Ala AlaIle IleHis His ValVal SerSer Gly Gly Thr Thr Asn Thr Asn Gly Gly Lys ThrArg LysPhe Arg AspPhe Asp

70 70 75 75 80 80

Asn Pro Asn Pro Val ValLeu LeuPro Pro PhePhe AsnAsn Asp Asp Gly Gly Val Phe Val Tyr Tyr Ala PheSer AlaThr Ser GluThr Glu 85 85 90 90 95 95

Lys Ser Lys Ser Asn AsnIle IleIle Ile ArgArg GlyGly Trp Trp Ile Ile Phe Thr Phe Gly Gly Thr ThrLeu ThrAsp Leu SerAsp Ser 100 100 105 105 110 110

Lys Thr Lys Thr Gln GlnSer SerLeu Leu LeuLeu IleIle Val Val Asn Asn Asn Thr Asn Ala Ala Asn ThrVal AsnVal Val IleVal Ile 115 115 120 120 125 125

Lys Val Lys Val Cys CysGlu GluPhe Phe GlnGln PhePhe Cys Cys Asn Asn Asp Phe Asp Pro Pro Leu PheGly LeuVal Gly TyrVal Tyr 130 130 135 135 140 140

Tyr His Tyr His Lys LysAsn AsnAsn Asn LysLys SerSer Trp Trp Met Met Glu Glu Glu Ser Ser Phe GluArg PheVal Arg TyrVal Tyr 145 145 150 150 155 155 160 160

Ser Ser Ser Ser Ala AlaAsn AsnAsn Asn CysCys ThrThr Phe Phe Glu Glu Tyr Ser Tyr Val Val Gln SerPro GlnPhe Pro LeuPhe Leu 165 165 170 170 175 175

Met Asp Met Asp Leu Leu Glu Glu Gly Gly Lys Lys Gln Gln Gly Gly Asn Asn Phe Phe Lys Lys Asn Asn Leu Leu Arg Arg Glu Glu Phe Phe 180 180 185 185 190 190

Val Phe Val Phe Lys LysAsn AsnIle Ile AspAsp GlyGly Tyr Tyr Phe Phe Lys Tyr Lys Ile Ile Ser TyrLys SerHis Lys ThrHis Thr

31

195 195 200 200 205 205

Pro Ile Pro Ile Asn AsnLeu LeuVal Val ArgArg AspAsp Leu Leu Pro Pro Gln Phe Gln Gly Gly Ser PheAla SerLeu Ala GluLeu Glu 210 210 215 215 220 220

Pro Leu Pro Leu Val ValAsp AspLeu Leu ProPro IleIle Gly Gly Ile Ile Asn Thr Asn Ile Ile Arg ThrPhe ArgGln Phe ThrGln Thr 225 225 230 230 235 235 240 240

Leu Leu Leu Leu Ala AlaLeu LeuHis His ArgArg SerSer Tyr Tyr Leu Leu Thr Gly Thr Pro Pro Asp GlySer AspSer Ser SerSer Ser 245 245 250 250 255 255

Gly Trp Gly Trp Thr ThrAla AlaGly Gly AlaAla AlaAla Ala Ala Tyr Tyr Tyr Gly Tyr Val Val Tyr GlyLeu TyrGln Leu ProGln Pro 260 260 265 265 270 270

Arg Thr Arg Thr Phe Phe Leu Leu Leu Leu Lys Lys Tyr Tyr Asn Asn Glu Glu Asn Asn Gly Gly Thr Thr Ile Ile Thr Thr Asp Asp Ala Ala 275 275 280 280 285 285

Val Asp Val Asp Cys CysAla AlaLeu Leu AspAsp ProPro Leu Leu Ser Ser Glu Lys Glu Thr Thr Cys LysThr CysLeu Thr LysLeu Lys 290 290 295 295 300 300

Ser Phe Ser Phe Thr ThrVal ValGlu Glu LysLys GlyGly Ile I le TyrTyr GlnGln Thr Thr Ser Ser Asn Arg Asn Phe PheVal Arg Val 305 305 310 310 315 315 320 320

Gln Pro Gln Pro Thr ThrGlu GluSer Ser IleIle ValVal Arg Arg Phe Phe Pro Ile Pro Asn Asn Thr IleAsn ThrLeu Asn CysLeu Cys 325 325 330 330 335 335

Pro Phe Pro Phe Gly GlyGlu GluVal Val PhePhe AsnAsn Ala Ala Thr Thr Arg Ala Arg Phe Phe Ser AlaVal SerTyr Val AlaTyr Ala 340 340 345 345 350 350

Trp Asn Trp Asn Arg ArgLys LysArg Arg IleIle SerSer Asn Asn Cys Cys Val Asp Val Ala Ala Tyr AspSer TyrVal Ser LeuVal Leu 355 355 360 360 365

32

Tyr Asn Tyr Asn Ser SerAla AlaSer Ser PhePhe SerSer Thr Thr Phe Phe Lys Tyr Lys Cys Cys Gly TyrVal GlySer Val ProSer Pro 370 370 375 375 380 380

Thr Lys Thr Lys Leu LeuAsn AsnAsp Asp LeuLeu CysCys Phe Phe Thr Thr Asn Tyr Asn Val Val Ala TyrAsp AlaSer Asp PheSer Phe 385 385 390 390 395 395 400 400

Val Ile Val Ile Arg Arg Gly Gly Asp Asp Glu Glu Val Val Arg Arg Gln Gln Ile Ile Ala Ala Pro Pro Gly Gly Gln Gln Thr Thr Gly Gly 405 405 410 410 415 415

Lys Ile Lys Ile Ala AlaAsp AspTyr Tyr AsnAsn TyrTyr Lys Lys Leu Leu Pro Asp Pro Asp Asp Phe AspThr PheGly Thr CysGly Cys 420 420 425 425 430 430

Val Ile Val Ile Ala Ala Trp Trp Asn Asn Ser Ser Asn Asn Asn Asn Leu Leu Asp Asp Ser Ser Lys Lys Val Val Gly Gly Gly Gly Asn Asn 435 435 440 440 445 445

Tyr Asn Tyr Asn Tyr TyrLeu LeuTyr Tyr ArgArg LeuLeu Phe Phe Arg Arg Lys Asn Lys Ser Ser Leu AsnLys LeuPro Lys PhePro Phe 450 450 455 455 460 460

Glu Arg Glu Arg Asp AspIle IleSer Ser ThrThr GluGlu Ile Ile Tyr Tyr Gln Gly Gln Ala Ala Ser GlyThr SerPro Thr CysPro Cys 465 465 470 470 475 475 480 480

Asn Gly Asn Gly Val Val Glu Glu Gly Gly Phe Phe Asn Asn Cys Cys Tyr Tyr Phe Phe Pro Pro Leu Leu Gln Gln Ser Ser Tyr Tyr Gly Gly 485 485 490 490 495 495

Phe Gln Phe Gln Pro ProThr ThrAsn Asn GlyGly ValVal Gly Gly Tyr Tyr Gln Tyr Gln Pro Pro Arg TyrVal ArgVal Val ValVal Val 500 500 505 505 510 510

Leu Ser Leu Ser Phe PheGlu GluLeu Leu LeuLeu HisHis Ala Ala Pro Pro Ala Val Ala Thr Thr Cys ValGly CysPro Gly LysPro Lys 515 515 520 520 525 525

Lys Ser Lys Ser Thr ThrAsn AsnLeu Leu ValVal LysLys Asn Asn Lys Lys Cys Asn Cys Val Val Phe AsnAsn PhePhe Asn AsnPhe Asn 530 530 535 535 540

33

Gly Leu Gly Leu Thr ThrGly GlyThr Thr GlyGly ValVal Leu Leu Thr Thr Glu Asn Glu Ser Ser Lys AsnLys LysPhe Lys LeuPhe Leu 545 545 550 550 555 555 560 560

Pro Phe Pro Phe Gln GlnGln GlnPhe Phe GlyGly ArgArg Asp Asp Ile Ile Ala Thr Ala Asp Asp Thr ThrAsp ThrAla Asp ValAla Val 565 565 570 570 575 575

Arg Asp Arg Asp Pro ProGln GlnThr Thr LeuLeu GluGlu Ile Ile Leu Leu Asp Thr Asp Ile Ile Pro ThrCys ProSer Cys PheSer Phe 580 580 585 585 590 590

Gly Gly Gly Gly Val ValSer SerVal Val IleIle ThrThr Pro Pro Gly Gly Thr Thr Thr Asn Asn Ser ThrAsn SerGln Asn ValGln Val 595 595 600 600 605 605

Ala Val Ala Val Leu LeuTyr TyrGln Gln AspAsp ValVal Asn Asn Cys Cys Thr Val Thr Glu Glu Pro ValVal ProAla Val IleAla Ile 610 610 615 615 620 620

His Ala His Ala Asp AspGln GlnLeu Leu ThrThr ProPro Thr Thr Trp Trp Arg Tyr Arg Val Val Ser TyrThr SerGly Thr SerGly Ser 625 625 630 630 635 635 640 640

Asn Val Asn Val Phe PheGln GlnThr Thr ArgArg AlaAla Gly Gly Cys Cys Leu Gly Leu Ile Ile Ala GlyGlu AlaHis Glu ValHis Val 645 645 650 650 655 655

Asn Asn Asn Asn Ser Ser Tyr Tyr Glu Glu Cys Cys Asp Asp Ile Ile Pro Pro Ile Ile Gly Gly Ala Ala Gly Gly Ile Ile Cys Cys Ala Ala 660 660 665 665 670 670

Ser Tyr Gln Ser Tyr GlnThr ThrGln GlnThrThr AsnAsn Ser Ser Pro Pro Arg Arg Arg Arg Arg Ala AlaSer ArgVal SerAlaVal Ala 675 675 680 680 685 685

Ser Gln Ser Gln Ser SerIle IleIle Ile AlaAla TyrTyr Thr Thr Met Met Ser Gly Ser Leu Leu Ala GlyGlu AlaAsn Glu SerAsn Ser 690 690 695 695 700 700

Val Ala Val Ala Tyr TyrSer SerAsn Asn AsnAsn SerSer Ile Ile Ala Ala Ile Thr Ile Pro Pro Asn ThrPhe AsnThr Phe IleThr Ile 705 705 710 710 715 715 720

34

Ser Val Ser Val Thr ThrThr ThrGlu Glu IleIle LeuLeu Pro Pro Val Val Ser Thr Ser Met Met Lys ThrThr LysSer Thr ValSer Val 725 725 730 730 735 735

Asp Cys Asp Cys Thr ThrMet MetTyr Tyr IleIle CysCys Gly Gly Asp Asp Ser Glu Ser Thr Thr Cys GluSer CysAsn Ser LeuAsn Leu 740 740 745 745 750 750

Leu Leu Leu Leu Gln GlnTyr TyrGly Gly SerSer PhePhe Cys Cys Thr Thr Gln Asn Gln Leu Leu Arg AsnAla ArgLeu Ala ThrLeu Thr 755 755 760 760 765 765

Gly Ile Gly Ile Ala AlaVal ValGlu Glu GlnGln AspAsp Lys Lys Asn Asn Thr Glu Thr Gln Gln Val GluPhe ValAla Phe GlnAla Gln 770 770 775 775 780 780

Val Lys Val Lys Gln GlnIle IleTyr Tyr LysLys ThrThr Pro Pro Pro Pro Ile Asp Ile Lys Lys Phe AspGly PheGly Gly PheGly Phe 785 785 790 790 795 795 800 800

Asn Phe Asn Phe Ser SerGln GlnIle Ile LeuLeu ProPro Asp Asp Pro Pro Ser Pro Ser Lys Lys Ser ProLys SerArg Lys SerArg Ser 805 805 810 810 815 815

Phe Ile Phe Ile Glu GluAsp AspLeu Leu LeuLeu PhePhe Asn Asn Lys Lys Val Leu Val Thr Thr Ala LeuAsp AlaAla Asp GlyAla Gly 820 820 825 825 830 830

Phe Ile Phe Ile Lys LysGln GlnTyr Tyr GlyGly AspAsp Cys Cys Leu Leu Gly Ile Gly Asp Asp Ala IleAla AlaArg Ala AspArg Asp 835 835 840 840 845 845

Leu Ile Leu Ile Cys CysAla AlaGln Gln LysLys PhePhe Asn Asn Gly Gly Leu Val Leu Thr Thr Leu ValPro LeuPro Pro LeuPro Leu 850 850 855 855 860 860

Leu Thr Leu Thr Asp AspGlu GluMet Met IleIle AlaAla Gln Gln Tyr Tyr Thr Ala Thr Ser Ser Leu AlaLeu LeuAla Leu GlyAla Gly 865 865 870 870 875 875 880 880

Thr Ile Thr Ile Thr ThrSer SerGly Gly TrpTrp ThrThr Phe Phe Gly Gly Ala Ala Ala Gly Gly Ala AlaLeu AlaGln Leu IleGln Ile

35

885 885 890 890 895 895

Pro Phe Pro Phe Ala AlaMet MetGln Gln MetMet AlaAla Tyr Tyr Arg Arg Phe Gly Phe Asn Asn Ile GlyGly IleVal Gly ThrVal Thr 900 900 905 905 910 910

Gln Asn Gln Asn Val ValLeu LeuTyr Tyr GluGlu AsnAsn Gln Gln Lys Lys Leu Ala Leu Ile Ile Asn AlaGln AsnPhe Gln AsnPhe Asn 915 915 920 920 925 925

Ser Ala Ser Ala Ile IleGly GlyLys Lys IleIle GlnGln Asp Asp Ser Ser Leu Ser Leu Ser Ser Thr SerAla ThrSer Ala AlaSer Ala 930 930 935 935 940 940

Leu Gly Leu Gly Lys LysLeu LeuGln Gln AspAsp ValVal Val Val Asn Asn Gln Ala Gln Asn Asn Gln AlaAla GlnLeu Ala AsnLeu Asn 945 945 950 950 955 955 960 960

Thr Leu Thr Leu Val ValLys LysGln Gln LeuLeu SerSer Ser Ser Asn Asn Phe Ala Phe Gly Gly Ile AlaSer IleSer Ser ValSer Val 965 965 970 970 975 975

Leu Asn Leu Asn Asp AspIle IleLeu Leu SerSer ArgArg Leu Leu Asp Asp Lys Glu Lys Val Val Ala GluGlu AlaVal Glu GlnVal Gln 980 980 985 985 990 990

Ile Ile Asp Asp Arg Arg Leu Leu Ile Ile Thr Thr Gly Gly Arg Leu Gln Arg Leu Gln Ser Ser Leu Leu Gln Gln Thr ThrTyr TyrVal Val 995 995 1000 1000 1005 1005

Thr Gln Thr Gln Gln GlnLeu LeuIle IleArg ArgAla AlaAlaAla Glu Glu Ile Ile Arg Arg Ala Ala SerSer AlaAla AsnAsn 1010 1010 1015 1015 1020 1020

Leu Ala Leu Ala Ala AlaThr ThrLys LysMet MetSer SerGluGlu Cys Cys Val Val Leu Leu Gly Gly GlnGln SerSer LysLys 1025 1025 1030 1030 1035 1035

Arg Val Arg Val Asp AspPhe PheCys CysGly GlyLys LysGlyGly Tyr Tyr His His Leu Leu Met Met Ser Ser Phe Phe Pro Pro 1040 1040 1045 1045 1050

36

Gln Ser Gln Ser Ala AlaPro ProHis HisGly GlyVal ValValVal Phe Phe Leu Leu His His Val Val ThrThr TyrTyr ValVal 1055 1055 1060 1060 1065 1065

Pro Ala Pro Ala Gln GlnGlu GluLys LysAsn AsnPhe PheThrThr Thr Thr Ala Ala Pro Pro Ala Ala IleIle CysCys HisHis 1070 1070 1075 1075 1080 1080

Asp Gly Asp Gly Lys LysAla AlaHis HisPhe PhePro ProArgArg Glu Glu Gly Gly Val Val Phe Phe Val Val Ser Ser Asn Asn 1085 1085 1090 1090 1095 1095

Gly Thr Gly Thr His HisTrp TrpPhe PheVal ValThr ThrGlnGln Arg Arg Asn Asn Phe Phe Tyr Tyr GluGlu ProPro GlnGln 1100 1100 1105 1105 1110 1110

Ile Ile Ile Thr Thr Ile Thr Thr Asp Asp Asn Asn Thr Thr Phe PheVal ValSer SerGly GlyAsn AsnCysCys AspAsp ValVal 1115 1115 1120 1120 1125 1125

Val Ile Val Ile Gly GlyIle IleVal ValAsn AsnAsn AsnThrThr Val Val Tyr Tyr Asp Asp Pro Pro Leu Leu Gln Gln Pro Pro 1130 1130 1135 1135 1140 1140

Glu Leu Glu Leu Asp AspSer SerPhe PheLys LysGlu GluGluGlu Leu Leu Asp Asp Lys Lys Tyr Tyr PhePhe LysLys AsnAsn 1145 1145 1150 1150 1155 1155

His Thr His Thr Ser SerPro ProAsp AspVal ValAsp AspLeuLeu Gly Gly Asp Asp Ile Ile Ser Ser GlyGly Ile I le Asn Asn 1160 1160 1165 1165 1170 1170

Ala Ser Ala Ser Val ValVal ValAsn AsnIle IleGln GlnLysLys Glu Glu Ile Ile Asp Asp Arg Arg Leu Leu Asn Asn Glu Glu 1175 1175 1180 1180 1185 1185

Val Ala Val Ala Lys LysAsn AsnLeu LeuAsn AsnGlu GluSerSer Leu Leu IleAsp I le AspLeu LeuGlnGln Glu Glu Leu Leu 1190 1190 1195 1195 1200 1200

Gly Lys Gly Lys Tyr TyrGlu GluGln GlnTyr TyrIle IleLysLys Trp Trp Pro Pro Trp Trp Tyr Tyr IleIle TrpTrp LeuLeu 1205 1205 1210 1210 1215

37

Gly Phe Gly Phe Ile IleAla AlaGly GlyLeu LeuIle IleAlaAla Ile Ile Val Val Met Met Val Val ThrThr IleIle MetMet 1220 1220 1225 1225 1230 1230

Leu Cys Leu Cys Cys CysMet MetThr ThrSer SerCys CysCysCys Ser Ser Cys Cys Leu Leu Lys Lys GlyGly CysCys CysCys 1235 1235 1240 1240 1245 1245

Ser Ser Cys GlySer Cys Gly SerCys CysCys CysLys LysPhe PheAsp AspGlu GluAsp AspAsp AspSerSer GluGlu ProPro 1250 1250 1255 1255 1260 1260

Val Leu Val Leu Lys LysGly GlyVal ValLys LysLeu LeuHisHis Tyr Tyr Thr Thr 1265 1265 1270 1270

<210> <210> 18 18 <211> <211> 75 75 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 18 18

Met Tyr Met Tyr Ser SerPhe PheVal Val SerSer GluGlu Glu Glu Thr Thr Gly Leu Gly Thr Thr Ile LeuVal IleAsn Val SerAsn Ser 1 1 55 10 10 15 15

Val Leu Val Leu Leu LeuPhe PheLeu Leu AlaAla PhePhe Val Val Val Val Phe Leu Phe Leu Leu Val LeuThr ValLeu Thr AlaLeu Ala 20 20 25 25 30 30

Ile Leu Thr Ile Leu ThrAla AlaLeu LeuArgArg LeuLeu CysCys Ala Ala Tyr Tyr Cys Asn Cys Cys CysIle AsnVal IleAsnVal Asn 35 35 40 40 45 45

Val Ser Val Ser Leu LeuVal ValLys Lys ProPro SerSer Phe Phe Tyr Tyr Val Ser Val Tyr Tyr Arg SerVal ArgLys Val AsnLys Asn 50 50 55 55 60 60

Leu Asn Leu Asn Ser SerSer SerArg Arg ValVal ProPro Asp Asp Leu Leu Leu Leu Val Val

70 70 75

38

<210> <210> 19 19 <211> <211> 222 222 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 19 19

Met Ala Met Ala Asp AspSer SerAsn Asn GlyGly ThrThr Ile Ile Thr Thr Val Glu Val Glu Glu Leu GluLys LeuLys Lys LeuLys Leu 1 1 55 10 10 15 15

Leu Glu Leu Glu Gln GlnTrp TrpAsn Asn LeuLeu ValVal Ile Ile Gly Gly Phe Phe Phe Leu Leu Leu PheThr LeuTrp Thr IleTrp Ile 20 20 25 25 30 30

Cys Leu Cys Leu Leu LeuGln GlnPhe Phe AlaAla TyrTyr Ala Ala Asn Asn Arg Arg Arg Asn Asn Phe ArgLeu PheTyr Leu IleTyr Ile 35 35 40 40 45 45

Ile Lys Leu Ile Lys LeuIle IlePhe PheLeuLeu TrpTrp LeuLeu Leu Leu Trp Trp Pro Thr Pro Val ValLeu ThrAla LeuCysAla Cys 50 50 55 55 60 60

Phe Val Phe Val Leu LeuAla AlaAla Ala ValVal TyrTyr Arg Arg Ile Ile Asn Ile Asn Trp Trp Thr IleGly ThrGly Gly IleGly Ile

70 70 75 75 80 80

Ala Ile Ala Ile Ala Ala Met Met Ala Ala Cys Cys Leu Leu Val Val Gly Gly Leu Leu Met Met Trp Trp Leu Leu Ser Ser Tyr Tyr Phe Phe 85 85 90 90 95 95

Ile Ala Ser Ile Ala SerPhe PheArg ArgLeuLeu PhePhe AlaAla Arg Arg Thr Thr Arg Met Arg Ser SerTrp MetSer TrpPheSer Phe 100 100 105 105 110 110

Asn Pro Asn Pro Glu Glu Thr Thr Asn Asn Ile Ile Leu Leu Leu Leu Asn Asn Val Val Pro Pro Leu Leu His His Gly Gly Thr Thr Ile Ile 115 115 120 120 125 125

Leu Thr Leu Thr Arg ArgPro ProLeu Leu LeuLeu GluGlu Ser Ser Glu Glu Leu Ile Leu Val Val Gly IleAla GlyVal Ala IleVal Ile 130 130 135 135 140

39

Leu Arg Leu Arg Gly GlyHis HisLeu Leu ArgArg IleIle Ala Ala Gly Gly His Leu His His His Gly LeuArg GlyCys Arg AspCys Asp 145 145 150 150 155 155 160 160

Ile Lys Asp Ile Lys AspLeu LeuPro ProLysLys GluGlu IleIle Thr Thr Val Val Ala Ser Ala Thr ThrArg SerThr ArgLeuThr Leu 165 165 170 170 175 175

Ser Tyr Ser Tyr Tyr TyrLys LysLeu Leu GlyGly AlaAla Ser Ser Gln Gln Arg Ala Arg Val Val Gly AlaAsp GlySer Asp GlySer Gly 180 180 185 185 190 190

Phe Ala Ala Phe Ala AlaTyr TyrSer SerArgArg TyrTyr Arg Arg Ile Ile Gly Gly Asn Lys Asn Tyr TyrLeu LysAsn LeuThrAsn Thr 195 195 200 200 205 205

Asp His Asp His Ser Ser Ser Ser Ser Ser Ser Ser Asp Asp Asn Asn Ile Ile Ala Ala Leu Leu Leu Leu Val Val Gln Gln 210 210 215 215 220 220

<210> <210> 20 20 <211> <211> 419 419 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 20 20

Met Ser Met Ser Asp AspAsn AsnGly Gly ProPro GlnGln Asn Asn Gln Gln Arg Ala Arg Asn Asn Pro AlaArg ProIle Arg ThrIle Thr 1 1 5 5 10 10 15 15

Phe Gly Gly Phe Gly GlyPro ProSer SerAspAsp SerSer Thr Thr Gly Gly Ser Ser Asn Asn Asn Gln GlnGly AsnGlu GlyArgGlu Arg 20 20 25 25 30 30

Ser Gly Ala Ser Gly AlaArg ArgSer SerLysLys GlnGln Arg Arg Arg Arg Pro Pro Gln Leu Gln Gly GlyPro LeuAsn ProAsnAsn Asn 35 35 40 40 45 45

Thr Ala Thr Ala Ser SerTrp TrpPhe Phe ThrThr AlaAla Leu Leu Thr Thr Gln Gly Gln His His Lys GlyGlu LysAsp Glu LeuAsp Leu 50 50 55 55 60

40

Lys Phe Lys Phe Pro ProArg ArgGly Gly GlnGln GlyGly Val Val Pro Pro Ile Thr Ile Asn Asn Asn ThrSer AsnSer Ser ProSer Pro

70 70 75 75 80 80

Asp Asp Asp Asp Gln GlnIle IleGly Gly TyrTyr TyrTyr Arg Arg Arg Arg Ala Arg Ala Thr Thr Arg ArgIle ArgArg Ile GlyArg Gly 85 85 90 90 95 95

Gly Asp Gly Asp Gly GlyLys LysMet Met LysLys AspAsp Leu Leu Ser Ser Pro Trp Pro Arg Arg Tyr TrpPhe TyrTyr Phe TyrTyr Tyr 100 100 105 105 110 110

Leu Gly Leu Gly Thr ThrGly GlyPro Pro GluGlu AlaAla Gly Gly Leu Leu Pro Gly Pro Tyr Tyr Ala GlyAsn AlaLys Asn AspLys Asp 115 115 120 120 125 125

Gly Ile Gly Ile Ile IleTrp TrpVal Val AlaAla ThrThr Glu Glu Gly Gly Ala Asn Ala Leu Leu Thr AsnPro ThrLys Pro AspLys Asp 130 130 135 135 140 140

His Ile His Ile Gly GlyThr ThrArg Arg AsnAsn ProPro Ala Ala Asn Asn Asn Ala Asn Ala Ala Ile AlaVal IleLeu Val GlnLeu Gln 145 145 150 150 155 155 160 160

Leu Pro Leu Pro Gln GlnGly GlyThr Thr ThrThr LeuLeu Pro Pro Lys Lys Gly Tyr Gly Phe Phe Ala TyrGlu AlaGly Glu SerGly Ser 165 165 170 170 175 175

Arg Gly Arg Gly Gly GlySer SerGln Gln AlaAla SerSer Ser Ser Arg Arg Ser Ser Ser Ser Ser Arg SerSer ArgArg Ser AsnArg Asn 180 180 185 185 190 190

Ser Ser Ser Ser Arg ArgAsn AsnSer Ser ThrThr ProPro Gly Gly Ser Ser Ser Gly Ser Arg Arg Thr GlySer ThrPro Ser AlaPro Ala 195 195 200 200 205 205

Arg Met Arg Met Ala AlaGly GlyAsn Asn GlyGly GlyGly Asp Asp Ala Ala Ala Ala Ala Leu Leu Leu AlaLeu LeuLeu Leu LeuLeu Leu 210 210 215 215 220 220

Asp Arg Asp Arg Leu LeuAsn AsnGln Gln LeuLeu GluGlu Ser Ser Lys Lys Met Gly Met Ser Ser Lys GlyGly LysGln Gly GlnGln Gln 225 225 230 230 235 235 240

41

Gln Gln Gln Gln Gly GlyGln GlnThr Thr ValVal ThrThr Lys Lys Lys Lys Ser Ala Ser Ala Ala Glu AlaAla GluSer Ala LysSer Lys 245 245 250 250 255 255

Lys Pro Lys Pro Arg ArgGln GlnLys Lys ArgArg ThrThr Ala Ala Thr Thr Lys Tyr Lys Ala Ala Asn TyrVal AsnThr Val GlnThr Gln 260 260 265 265 270 270

Ala Phe Ala Phe Gly GlyArg ArgArg Arg GlyGly ProPro Glu Glu Gln Gln Thr Gly Thr Gln Gln Asn GlyPhe AsnGly Phe AspGly Asp 275 275 280 280 285 285

Gln Glu Gln Glu Leu LeuIle IleArg Arg GlnGln GlyGly Thr Thr Asp Asp Tyr His Tyr Lys Lys Trp HisPro TrpGln Pro IleGln Ile 290 290 295 295 300 300

Ala Gln Ala Gln Phe PheAla AlaPro Pro SerSer AlaAla Ser Ser Ala Ala Phe Gly Phe Phe Phe Met GlySer MetArg Ser IleArg Ile 305 305 310 310 315 315 320 320

Gly Met Gly Met Glu GluVal ValThr Thr ProPro SerSer Gly Gly Thr Thr Trp Thr Trp Leu Leu Tyr ThrThr TyrGly Thr AlaGly Ala 325 325 330 330 335 335

Ile Lys Leu Ile Lys LeuAsp AspAsp AspLysLys AspAsp ProPro Asn Asn Phe Phe Lys Gln Lys Asp AspVal GlnIle ValLeuIle Leu 340 340 345 345 350 350

Leu Asn Leu Asn Lys LysHis HisIle Ile AspAsp AlaAla Tyr Tyr Lys Lys Thr Pro Thr Phe Phe Pro ProThr ProGlu Thr ProGlu Pro 355 355 360 360 365 365

Lys Lys Lys Lys Asp AspLys LysLys Lys LysLys LysLys Ala Ala Asp Asp Glu Gln Glu Thr Thr Ala GlnLeu AlaPro Leu GlnPro Gln 370 370 375 375 380 380

Arg Gln Arg Gln Lys LysLys LysGln Gln GlnGln ThrThr Val Val Thr Thr Leu Pro Leu Leu Leu Ala ProAla AlaAsp Ala LeuAsp Leu 385 385 390 390 395 395 400 400

Asp Asp Asp Asp Phe PheSer SerLys Lys GlnGln LeuLeu Gln Gln Gln Gln Ser Ser Ser Met Met Ser SerAla SerAsp Ala SerAsp Ser

42

405 405 410 410 415 415

Thr Gln Thr Gln Ala Ala

<210> <210> 21 21

<211> <211> 7096 7096 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 21 21

Met Glu Met Glu Ser SerLeu LeuVal Val ProPro GlyGly Phe Phe Asn Asn Glu Thr Glu Lys Lys His ThrVal HisGln Val LeuGln Leu 1 1 55 10 10 15 15

Ser Leu Ser Leu Pro ProVal ValLeu Leu GlnGln ValVal Arg Arg Asp Asp Val Val Val Leu Leu Arg ValGly ArgPhe Gly GlyPhe Gly 20 20 25 25 30 30

Asp Ser Asp Ser Val ValGlu GluGlu Glu ValVal LeuLeu Ser Ser Glu Glu Ala Gln Ala Arg Arg His GlnLeu HisLys Leu AspLys Asp 35 35 40 40 45 45

Gly Thr Cys Gly Thr CysGly GlyLeu LeuValVal GluGlu Val Val Glu Glu Lys Lys Gly Leu Gly Val ValPro LeuGln ProLeuGln Leu 50 50 55 55 60 60

Glu Gln Glu Gln Pro ProTyr TyrVal Val PhePhe IleIle Lys Lys Arg Arg Ser Ala Ser Asp Asp Arg AlaThr ArgAla Thr ProAla Pro

70 70 75 75 80 80

His Gly His Gly His HisVal ValMet Met ValVal GluGlu Leu Leu Val Val Ala Leu Ala Glu Glu Glu LeuGly GluIle Gly GlnIle Gln 85 85 90 90 95 95

Tyr Gly Tyr Gly Arg ArgSer SerGly Gly GluGlu ThrThr Leu Leu Gly Gly Val Val Val Leu Leu Pro ValHis ProVal His GlyVal Gly 100 100 105 105 110 110

Glu Ile Glu Ile Pro ProVal ValAla Ala TyrTyr ArgArg Lys Lys Val Val Leu Arg Leu Leu Leu Lys ArgAsn LysGly Asn AsnGly Asn

43

115 115 120 120 125 125

Lys Gly Lys Gly Ala AlaGly GlyGly Gly HisHis SerSer Tyr Tyr Gly Gly Ala Leu Ala Asp Asp Lys LeuSer LysPhe Ser AspPhe Asp 130 130 135 135 140 140

Leu Gly Leu Gly Asp AspGlu GluLeu Leu GlyGly ThrThr Asp Asp Pro Pro Tyr Asp Tyr Glu Glu Phe AspGln PheGlu Gln AsnGlu Asn 145 145 150 150 155 155 160 160

Trp Asn Trp Asn Thr ThrLys LysHis His SerSer SerSer Gly Gly Val Val Thr Glu Thr Arg Arg Leu GluMet LeuArg Met GluArg Glu 165 165 170 170 175 175

Leu Asn Leu Asn Gly GlyGly GlyAla Ala TyrTyr ThrThr Arg Arg Tyr Tyr Val Asn Val Asp Asp Asn AsnPhe AsnCys Phe GlyCys Gly 180 180 185 185 190 190

Pro Asp Pro Asp Gly GlyTyr TyrPro Pro LeuLeu GluGlu Cys Cys Ile Ile Lys Leu Lys Asp Asp Leu LeuAla LeuArg Ala AlaArg Ala 195 195 200 200 205 205

Gly Lys Gly Lys Ala AlaSer SerCys Cys ThrThr LeuLeu Ser Ser Glu Glu Gln Asp Gln Leu Leu Phe AspIle PheAsp Ile ThrAsp Thr 210 210 215 215 220 220

Lys Arg Lys Arg Gly GlyVal ValTyr Tyr CysCys CysCys Arg Arg Glu Glu His His His Glu Glu Glu HisIle GluAla Ile TrpAla Trp 225 225 230 230 235 235 240 240

Tyr Thr Tyr Thr Glu GluArg ArgSer Ser GluGlu LysLys Ser Ser Tyr Tyr Glu Gln Glu Leu Leu Thr GlnPro ThrPhe Pro GluPhe Glu 245 245 250 250 255 255

Ile Lys Leu Ile Lys LeuAla AlaLys LysLysLys PhePhe AspAsp Thr Thr Phe Phe Asn Glu Asn Gly GlyCys GluPro CysAsnPro Asn 260 260 265 265 270 270

Phe Val Phe Val Phe PhePro ProLeu Leu AsnAsn SerSer Ile Ile Ile Ile Lys Ile Lys Thr Thr Gln IlePro GlnArg Pro ValArg Val 275 275 280 280 285

44

Glu Lys Glu Lys Lys LysLys LysLeu Leu AspAsp GlyGly Phe Phe Met Met Gly Ile Gly Arg Arg Arg IleSer ArgVal Ser TyrVal Tyr 290 290 295 295 300 300

Pro Val Pro Val Ala AlaSer SerPro Pro AsnAsn GluGlu Cys Cys Asn Asn Gln Cys Gln Met Met Leu CysSer LeuThr Ser LeuThr Leu 305 305 310 310 315 315 320 320

Met Lys Met Lys Cys CysAsp AspHis His CysCys GlyGly Glu Glu Thr Thr Ser Gln Ser Trp Trp Thr GlnGly ThrAsp Gly PheAsp Phe 325 325 330 330 335 335

Val Lys Val Lys Ala AlaThr ThrCys Cys GluGlu PhePhe Cys Cys Gly Gly Thr Asn Thr Glu Glu Leu AsnThr LeuLys Thr GluLys Glu 340 340 345 345 350 350

Gly Ala Gly Ala Thr ThrThr ThrCys Cys GlyGly TyrTyr Leu Leu Pro Pro Gln Ala Gln Asn Asn Val AlaVal ValLys Val IleLys Ile 355 355 360 360 365 365

Tyr Cys Tyr Cys Pro ProAla AlaCys Cys HisHis AsnAsn Ser Ser Glu Glu Val Pro Val Gly Gly Glu ProHis GluSer His LeuSer Leu 370 370 375 375 380 380

Ala Glu Ala Glu Tyr TyrHis HisAsn Asn GluGlu SerSer Gly Gly Leu Leu Lys Ile Lys Thr Thr Leu IleArg LeuLys Arg GlyLys Gly 385 385 390 390 395 395 400 400

Gly Arg Thr Gly Arg ThrIle IleAla AlaPhePhe GlyGly Gly Gly Cys Cys Val Val Phe Tyr Phe Ser SerVal TyrGly ValCysGly Cys 405 405 410 410 415 415

His Asn His Asn Lys LysCys CysAla Ala TyrTyr TrpTrp Val Val Pro Pro Arg Ser Arg Ala Ala Ala SerAsn AlaIle Asn GlyIle Gly 420 420 425 425 430 430

Cys Asn Cys Asn His HisThr ThrGly Gly ValVal ValVal Gly Gly Glu Glu Gly Glu Gly Ser Ser Gly GluLeu GlyAsn Leu AspAsn Asp 435 435 440 440 445 445

Asn Leu Asn Leu Leu LeuGlu GluIle Ile LeuLeu GlnGln Lys Lys Glu Glu Lys Asn Lys Val Val Ile AsnAsn IleIle Asn ValIle Val 450 450 455 455 460

45

Gly Asp Phe Gly Asp PheLys LysLeu LeuAsnAsn GluGlu Glu Glu Ile Ile Ala Ala Ile Leu Ile Ile IleAla LeuSer AlaPheSer Phe 465 465 470 470 475 475 480 480

Ser Ala Ser Ala Ser SerThr ThrSer Ser AlaAla PhePhe Val Val Glu Glu Thr Lys Thr Val Val Gly LysLeu GlyAsp Leu TyrAsp Tyr 485 485 490 490 495 495

Lys Ala Lys Ala Phe PheLys LysGln Gln IleIle ValVal Glu Glu Ser Ser Cys Asn Cys Gly Gly Phe AsnLys PheVal Lys ThrVal Thr 500 500 505 505 510 510

Lys Gly Lys Gly Lys LysAla AlaLys Lys LysLys GlyGly Ala Ala Trp Trp Asn Gly Asn Ile Ile Glu GlyGln GluLys Gln SerLys Ser 515 515 520 520 525 525

Ile Leu Ser Ile Leu SerPro ProLeu LeuTyrTyr AlaAla PhePhe Ala Ala Ser Ser Glu Ala Glu Ala AlaArg AlaVal ArgValVal Val 530 530 535 535 540 540

Arg Ser Arg Ser Ile IlePhe PheSer Ser ArgArg ThrThr Leu Leu Glu Glu Thr Gln Thr Ala Ala Asn GlnSer AsnVal Ser ArgVal Arg 545 545 550 550 555 555 560 560

Val Leu Val Leu Gln GlnLys LysAla Ala AlaAla IleIle Thr Thr I leIle Leu Leu Asp Asp Gly Gly Ile Gln Ile Ser SerTyr Gln Tyr 565 565 570 570 575 575

Ser Leu Ser Leu Arg ArgLeu LeuIle Ile AspAsp AlaAla Met Met Met Met Phe Ser Phe Thr Thr Asp SerLeu AspAla Leu ThrAla Thr 580 580 585 585 590 590

Asn Asn Asn Asn Leu LeuVal ValVal Val MetMet AlaAla Tyr Tyr Ile Ile Thr Gly Thr Gly Gly Val GlyVal ValGln Val LeuGln Leu 595 595 600 600 605 605

Thr Ser Thr Ser Gln GlnTrp TrpLeu Leu ThrThr AsnAsn Ile Ile Phe Phe Gly Val Gly Thr Thr Tyr ValGlu TyrLys Glu LeuLys Leu 610 610 615 615 620 620

Lys Pro Lys Pro Val ValLeu LeuAsp Asp TrpTrp LeuLeu Glu Glu Glu Glu Lys Lys Lys Phe Phe Glu LysGly GluVal Gly GluVal Glu 625 625 630 630 635 635 640

46

Phe Leu Phe Leu Arg ArgAsp AspGly Gly TrpTrp GluGlu Ile Ile Val Val Lys Ile Lys Phe Phe Ser IleThr SerCys Thr AlaCys Ala 645 645 650 650 655 655

Cys Glu Cys Glu Ile IleVal ValGly Gly GlyGly GlnGln Ile Ile Val Val Thr Ala Thr Cys Cys Lys AlaGlu LysIle Glu LysIle Lys 660 660 665 665 670 670

Glu Ser Glu Ser Val ValGln GlnThr Thr PhePhe PhePhe Lys Lys Leu Leu Val Lys Val Asn Asn Phe LysLeu PheAla Leu LeuAla Leu 675 675 680 680 685 685

Cys Ala Cys Ala Asp AspSer SerIle Ile IleIle IleIle Gly Gly Gly Gly Ala Leu Ala Lys Lys Lys LeuAla LysLeu Ala AsnLeu Asn 690 690 695 695 700 700

Leu Gly Leu Gly Glu GluThr ThrPhe Phe ValVal ThrThr His His Ser Ser Lys Leu Lys Gly Gly Tyr LeuArg TyrLys Arg CysLys Cys 705 705 710 710 715 715 720 720

Val Lys Val Lys Ser SerArg ArgGlu Glu GluGlu ThrThr Gly Gly Leu Leu Leu Pro Leu Met Met Leu ProLys LeuAla Lys ProAla Pro 725 725 730 730 735 735

Lys Glu Lys Glu Ile IleIle IlePhe Phe LeuLeu GluGlu Gly Gly Glu Glu Thr Pro Thr Leu Leu Thr ProGlu ThrVal Glu LeuVal Leu 740 740 745 745 750 750

Thr Glu Thr Glu Glu GluVal ValVal Val LeuLeu LysLys Thr Thr Gly Gly Asp Gln Asp Leu Leu Pro GlnLeu ProGlu Leu GlnGlu Gln 755 755 760 760 765 765

Pro Thr Pro Thr Ser SerGlu GluAla Ala ValVal GluGlu Ala Ala Pro Pro Leu Gly Leu Val Val Thr GlyPro ThrVal Pro CysVal Cys 770 770 775 775 780 780

Ile Asn Gly Ile Asn GlyLeu LeuMet MetLeuLeu LeuLeu GluGlu Ile Ile Lys Lys Asp Glu Asp Thr ThrLys GluTyr LysCysTyr Cys 785 785 790 790 795 795 800 800

Ala Leu Ala Leu Ala AlaPro ProAsn Asn MetMet MetMet Val Val Thr Thr Asn Thr Asn Asn Asn Phe ThrThr PheLeu Thr LysLeu Lys

47

805 805 810 810 815 815

Gly Gly Gly Gly Ala AlaPro ProThr Thr LysLys ValVal Thr Thr Phe Phe Gly Asp Gly Asp Asp Thr AspVal ThrIle Val GluIle Glu 820 820 825 825 830 830

Val Gln Val Gln Gly Gly Tyr Tyr Lys Lys Ser Ser Val Val Asn Asn Ile Ile Thr Thr Phe Phe Glu Glu Leu Leu Asp Asp Glu Glu Arg Arg 835 835 840 840 845 845

Ile Asp Lys Ile Asp LysVal ValLeu LeuAsnAsn GluGlu LysLys Cys Cys Ser Ser Ala Thr Ala Tyr TyrVal ThrGlu ValLeuGlu Leu 850 850 855 855 860 860

Gly Thr Glu Gly Thr GluVal ValAsn AsnGluGlu PhePhe Ala Ala Cys Cys Val Val Val Asp Val Ala AlaAla AspVal AlaIleVal Ile 865 865 870 870 875 875 880 880

Lys Thr Lys Thr Leu LeuGln GlnPro Pro ValVal SerSer Glu Glu Leu Leu Leu Pro Leu Thr Thr Leu ProGly LeuIle Gly AspIle Asp 885 885 890 890 895 895

Leu Asp Leu Asp Glu GluTrp TrpSer Ser MetMet AlaAla Thr Thr Tyr Tyr Tyr Phe Tyr Leu Leu Asp PheGlu AspSer Glu GlySer Gly 900 900 905 905 910 910

Glu Phe Glu Phe Lys LysLeu LeuAla Ala SerSer HisHis Met Met Tyr Tyr Cys Phe Cys Ser Ser Tyr PhePro TyrPro Pro AspPro Asp 915 915 920 920 925 925

Glu Asp Glu Asp Glu GluGlu GluGlu Glu GlyGly AspAsp Cys Cys Glu Glu Glu Glu Glu Glu Glu Phe GluGlu PhePro Glu SerPro Ser 930 930 935 935 940 940

Thr Gln Thr Gln Tyr TyrGlu GluTyr Tyr GlyGly ThrThr Glu Glu Asp Asp Asp Gln Asp Tyr Tyr Gly GlnLys GlyPro Lys LeuPro Leu 945 945 950 950 955 955 960 960

Glu Phe Glu Phe Gly GlyAla AlaThr Thr SerSer AlaAla Ala Ala Leu Leu Gln Glu Gln Pro Pro Glu GluGlu GluGln Glu GluGln Glu 965 965 970 970 975

48

Glu Asp Trp Glu Asp TrpLeu LeuAsp AspAspAsp AspAsp Ser Ser Gln Gln Gln Gln Thr Gly Thr Val ValGln GlyGln GlnAspGln Asp 980 980 985 985 990 990

Gly Ser Gly Ser Glu Glu Asp Asp Asn Asn Gln Gln Thr Thr Thr Thr Thr ThrIle IleGln GlnThr ThrIle IleValVal Glu Glu Val Val 995 995 1000 1000 1005 1005

Gln Pro Gln Pro Gln GlnLeu LeuGlu GluMet MetGlu GluLeuLeu Thr Thr Pro Pro Val Val Val Val GlnGln ThrThr IleIle 1010 1010 1015 1015 1020 1020

Glu Val Glu Val Asn AsnSer SerPhe PheSer SerGly GlyTyrTyr Leu Leu Lys Lys Leu Leu Thr Thr AspAsp AsnAsn ValVal 1025 1025 1030 1030 1035 1035

Tyr Ile Tyr Ile Lys LysAsn AsnAla AlaAsp AspIle IleValVal Glu Glu Glu Glu Ala Ala Lys Lys LysLys ValVal LysLys 1040 1040 1045 1045 1050 1050

Pro Thr Pro Thr Val ValVal ValVal ValAsn AsnAla AlaAlaAla Asn Asn Val Val Tyr Tyr Leu Leu LysLys HisHis GlyGly 1055 1055 1060 1060 1065 1065

Gly Gly Gly Gly Val ValAla AlaGly GlyAla AlaLeu LeuAsnAsn Lys Lys Ala Ala Thr Thr Asn Asn AsnAsn AlaAla MetMet 1070 1070 1075 1075 1080 1080

Gln Val Gln Val Glu GluSer SerAsp AspAsp AspTyr TyrIleIle Ala Ala Thr Thr Asn Asn Gly Gly ProPro LeuLeu LysLys 1085 1085 1090 1090 1095 1095

Val Gly Val Gly Gly GlySer SerCys CysVal ValLeu LeuSerSer Gly Gly His His Asn Asn Leu Leu Ala Ala Lys Lys His His 1100 1100 1105 1105 1110 1110

Cys Leu Cys Leu His HisVal ValVal ValGly GlyPro ProAsnAsn Val Val Asn Asn Lys Lys Gly Gly GluGlu AspAsp IleIle 1115 1115 1120 1120 1125 1125

Gln Leu Gln Leu Leu LeuLys LysSer SerAla AlaTyr TyrGluGlu Asn Asn Phe Phe Asn Asn Gln Gln HisHis GluGlu ValVal 1130 1130 1135 1135 1140

49

Leu Leu Leu Leu Ala AlaPro ProLeu LeuLeu LeuSer SerAlaAla Gly Gly Ile Ile Phe Phe Gly Gly AlaAla AspAsp ProPro 1145 1145 1150 1150 1155 1155

Ile Ile His Ser Leu His Ser Leu Arg Arg Val Val Cys Cys Val ValAsp AspThr ThrVal ValArg ArgThrThr AsnAsn ValVal 1160 1160 1165 1165 1170 1170

Tyr Leu Tyr Leu Ala AlaVal ValPhe PheAsp AspLys LysAsnAsn Leu Leu Tyr Tyr Asp Asp Lys Lys LeuLeu ValVal SerSer 1175 1175 1180 1180 1185 1185

Ser Ser Phe LeuGlu Phe Leu GluMet MetLys LysSer SerGlu GluLys LysGln GlnVal ValGlu GluGlnGln LysLys IleIle 1190 1190 1195 1195 1200 1200

Ala Glu Ala Glu Ile IlePro ProLys LysGlu GluGlu GluValVal Lys Lys Pro Pro Phe Phe Ile Ile Thr Thr Glu Glu Ser Ser 1205 1205 1210 1210 1215 1215

Lys Pro Lys Pro Ser SerVal ValGlu GluGln GlnArg ArgLysLys Gln Gln Asp Asp Asp Asp Lys Lys LysLys IleIle LysLys 1220 1220 1225 1225 1230 1230

Ala Cys Ala Cys Val ValGlu GluGlu GluVal ValThr ThrThrThr Thr Thr Leu Leu Glu Glu Glu Glu Thr Thr Lys Lys Phe Phe 1235 1235 1240 1240 1245 1245

Leu Thr Leu Thr Glu GluAsn AsnLeu LeuLeu LeuLeu LeuTyrTyr Ile Ile Asp Asp Ile Ile Asn Asn Gly Gly Asn Asn Leu Leu 1250 1250 1255 1255 1260 1260

His Pro His Pro Asp AspSer SerAla AlaThr ThrLeu LeuValVal Ser Ser Asp Asp IleAsp I le AspIleIle Thr Thr Phe Phe 1265 1265 1270 1270 1275 1275

Leu Lys Leu Lys Lys LysAsp AspAla AlaPro ProTyr TyrIleIle Val Val Gly Gly Asp Asp Val Val ValVal GlnGln GluGlu 1280 1280 1285 1285 1290 1290

Gly Val Gly Val Leu LeuThr ThrAla AlaVal ValVal ValIleIle Pro Pro Thr Thr Lys Lys Lys Lys AlaAla GlyGly GlyGly 1295 1295 1300 1300 1305

50

Thr Thr Thr Thr Glu GluMet MetLeu LeuAla AlaLys LysAlaAla Leu Leu Arg Arg Lys Lys Val Val ProPro ThrThr AspAsp 1310 1310 1315 1315 1320 1320

Asn Tyr Asn Tyr Ile IleThr ThrThr ThrTyr TyrPro ProGlyGly Gln Gln Gly Gly Leu Leu Asn Asn Gly Gly Tyr Tyr Thr Thr 1325 1325 1330 1330 1335 1335

Val Glu Val Glu Glu GluAla AlaLys LysThr ThrVal ValLeuLeu Lys Lys Lys Lys Cys Cys Lys Lys Ser Ser Ala Ala Phe Phe 1340 1340 1345 1345 1350 1350

Tyr Ile Tyr Ile Leu LeuPro ProSer SerIle IleIle IleSerSer Asn Asn Glu Glu Lys Lys Gln Gln GluGlu IleIle LeuLeu 1355 1355 1360 1360 1365 1365

Gly Thr Gly Thr Val ValSer SerTrp TrpAsn AsnLeu LeuArgArg Glu Glu Met Met Leu Leu Ala Ala HisHis AlaAla GluGlu 1370 1370 1375 1375 1380 1380

Glu Thr Glu Thr Arg ArgLys LysLeu LeuMet MetPro ProValVal Cys Cys Val Val Glu Glu Thr Thr LysLys AlaAla IleIle 1385 1385 1390 1390 1395 1395

Val Ser Val Ser Thr ThrIle IleGln GlnArg ArgLys LysTyrTyr Lys Lys Gly Gly Ile Ile Lys Lys Ile Ile Gln Gln Glu Glu 1400 1400 1405 1405 1410 1410

Gly Val Gly Val Val ValAsp AspTyr TyrGly GlyAla AlaArgArg Phe Phe Tyr Tyr Phe Phe Tyr Tyr ThrThr SerSer LysLys 1415 1415 1420 1420 1425 1425

Thr Thr Thr Thr Val ValAla AlaSer SerLeu LeuIle IleAsnAsn Thr Thr Leu Leu Asn Asn Asp Asp Leu Leu Asn Asn Glu Glu 1430 1430 1435 1435 1440 1440

Thr Leu Thr Leu Val ValThr ThrMet MetPro ProLeu LeuGlyGly Tyr Tyr Val Val Thr Thr His His Gly Gly Leu Leu Asn Asn 1445 1445 1450 1450 1455 1455

Leu Glu Leu Glu Glu GluAla AlaAla AlaArg ArgTyr TyrMetMet Arg Arg Ser Ser Leu Leu Lys Lys ValVal ProPro AlaAla

51

1460 1460 1465 1465 1470 1470

Thr Val Thr Val Ser SerVal ValSer SerSer SerPro ProAspAsp Ala Ala Val Val Thr Thr Ala Ala Tyr Tyr Asn Asn Gly Gly 1475 1475 1480 1480 1485 1485

Tyr Leu Tyr Leu Thr ThrSer SerSer SerSer SerLys LysThrThr Pro Pro Glu Glu Glu Glu His His PhePhe Ile I le Glu Glu 1490 1490 1495 1495 1500 1500

Thr Ile Thr Ile Ser SerLeu LeuAla AlaGly GlySer SerTyrTyr Lys Lys Asp Asp Trp Trp Ser Ser TyrTyr SerSer GlyGly 1505 1505 1510 1510 1515 1515

Gln Ser Gln Ser Thr ThrGln GlnLeu LeuGly GlyIle IleGluGlu Phe Phe Leu Leu Lys Lys Arg Arg GlyGly AspAsp LysLys 1520 1520 1525 1525 1530 1530

Ser Val Ser Val Tyr TyrTyr TyrThr ThrSer SerAsn AsnProPro Thr Thr Thr Thr Phe Phe His His LeuLeu AspAsp GlyGly 1535 1535 1540 1540 1545 1545

Glu Val Glu Val Ile IleThr ThrPhe PheAsp AspAsn AsnLeuLeu Lys Lys Thr Thr Leu Leu Leu Leu SerSer LeuLeu ArgArg 1550 1550 1555 1555 1560 1560

Glu Val Glu Val Arg ArgThr ThrIle IleLys LysVal ValPhePhe Thr Thr Thr Thr Val Val Asp Asp AsnAsn Ile I le Asn Asn 1565 1565 1570 1570 1575 1575

Leu His Leu His Thr ThrGln GlnVal ValVal ValAsp AspMetMet Ser Ser Met Met Thr Thr Tyr Tyr GlyGly GlnGln GlnGln 1580 1580 1585 1585 1590 1590

Phe Gly Phe Gly Pro ProThr ThrTyr TyrLeu LeuAsp AspGlyGly Ala Ala Asp Asp Val Val Thr Thr LysLys Ile I le Lys Lys 1595 1595 1600 1600 1605 1605

Pro His Pro His Asn AsnSer SerHis HisGlu GluGly GlyLysLys Thr Thr Phe Phe Tyr Tyr Val Val LeuLeu ProPro AsnAsn 1610 1610 1615 1615 1620

52

Asp Asp Asp Asp Thr ThrLeu LeuArg ArgVal ValGlu GluAlaAla Phe Phe Glu Glu Tyr Tyr Tyr Tyr His His Thr Thr Thr Thr 1625 1625 1630 1630 1635 1635

Asp Pro Asp Pro Ser SerPhe PheLeu LeuGly GlyArg ArgTyrTyr Met Met Ser Ser Ala Ala Leu Leu Asn Asn His His Thr Thr 1640 1640 1645 1645 1650 1650

Lys Lys Lys Lys Trp TrpLys LysTyr TyrPro ProGln GlnValVal Asn Asn Gly Gly Leu Leu Thr Thr SerSer IleIle LysLys 1655 1655 1660 1660 1665 1665

Trp Ala Trp Ala Asp AspAsn AsnAsn AsnCys CysTyr TyrLeuLeu Ala Ala Thr Thr Ala Ala Leu Leu Leu Leu Thr Thr Leu Leu 1670 1670 1675 1675 1680 1680

Gln Gln Gln Gln Ile IleGlu GluLeu LeuLys LysPhe PheAsnAsn Pro Pro Pro Pro Ala Ala Leu Leu Gln Gln Asp Asp Ala Ala 1685 1685 1690 1690 1695 1695

Tyr Tyr Tyr Tyr Arg ArgAla AlaArg ArgAla AlaGly GlyGluGlu Ala Ala Ala Ala Asn Asn Phe Phe CysCys AlaAla LeuLeu 1700 1700 1705 1705 1710 1710

Ile Ile Leu Ala Tyr Leu Ala Tyr Cys Cys Asn Asn Lys Lys Thr ThrVal ValGly GlyGlu GluLeu LeuGlyGly AspAsp ValVal 1715 1715 1720 1720 1725 1725

Arg Glu Arg Glu Thr ThrMet MetSer SerTyr TyrLeu LeuPhePhe Gln Gln His His Ala Ala Asn Asn Leu Leu Asp Asp Ser Ser 1730 1730 1735 1735 1740 1740

Cys Lys Cys Lys Arg ArgVal ValLeu LeuAsn AsnVal ValValVal Cys Cys Lys Lys Thr Thr Cys Cys GlyGly GlnGln GlnGln 1745 1745 1750 1750 1755 1755

Gln Thr Gln Thr Thr ThrLeu LeuLys LysGly GlyVal ValGluGlu Ala Ala Val Val Met Met Tyr Tyr MetMet GlyGly ThrThr 1760 1760 1765 1765 1770 1770

Leu Ser Leu Ser Tyr TyrGlu GluGln GlnPhe PheLys LysLysLys Gly Gly Val Val Gln Gln Ile Ile ProPro CysCys ThrThr 1775 1775 1780 1780 1785

53

Cys Gly Cys Gly Lys LysGln GlnAla AlaThr ThrLys LysTyrTyr Leu Leu Val Val Gln Gln Gln Gln GluGlu SerSer ProPro 1790 1790 1795 1795 1800 1800

Phe Val Phe Val Met MetMet MetSer SerAla AlaPro ProProPro Ala Ala Gln Gln Tyr Tyr Glu Glu LeuLeu LysLys HisHis 1805 1805 1810 1810 1815 1815

Gly Thr Gly Thr Phe PheThr ThrCys CysAla AlaSer SerGluGlu Tyr Tyr Thr Thr Gly Gly Asn Asn TyrTyr GlnGln CysCys 1820 1820 1825 1825 1830 1830

Gly His Gly His Tyr TyrLys LysHis HisIle IleThr ThrSerSer Lys Lys Glu Glu Thr Thr Leu Leu TyrTyr CysCys IleIle 1835 1835 1840 1840 1845 1845

Asp Gly Asp Gly Ala AlaLeu LeuLeu LeuThr ThrLys LysSerSer Ser Ser Glu Glu Tyr Tyr Lys Lys Gly Gly Pro Pro Ile Ile 1850 1850 1855 1855 1860 1860

Thr Asp Thr Asp Val ValPhe PheTyr TyrLys LysGlu GluAsnAsn Ser Ser Tyr Tyr Thr Thr Thr Thr ThrThr IleIle LysLys 1865 1865 1870 1870 1875 1875

Pro Val Pro Val Thr ThrTyr TyrLys LysLeu LeuAsp AspGlyGly Val Val Val Val Cys Cys Thr Thr GluGlu IleIle AspAsp 1880 1880 1885 1885 1890 1890

Pro Lys Pro Lys Leu LeuAsp AspAsn AsnTyr TyrTyr TyrLysLys Lys Lys Asp Asp Asn Asn Ser Ser Tyr Tyr Phe Phe Thr Thr 1895 1895 1900 1900 1905 1905

Glu Gln Glu Gln Pro ProIle IleAsp AspLeu LeuVal ValProPro Asn Asn Gln Gln Pro Pro Tyr Tyr ProPro AsnAsn AlaAla 1910 1910 1915 1915 1920 1920

Ser Phe Ser Phe Asp AspAsn AsnPhe PheLys LysPhe PheValVal Cys Cys Asp Asp Asn Asn Ile Ile LysLys PhePhe AlaAla 1925 1925 1930 1930 1935 1935

Asp Asp Asp Asp Leu LeuAsn AsnGln GlnLeu LeuThr ThrGlyGly Tyr Tyr Lys Lys Lys Lys Pro Pro Ala Ala Ser Ser Arg Arg 1940 1940 1945 1945 1950

54

Glu Leu Glu Leu Lys LysVal ValThr ThrPhe PhePhe PheProPro Asp Asp Leu Leu Asn Asn Gly Gly AspAsp ValVal ValVal 1955 1955 1960 1960 1965 1965

Ala Ile Ala Ile Asp AspTyr TyrLys LysHis HisTyr TyrThrThr Pro Pro Ser Ser Phe Phe Lys Lys Lys Lys Gly Gly Ala Ala 1970 1970 1975 1975 1980 1980

Lys Leu Lys Leu Leu LeuHis HisLys LysPro ProIle IleValVal Trp Trp His His Val Val Asn Asn AsnAsn AlaAla ThrThr 1985 1985 1990 1990 1995 1995

Asn Lys Asn Lys Ala AlaThr ThrTyr TyrLys LysPro ProAsnAsn Thr Thr Trp Trp Cys Cys Ile Ile Arg Arg Cys Cys Leu Leu 2000 2000 2005 2005 2010 2010

Trp Ser Trp Ser Thr ThrLys LysPro ProVal ValGlu GluThrThr Ser Ser Asn Asn Ser Ser Phe Phe Asp Asp Val Val Leu Leu 2015 2015 2020 2020 2025 2025

Lys Ser Lys Ser Glu GluAsp AspAla AlaGln GlnGly GlyMetMet Asp Asp Asn Asn Leu Leu Ala Ala CysCys GluGlu AspAsp 2030 2030 2035 2035 2040 2040

Leu Lys Leu Lys Pro ProVal ValSer SerGlu GluGlu GluValVal Val Val Glu Glu Asn Asn Pro Pro ThrThr IleGln I (le Gln 2045 2045 2050 2050 2055 2055

Lys Asp Lys Asp Val ValLeu LeuGlu GluCys CysAsn AsnValVal Lys Lys Thr Thr Thr Thr Glu Glu ValVal ValVal GlyGly 2060 2060 2065 2065 2070 2070

Asp Ile Asp Ile Ile IleLeu LeuLys LysPro ProAla AlaAsnAsn Asn Asn Ser Ser Leu Leu Lys Lys Ile Ile Thr Thr Glu Glu 2075 2075 2080 2080 2085 2085

Glu Val Glu Val Gly GlyHis HisThr ThrAsp AspLeu LeuMetMet Ala Ala Ala Ala Tyr Tyr Val Val AspAsp AsnAsn SerSer 2090 2090 2095 2095 2100 2100

Ser Ser Leu ThrIle Leu Thr IleLys LysLys LysPro ProAsn AsnGlu GluLeu LeuSer SerArg ArgValVal LeuLeu GlyGly

55

2105 2105 2110 2110 2115 2115

Leu Lys Leu Lys Thr ThrLeu LeuAla AlaThr ThrHis HisGlyGly Leu Leu Ala Ala Ala Ala Val Val AsnAsn SerSer ValVal 2120 2120 2125 2125 2130 2130

Pro Trp Pro Trp Asp AspThr ThrIle IleAla AlaAsn AsnTyrTyr Ala Ala Lys Lys Pro Pro Phe Phe LeuLeu AsnAsn LysLys 2135 2135 2140 2140 2145 2145

Val Val Val Val Ser SerThr ThrThr ThrThr ThrAsn AsnIleIle Val Val Thr Thr Arg Arg Cys Cys Leu Leu Asn Asn Arg Arg 2150 2150 2155 2155 2160 2160

Val Cys Val Cys Thr ThrAsn AsnTyr TyrMet MetPro ProTyrTyr Phe Phe Phe Phe Thr Thr Leu Leu Leu Leu Leu Leu Gln Gln 2165 2165 2170 2170 2175 2175

Leu Cys Leu Cys Thr ThrPhe PheThr ThrArg ArgSer SerThrThr Asn Asn Ser Ser Arg Arg Ile Ile Lys Lys Ala Ala Ser Ser 2180 2180 2185 2185 2190 2190

Met Pro Met Pro Thr ThrThr ThrIle IleAla AlaLys LysAsnAsn Thr Thr Val Val Lys Lys Ser Ser Val Val Gly Gly Lys Lys 2195 2195 2200 2200 2205 2205

Phe Cys Phe Cys Leu LeuGlu GluAla AlaSer SerPhe PheAsnAsn Tyr Tyr Leu Leu Lys Lys Ser Ser ProPro AsnAsn PhePhe 2210 2210 2215 2215 2220 2220

Ser Lys Ser Lys Leu LeuIle IleAsn AsnIle IleIle IleIleIle Trp Trp Phe Phe Leu Leu Leu Leu LeuLeu SerSer ValVal 2225 2225 2230 2230 2235 2235

Cys Leu Cys Leu Gly GlySer SerLeu LeuIle IleTyr TyrSerSer Thr Thr Ala Ala Ala Ala Leu Leu GlyGly ValVal LeuLeu 2240 2240 2245 2245 2250 2250

Met Ser Met Ser Asn AsnLeu LeuGly GlyMet MetPro ProSerSer Tyr Tyr Cys Cys Thr Thr Gly Gly Tyr Tyr Arg Arg Glu Glu 2255 2255 2260 2260 2265

56

Gly Tyr Gly Tyr Leu LeuAsn AsnSer SerThr ThrAsn AsnValVal Thr Thr Ile Ile Ala Ala Thr Thr TyrTyr CysCys ThrThr 2270 2270 2275 2275 2280 2280

Gly Ser Gly Ser Ile IlePro ProCys CysSer SerVal ValCysCys Leu Leu Ser Ser Gly Gly Leu Leu AspAsp SerSer LeuLeu 2285 2285 2290 2290 2295 2295

Asp Thr Asp Thr Tyr TyrPro ProSer SerLeu LeuGlu GluThrThr Ile Ile Gln Gln Ile Ile Thr Thr Ile Ile Ser Ser Ser Ser 2300 2300 2305 2305 2310 2310

Phe Lys Phe Lys Trp TrpAsp AspLeu LeuThr ThrAla AlaPhePhe Gly Gly Leu Leu Val Val Ala Ala GluGlu TrpTrp PhePhe 2315 2315 2320 2320 2325 2325

Leu Ala Leu Ala Tyr TyrI Ile Leu Phe le Leu Phe Thr Thr Arg ArgPhe PhePhe PheTyr TyrVal ValLeuLeu Gly Gly Leu Leu 2330 2330 2335 2335 2340 2340

Ala Ala Ala Ala Ile IleMet MetGln GlnLeu LeuPhe PhePhePhe Ser Ser Tyr Tyr Phe Phe Ala Ala Val Val His His Phe Phe 2345 2345 2350 2350 2355 2355

Ile Ile Ser Asn Ser Ser Asn Ser Trp Trp Leu Leu Met Met Trp TrpLeu LeuIle IleI Ile Asn Leu le Asn LeuVal ValGln Gln 2360 2360 2365 2365 2370 2370

Met Ala Met Ala Pro ProI Ile Ser Ala le Ser Ala Met Met Val ValArg ArgMet MetTyr TyrIle IlePhePhe Phe Phe Ala Ala 2375 2375 2380 2380 2385 2385

Ser Ser Phe TyrTyr Phe Tyr TyrVal ValTrp TrpLys LysSer SerTyr TyrVal ValHis HisVal ValValVal AspAsp GlyGly 2390 2390 2395 2395 2400 2400

Cys Asn Cys Asn Ser SerSer SerThr ThrCys CysMet MetMetMet Cys Cys Tyr Tyr Lys Lys Arg Arg AsnAsn ArgArg AlaAla 2405 2405 2410 2410 2415 2415

Thr Arg Thr Arg Val ValGlu GluCys CysThr ThrThr ThrIleIle Val Val Asn Asn Gly Gly Val Val ArgArg ArgArg SerSer 2420 2420 2425 2425 2430

57

Phe Tyr Phe Tyr Val ValTyr TyrAla AlaAsn AsnGly GlyGlyGly Lys Lys Gly Gly Phe Phe Cys Cys LysLys LeuLeu HisHis 2435 2435 2440 2440 2445 2445

Asn Trp Asn Trp Asn AsnCys CysVal ValAsn AsnCys CysAspAsp Thr Thr Phe Phe Cys Cys Ala Ala Gly Gly Ser Ser Thr Thr 2450 2450 2455 2455 2460 2460

Phe Ile Phe Ile Ser SerAsp AspGlu GluVal ValAla AlaArgArg Asp Asp Leu Leu Ser Ser Leu Leu GlnGln PhePhe LysLys 2465 2465 2470 2470 2475 2475

Arg Pro Arg Pro Ile IleAsn AsnPro ProThr ThrAsp AspGlnGln Ser Ser Ser Ser Tyr Tyr Ile Ile Val Val Asp Asp Ser Ser 2480 2480 2485 2485 2490 2490

Val Thr Val Thr Val ValLys LysAsn AsnGly GlySer SerIleIle His His Leu Leu Tyr Tyr Phe Phe Asp Asp Lys Lys Ala Ala 2495 2495 2500 2500 2505 2505

Gly Gln Gly Gln Lys LysThr ThrTyr TyrGlu GluArg ArgHisHis Ser Ser Leu Leu Ser Ser His His PhePhe ValVal AsnAsn 2510 2510 2515 2515 2520 2520

Leu Asp Leu Asp Asn AsnLeu LeuArg ArgAla AlaAsn AsnAsnAsn Thr Thr Lys Lys Gly Gly Ser Ser LeuLeu ProPro IleIle 2525 2525 2530 2530 2535 2535

Asn Val Asn Val Ile IleVal ValPhe PheAsp AspGly GlyLysLys Ser Ser Lys Lys Cys Cys Glu Glu Glu Glu Ser Ser Ser Ser 2540 2540 2545 2545 2550 2550

Ala Lys Ala Lys Ser SerAla AlaSer SerVal ValTyr TyrTyrTyr Ser Ser Gln Gln Leu Leu Met Met Cys Cys Gln Gln Pro Pro 2555 2555 2560 2560 2565 2565

Ile Ile Leu Leu Leu Leu Leu Leu Asp Asp Gln Gln Ala Ala Leu LeuVal ValSer SerAsp AspVal ValGlyGly AspAsp SerSer 2570 2570 2575 2575 2580 2580

Ala Glu Ala Glu Val ValAla AlaVal ValLys LysMet MetPhePhe Asp Asp Ala Ala Tyr Tyr Val Val Asn Asn Thr Thr Phe Phe 2585 2585 2590 2590 2595

58

Ser Ser Ser Ser Thr ThrPhe PheAsn AsnVal ValPro ProMetMet Glu Glu Lys Lys Leu Leu Lys Lys ThrThr LeuLeu ValVal 2600 2600 2605 2605 2610 2610

Ala Thr Ala Thr Ala AlaGlu GluAla AlaGlu GluLeu LeuAlaAla Lys Lys Asn Asn Val Val Ser Ser Leu Leu Asp Asp Asn Asn 2615 2615 2620 2620 2625 2625

Val Leu Val Leu Ser SerThr ThrPhe PheI Ile Ser Ala le Ser AlaAla AlaArg ArgGln GlnGly GlyPhePhe Val Val Asp Asp 2630 2630 2635 2635 2640 2640

Ser Asp Ser Asp Val ValGlu GluThr ThrLys LysAsp AspValVal Val Val Glu Glu Cys Cys Leu Leu LysLys LeuLeu SerSer 2645 2645 2650 2650 2655 2655

His Gln His Gln Ser SerAsp AspIle IleGlu GluVal ValThrThr Gly Gly Asp Asp Ser Ser Cys Cys Asn Asn Asn Asn Tyr Tyr 2660 2660 2665 2665 2670 2670

Met Leu Met Leu Thr ThrTyr TyrAsn AsnLys LysVal ValGluGlu Asn Asn Met Met Thr Thr Pro Pro Arg Arg Asp Asp Leu Leu 2675 2675 2680 2680 2685 2685

Gly Ala Gly Ala Cys CysIle IleAsp AspCys CysSer SerAlaAla Arg Arg His His Ile Ile Asn Asn AlaAla GlnGln ValVal 2690 2690 2695 2695 2700 2700

Ala Lys Ala Lys Ser SerHis HisAsn AsnI Ile lle Ala Leu Ile Ala Leu Ile Trp Trp Asn Asn Val Val Lys LysAsp AspPhe Phe 2705 2705 2710 2710 2715 2715

Met Ser Met Ser Leu LeuSer SerGlu GluGln GlnLeu LeuArgArg Lys Lys Gln Gln Ile Ile Arg Arg Ser Ser Ala Ala Ala Ala 2720 2720 2725 2725 2730 2730

Lys Lys Lys Lys Asn AsnAsn AsnLeu LeuPro ProPhe PheLysLys Leu Leu Thr Thr Cys Cys Ala Ala ThrThr ThrThr ArgArg 2735 2735 2740 2740 2745 2745

Gln Val Gln Val Val ValAsn AsnVal ValVal ValThr ThrThrThr Lys Lys Ile Ile Ala Ala Leu Leu Lys Lys Gly Gly Gly Gly

59

2750 2750 2755 2755 2760 2760

Lys Ile Lys Ile Val ValAsn AsnAsn AsnTrp TrpLeu LeuLysLys Gln Gln Leu Leu Ile Ile Lys Lys ValVal ThrThr LeuLeu 2765 2765 2770 2770 2775 2775

Val Phe Val Phe Leu LeuPhe PheVal ValAla AlaAla AlaIleIle Phe Phe Tyr Tyr Leu Leu Ile Ile Thr Thr Pro Pro Val Val 2780 2780 2785 2785 2790 2790

His Val His Val Met MetSer SerLys LysHis HisThr ThrAspAsp Phe Phe Ser Ser Ser Ser Glu Glu IleIle IleIle GlyGly 2795 2795 2800 2800 2805 2805

Tyr Lys Tyr Lys Ala AlaIle IleAsp AspGly GlyGly GlyValVal Thr Thr Arg Arg Asp Asp Ile Ile AlaAla SerSer ThrThr 2810 2810 2815 2815 2820 2820

Asp Thr Asp Thr Cys CysPhe PheAla AlaAsn AsnLys LysHisHis Ala Ala Asp Asp Phe Phe Asp Asp Thr Thr Trp Trp Phe Phe 2825 2825 2830 2830 2835 2835

Ser Gln Ser Gln Arg ArgGly GlyGly GlySer SerTyr TyrThrThr Asn Asn Asp Asp Lys Lys Ala Ala CysCys ProPro LeuLeu 2840 2840 2845 2845 2850 2850

Ile Ile Ala Ala Val Ala Ala Val Ile Ile Thr Thr Arg Arg Glu GluVal ValGly GlyPhe PheVal ValValVal ProPro GlyGly 2855 2855 2860 2860 2865 2865

Leu Pro Leu Pro Gly GlyThr ThrIle IleLeu LeuArg ArgThrThr Thr Thr Asn Asn Gly Gly Asp Asp PhePhe LeuLeu HisHis 2870 2870 2875 2875 2880 2880

Phe Leu Phe Leu Pro ProArg ArgVal ValPhe PheSer SerAlaAla Val Val Gly Gly Asn Asn Ile Ile CysCys TyrTyr ThrThr 2885 2885 2890 2890 2895 2895

Pro Ser Pro Ser Lys LysLeu LeuIle IleGlu GluTyr TyrThrThr Asp Asp Phe Phe Ala Ala Thr Thr SerSer AlaAla CysCys 2900 2900 2905 2905 2910

60

Val Leu Val Leu Ala AlaAla AlaGlu GluCys CysThr ThrIleIle Phe Phe Lys Lys Asp Asp Ala Ala Ser Ser Gly Gly Lys Lys 2915 2915 2920 2920 2925 2925

Pro Val Pro Val Pro ProTyr TyrCys CysTyr TyrAsp AspThrThr Asn Asn Val Val Leu Leu Glu Glu GlyGly SerSer ValVal 2930 2930 2935 2935 2940 2940

Ala Tyr Ala Tyr Glu GluSer SerLeu LeuArg ArgPro ProAspAsp Thr Thr Arg Arg Tyr Tyr Val Val Leu Leu Met Met Asp Asp 2945 2945 2950 2950 2955 2955

Gly Ser Gly Ser Ile IleIle IleGln GlnPhe PhePro ProAsnAsn Thr Thr Tyr Tyr Leu Leu Glu Glu GlyGly SerSer ValVal 2960 2960 2965 2965 2970 2970

Arg Val Arg Val Val ValThr ThrThr ThrPhe PheAsp AspSerSer Glu Glu Tyr Tyr Cys Cys Arg Arg His His Gly Gly Thr Thr 2975 2975 2980 2980 2985 2985

Cys Glu Cys Glu Arg ArgSer SerGlu GluAla AlaGly GlyValVal Cys Cys Val Val Ser Ser Thr Thr SerSer GlyGly ArgArg 2990 2990 2995 2995 3000 3000

Trp Val Trp Val Leu LeuAsn AsnAsn AsnAsp AspTyr TyrTyrTyr Arg Arg Ser Ser Leu Leu Pro Pro GlyGly ValVal Phe I Phe 3005 3005 3010 3010 3015 3015

Cys Gly Cys Gly Val ValAsp AspAla AlaVal ValAsn AsnLeuLeu Leu Leu Thr Thr Asn Asn Met Met PhePhe ThrThr ProPro 3020 3020 3025 3025 3030 3030

Leu Ile Leu Ile Gln GlnPro ProIle IleGly GlyAla AlaLeuLeu Asp Asp Ile Ile Ser Ser Ala Ala Ser Ser Ile Ile Val Val 3035 3035 3040 3040 3045 3045

Ala Gly Ala Gly Gly GlyIle IleVal ValAla AlaIle IleValVal Val Val Thr Thr Cys Cys Leu Leu Ala Ala Tyr Tyr Tyr Tyr 3050 3050 3055 3055 3060 3060

Phe Met Phe Met Arg ArgPhe PheArg ArgArg ArgAla AlaPhePhe Gly Gly Glu Glu Tyr Tyr Ser Ser HisHis ValVal ValVal 3065 3065 3070 3070 3075

61

Ala Phe Ala Phe Asn AsnThr ThrLeu LeuLeu LeuPhe PheLeuLeu Met Met Ser Ser Phe Phe Thr Thr Val Val Leu Leu Cys Cys 3080 3080 3085 3085 3090 3090

Leu Thr Leu Thr Pro ProVal ValTyr TyrSer SerPhe PheLeuLeu Pro Pro Gly Gly Val Val Tyr Tyr SerSer ValVal IleIle 3095 3095 3100 3100 3105 3105

Tyr Leu Tyr Leu Tyr TyrLeu LeuThr ThrPhe PheTyr TyrLeuLeu Thr Thr Asn Asn Asp Asp Val Val SerSer PhePhe LeuLeu 3110 3110 3115 3115 3120 3120

Ala His Ala His Ile IleGln GlnTrp TrpMet MetVal ValMetMet Phe Phe Thr Thr Pro Pro Leu Leu Val Val Pro Pro Phe Phe 3125 3125 3130 3130 3135 3135

Trp Ile Trp Ile Thr ThrIle IleAla AlaTyr TyrIle IleIleIle Cys Cys Ile Ile Ser Ser Thr Thr LysLys HisHis PhePhe 3140 3140 3145 3145 3150 3150

Tyr Trp Tyr Trp Phe PhePhe PheSer SerAsn AsnTyr TyrLeuLeu Lys Lys Arg Arg Arg Arg Val Val ValVal PhePhe AsnAsn 3155 3155 3160 3160 3165 3165

Gly Val Gly Val Ser SerPhe PheSer SerThr ThrPhe PheGluGlu Glu Glu Ala Ala Ala Ala Leu Leu CysCys ThrThr PhePhe 3170 3170 3175 3175 3180 3180

Leu Leu Leu Leu Asn AsnLys LysGlu GluMet MetTyr TyrLeuLeu Lys Lys Leu Leu Arg Arg Ser Ser Asp Asp Val Val Leu Leu 3185 3185 3190 3190 3195 3195

Leu Pro Leu Pro Leu LeuThr ThrGln GlnTyr TyrAsn AsnArgArg Tyr Tyr Leu Leu Ala Ala Leu Leu TyrTyr AsnAsn LysLys 3200 3200 3205 3205 3210 3210

Tyr Lys Tyr Lys Tyr TyrPhe PheSer SerGly GlyAla AlaMetMet Asp Asp Thr Thr Thr Thr Ser Ser TyrTyr ArgArg GluGlu 3215 3215 3220 3220 3225 3225

Ala Ala Ala Ala Cys CysCys CysHis HisLeu LeuAla AlaLysLys Ala Ala Leu Leu Asn Asn Asp Asp Phe Phe Ser Ser Asn Asn 3230 3230 3235 3235 3240

62

Ser Gly Ser Gly Ser SerAsp AspVal ValLeu LeuTyr TyrGlnGln Pro Pro Pro Pro Gln Gln Thr Thr SerSer IleIle ThrThr 3245 3245 3250 3250 3255 3255

Ser Ser Ala ValLeu Ala Val LeuGln GlnSer SerGly GlyPhe PheArg ArgLys LysMet MetAla AlaPhePhe ProPro SerSer 3260 3260 3265 3265 3270 3270

Gly Lys Gly Lys Val ValGlu GluGly GlyCys CysMet MetValVal Gln Gln Val Val Thr Thr Cys Cys GlyGly ThrThr ThrThr 3275 3275 3280 3280 3285 3285

Thr Leu Thr Leu Asn AsnGly GlyLeu LeuTrp TrpLeu LeuAspAsp Asp Asp Val Val Val Val Tyr Tyr CysCys ProPro ArgArg 3290 3290 3295 3295 3300 3300

His Val His Val IIle Cys Thr le Cys Thr Ser Ser Glu Glu Asp AspMet MetLeu LeuAsn AsnPro ProAsnAsn Tyr Tyr Glu Glu 3305 3305 3310 3310 3315 3315

Asp Leu Asp Leu Leu LeuIle IleArg ArgLys LysSer SerAsnAsn His His Asn Asn Phe Phe Leu Leu Val Val Gln Gln Ala Ala 3320 3320 3325 3325 3330 3330

Gly Asn Gly Asn Val ValGln GlnLeu LeuArg ArgVal ValIleIle Gly Gly His His Ser Ser Met Met GlnGln AsnAsn CysCys 3335 3335 3340 3340 3345 3345

Val Leu Val Leu Lys LysLeu LeuLys LysVal ValAsp AspThrThr Ala Ala Asn Asn Pro Pro Lys Lys Thr Thr Pro Pro Lys Lys 3350 3350 3355 3355 3360 3360

Tyr Lys Tyr Lys Phe PheVal ValArg ArgIle IleGln GlnProPro Gly Gly Gln Gln Thr Thr Phe Phe SerSer ValVal LeuLeu 3365 3365 3370 3370 3375 3375

Ala Cys Ala Cys Tyr TyrAsn AsnGly GlySer SerPro ProSerSer Gly Gly Val Val Tyr Tyr Gln Gln Cys Cys Ala Ala Met Met 3380 3380 3385 3385 3390 3390

Arg Pro Arg Pro Asn AsnPhe PheThr ThrIle IleLys LysGlyGly Ser Ser Phe Phe Leu Leu Asn Asn Gly Gly Ser Ser Cys Cys

63

3395 3395 3400 3400 3405 3405

Gly Ser Gly Ser Val ValGly GlyPhe PheAsn AsnIle IleAspAsp Tyr Tyr Asp Asp Cys Cys Val Val SerSer PhePhe CysCys 3410 3410 3415 3415 3420 3420

Tyr Met Tyr Met His HisHis HisMet MetGlu GluLeu LeuProPro Thr Thr Gly Gly Val Val His His AlaAla GlyGly ThrThr 3425 3425 3430 3430 3435 3435

Asp Leu Asp Leu Glu GluGly GlyAsn AsnPhe PheTyr TyrGlyGly Pro Pro Phe Phe Val Val Asp Asp Arg Arg Gln Gln Thr Thr 3440 3440 3445 3445 3450 3450

Ala Gln Ala Gln Ala AlaAla AlaGly GlyThr ThrAsp AspThrThr Thr Thr Ile Ile Thr Thr Val Val Asn Asn Val Val Leu Leu 3455 3455 3460 3460 3465 3465

Ala Trp Ala Trp Leu LeuTyr TyrAla AlaAla AlaVal ValIleIle Asn Asn Gly Gly Asp Asp Arg Arg Trp Trp Phe Phe Leu Leu 3470 3470 3475 3475 3480 3480

Asn Arg Asn Arg Phe PheThr ThrThr ThrThr ThrLeu LeuAsnAsn Asp Asp Phe Phe Asn Asn Leu Leu Val Val Ala Ala Met Met 3485 3485 3490 3490 3495 3495

Lys Tyr Lys Tyr Asn AsnTyr TyrGlu GluPro ProLeu LeuThrThr Gln Gln Asp Asp His His Val Val AspAsp Ile I le Leu Leu 3500 3500 3505 3505 3510 3510

Gly Pro Gly Pro Leu LeuSer SerAla AlaGln GlnThr ThrGlyGly IleAla I le AlaVal ValLeu LeuAspAsp Met Met Cys Cys 3515 3515 3520 3520 3525 3525

Ala Ser Ala Ser Leu LeuLys LysGlu GluLeu LeuLeu LeuGlnGln Asn Asn Gly Gly Met Met Asn Asn Gly Gly Arg Arg Thr Thr 3530 3530 3535 3535 3540 3540

Ile Ile Leu Gly Ser Leu Gly Ser Ala Ala Leu Leu Leu Leu Glu GluAsp AspGlu GluPhe PheThr ThrProPro PhePhe AspAsp 3545 3545 3550 3550 3555

64

Val Val Val Val Arg ArgGln GlnCys CysSer SerGly GlyValVal Thr Thr Phe Phe Gln Gln Ser Ser Ala Ala Val Val Lys Lys 3560 3560 3565 3565 3570 3570

Arg Thr Arg Thr Ile IleLys LysGly GlyThr ThrHis HisHisHis Trp Trp Leu Leu Leu Leu Leu Leu Thr Thr Ile Ile Leu Leu 3575 3575 3580 3580 3585 3585

Thr Ser Thr Ser Leu LeuLeu LeuVal ValLeu LeuVal ValGlnGln Ser Ser Thr Thr Gln Gln Trp Trp SerSer LeuLeu PhePhe 3590 3590 3595 3595 3600 3600

Phe Phe Phe LeuTyr Phe Leu TyrGlu GluAsn AsnAla AlaPhe PheLeu LeuPro ProPhe PheAla AlaMetMet GlyGly IleIle 3605 3605 3610 3610 3615 3615

Ile Ile Ala Met Ser Ala Met Ser Ala Ala Phe Phe Ala Ala Met MetMet MetPhe PheVal ValLys LysHisHis LysLys HisHis 3620 3620 3625 3625 3630 3630

Ala Phe Ala Phe Leu LeuCys CysLeu LeuPhe PheLeu LeuLeuLeu Pro Pro Ser Ser Leu Leu Ala Ala Thr Thr Val Val Ala Ala 3635 3635 3640 3640 3645 3645

Tyr Phe Tyr Phe Asn AsnMet MetVal ValTyr TyrMet MetProPro Ala Ala Ser Ser Trp Trp Val Val MetMet ArgArg Ile I le 3650 3650 3655 3655 3660 3660

Met Thr Met Thr Trp TrpLeu LeuAsp AspMet MetVal ValAspAsp Thr Thr Ser Ser Leu Leu Ser Ser Gly Gly Phe Phe Lys Lys 3665 3665 3670 3670 3675 3675

Leu Lys Leu Lys Asp AspCys CysVal ValMet MetTyr TyrAlaAla Ser Ser Ala Ala Val Val Val Val LeuLeu LeuLeu IleIle 3680 3680 3685 3685 3690 3690

Leu Met Leu Met Thr ThrAla AlaArg ArgThr ThrVal ValTyrTyr Asp Asp Asp Asp Gly Gly Ala Ala ArgArg ArgArg ValVal 3695 3695 3700 3700 3705 3705

Trp Thr Trp Thr Leu LeuMet MetAsn AsnVal ValLeu LeuThrThr Leu Leu Val Val Tyr Tyr Lys Lys ValVal TyrTyr TyrTyr 3710 3710 3715 3715 3720

65

Gly Asn Gly Asn Ala AlaLeu LeuAsp AspGln GlnAla AlaIleIle Ser Ser Met Met Trp Trp Ala Ala LeuLeu IleIle IleIle 3725 3725 3730 3730 3735 3735

Ser Val Thr Ser Val ThrSer SerAsn AsnTyr TyrSer SerGlyGly Val Val Val Val Thr Thr Thr Thr Val Val Met Met Phe Phe 3740 3740 3745 3745 3750 3750

Leu Ala Leu Ala Arg ArgGly GlyIle IleVal ValPhe PheMetMet Cys Cys Val Val Glu Glu Tyr Tyr CysCys ProPro IleIle 3755 3755 3760 3760 3765 3765

Phe Phe Phe IleThr Phe Ile ThrGly GlyAsn AsnThr ThrLeu LeuGln GlnCys CysI Ile MetLeu le Met LeuVal ValTyr Tyr 3770 3770 3775 3775 3780 3780

Cys Phe Cys Phe Leu LeuGly GlyTyr TyrPhe PheCys CysThrThr Cys Cys Tyr Tyr Phe Phe Gly Gly LeuLeu PhePhe CysCys 3785 3785 3790 3790 3795 3795

Leu Leu Leu Leu Asn AsnArg ArgTyr TyrPhe PheArg ArgLeuLeu Thr Thr Leu Leu Gly Gly Val Val TyrTyr AspAsp TyrTyr 3800 3800 3805 3805 3810 3810

Leu Val Leu Val Ser SerThr ThrGln GlnGlu GluPhe PheArgArg Tyr Tyr Met Met Asn Asn Ser Ser GlnGln GlyGly LeuLeu 3815 3815 3820 3820 3825 3825

Leu Pro Leu Pro Pro ProLys LysAsn AsnSer SerIle IleAspAsp Ala Ala Phe Phe Lys Lys Leu Leu Asn Asn Ile Ile Lys Lys 3830 3830 3835 3835 3840 3840

Leu Leu Leu Leu Gly GlyVal ValGly GlyGly GlyLys LysProPro Cys Cys IleLys I le LysVal ValAlaAla Thr Thr Val Val 3845 3845 3850 3850 3855 3855

Gln Ser Gln Ser Lys LysMet MetSer SerAsp AspVal ValLysLys Cys Cys Thr Thr Ser Ser Val Val ValVal LeuLeu LeuLeu 3860 3860 3865 3865 3870 3870

Ser Val Ser Val Leu LeuGln GlnGln GlnLeu LeuArg ArgValVal Glu Glu Ser Ser Ser Ser Ser Ser LysLys LeuLeu TrpTrp 3875 3875 3880 3880 3885

66

Ala Gln Ala Gln Cys CysVal ValGln GlnLeu LeuHis HisAsnAsn Asp Asp Ile Ile Leu Leu Leu Leu Ala Ala Lys Lys Asp Asp 3890 3890 3895 3895 3900 3900

Thr Thr Thr Thr Glu GluAla AlaPhe PheGlu GluLys LysMetMet Val Val Ser Ser Leu Leu Leu Leu SerSer ValVal LeuLeu 3905 3905 3910 3910 3915 3915

Leu Ser Leu Ser Met MetGln GlnGly GlyAla AlaVal ValAspAsp IleAsn I le AsnLys LysLeu LeuCysCys Glu Glu Glu Glu 3920 3920 3925 3925 3930 3930

Met Leu Met Leu Asp AspAsn AsnArg ArgAla AlaThr ThrLeuLeu Gln Gln Ala Ala IleAla I le AlaSerSer Glu Glu Phe Phe 3935 3935 3940 3940 3945 3945

Ser Ser Ser Ser Leu LeuPro ProSer SerTyr TyrAla AlaAlaAla Phe Phe Ala Ala Thr Thr Ala Ala GlnGln GluGlu AlaAla 3950 3950 3955 3955 3960 3960

Tyr Glu Tyr Glu Gln GlnAla AlaVal ValAla AlaAsn AsnGlyGly Asp Asp Ser Ser Glu Glu Val Val ValVal LeuLeu LysLys 3965 3965 3970 3970 3975 3975

Lys Leu Lys Leu Lys LysLys LysSer SerLeu LeuAsn AsnValVal Ala Ala Lys Lys Ser Ser Glu Glu PhePhe AspAsp ArgArg 3980 3980 3985 3985 3990 3990

Asp Ala Asp Ala Ala AlaMet MetGln GlnArg ArgLys LysLeuLeu Glu Glu Lys Lys Met Met Ala Ala Asp Asp Gln Gln Ala Ala 3995 3995 4000 4000 4005 4005

Met Thr Met Thr Gln GlnMet MetTyr TyrLys LysGln GlnAlaAla Arg Arg Ser Ser Glu Glu Asp Asp Lys Lys Arg Arg Ala Ala 4010 4010 4015 4015 4020 4020

Lys Val Lys Val Thr ThrSer SerAla AlaMet MetGln GlnThrThr Met Met Leu Leu Phe Phe Thr Thr MetMet LeuLeu ArgArg 4025 4025 4030 4030 4035 4035

Lys Leu Lys Leu Asp AspAsn AsnAsp AspAla AlaLeu LeuAsnAsn Asn Asn Ile Ile Ile Ile Asn Asn AsnAsn AlaAla ArgArg

67

4040 4040 4045 4045 4050 4050

Asp Gly Asp Gly Cys CysVal ValPro ProLeu LeuAsn AsnIleIle Ile Ile Pro Pro Leu Leu Thr Thr Thr Thr Ala Ala Ala Ala 4055 4055 4060 4060 4065 4065

Lys Leu Lys Leu Met MetVal ValVal ValIle IlePro ProAspAsp Tyr Tyr Asn Asn Thr Thr Tyr Tyr LysLys AsnAsn ThrThr 4070 4070 4075 4075 4080 4080

Cys Asp Cys Asp Gly GlyThr ThrThr ThrPhe PheThr ThrTyrTyr Ala Ala Ser Ser Ala Ala Leu Leu TrpTrp GluGlu IleIle 4085 4085 4090 4090 4095 4095

Gln Gln Gln Gln Val ValVal ValAsp AspAla AlaAsp AspSerSer Lys Lys Ile Ile Val Val Gln Gln LeuLeu SerSer GluGlu 4100 4100 4105 4105 4110 4110

Ile Ile Ser Met Asp Ser Met Asp Asn Asn Ser Ser Pro Pro Asn AsnLeu LeuAla AlaTrp TrpPro ProLeuLeu Ile Val I (le Val 4115 4115 4120 4120 4125 4125

Thr Ala Thr Ala Leu LeuArg ArgAla AlaAsn AsnSer SerAlaAla Val Val Lys Lys Leu Leu Gln Gln AsnAsn AsnAsn GluGlu 4130 4130 4135 4135 4140 4140

Leu Ser Leu Ser Pro ProVal ValAla AlaLeu LeuArg ArgGlnGln Met Met Ser Ser Cys Cys Ala Ala AlaAla GlyGly ThrThr 4145 4145 4150 4150 4155 4155

Thr Gln Thr Gln Thr ThrAla AlaCys CysThr ThrAsp AspAspAsp Asn Asn Ala Ala Leu Leu Ala Ala TyrTyr TyrTyr AsnAsn 4160 4160 4165 4165 4170 4170

Thr Thr Thr Thr Lys LysGly GlyGly GlyArg ArgPhe PheValVal Leu Leu Ala Ala Leu Leu Leu Leu SerSer AspAsp LeuLeu 4175 4175 4180 4180 4185 4185

Gln Asp Gln Asp Leu LeuLys LysTrp TrpAla AlaArg ArgPhePhe Pro Pro Lys Lys Ser Ser Asp Asp Gly Gly Thr Thr Gly Gly 4190 4190 4195 4195 4200

68

Thr Ile Thr Ile Tyr TyrThr ThrGlu GluLeu LeuGlu GluProPro Pro Pro Cys Cys Arg Arg Phe Phe ValVal ThrThr AspAsp 4205 4205 4210 4210 4215 4215

Thr Pro Thr Pro Lys LysGly GlyPro ProLys LysVal ValLysLys Tyr Tyr Leu Leu Tyr Tyr Phe Phe Ile Ile Lys Lys Gly Gly 4220 4220 4225 4225 4230 4230

Leu Asn Leu Asn Asn AsnLeu LeuAsn AsnArg ArgGly GlyMetMet Val Val Leu Leu Gly Gly Ser Ser LeuLeu AlaAla AlaAla 4235 4235 4240 4240 4245 4245

Thr Val Thr Val Arg ArgLeu LeuGln GlnAla AlaGly GlyAsnAsn Ala Ala Thr Thr Glu Glu Val Val ProPro AlaAla AsnAsn 4250 4250 4255 4255 4260 4260

Ser Thr Ser Thr Val ValLeu LeuSer SerPhe PheCys CysAlaAla Phe Phe Ala Ala Val Val Asp Asp AlaAla AlaAla LysLys 4265 4265 4270 4270 4275 4275

Ala Tyr Ala Tyr Lys LysAsp AspTyr TyrLeu LeuAla AlaSerSer Gly Gly Gly Gly Gln Gln Pro Pro Ile Ile Thr Thr Asn Asn 4280 4280 4285 4285 4290 4290

Cys Val Cys Val Lys LysMet MetLeu LeuCys CysThr ThrHisHis Thr Thr Gly Gly Thr Thr Gly Gly GlnGln AlaAla IleIle 4295 4295 4300 4300 4305 4305

Thr Val Thr Val Thr ThrPro ProGlu GluAla AlaAsn AsnMetMet Asp Asp Gln Gln Glu Glu Ser Ser Phe Phe Gly Gly Gly Gly 4310 4310 4315 4315 4320 4320

Ala Ser Ala Ser Cys CysCys CysLeu LeuTyr TyrCys CysArgArg Cys Cys His His Ile Ile Asp Asp His His Pro Pro Asn Asn 4325 4325 4330 4330 4335 4335

Pro Lys Pro Lys Gly GlyPhe PheCys CysAsp AspLeu LeuLysLys Gly Gly Lys Lys Tyr Tyr Val Val GlnGln IleIle ProPro 4340 4340 4345 4345 4350 4350

Thr Thr Thr Thr Cys CysAla AlaAsn AsnAsp AspPro ProValVal Gly Gly Phe Phe Thr Thr Leu Leu LysLys AsnAsn ThrThr 4355 4355 4360 4360 4365

69

Val Cys Val Cys Thr ThrVal ValCys CysGly GlyMet MetTrpTrp Lys Lys Gly Gly Tyr Tyr Gly Gly Cys Cys Ser Ser Cys Cys 4370 4370 4375 4375 4380 4380

Asp Gln Asp Gln Leu LeuArg ArgGlu GluPro ProMet MetLeuLeu Gln Gln Ser Ser Ala Ala Asp Asp Ala Ala Gln Gln Ser Ser 4385 4385 4390 4390 4395 4395

Phe Leu Phe Leu Asn AsnArg ArgVal ValCys CysGly GlyValVal Ser Ser Ala Ala Ala Ala Arg Arg LeuLeu ThrThr ProPro 4400 4400 4405 4405 4410 4410

Cys Gly Cys Gly Thr ThrGly GlyThr ThrSer SerThr ThrAspAsp Val Val Val Val Tyr Tyr Arg Arg AlaAla PhePhe AspAsp 4415 4415 4420 4420 4425 4425

Ile Ile Tyr Asn Asp Tyr Asn Asp Lys Lys Val Val Ala Ala Gly GlyPhe PheAla AlaLys LysPhe PheLeuLeu LysLys ThrThr 4430 4430 4435 4435 4440 4440

Asn Cys Asn Cys Cys CysArg ArgPhe PheGln GlnGlu GluLysLys Asp Asp Glu Glu Asp Asp Asp Asp Asn Asn Leu Leu Ile Ile 4445 4445 4450 4450 4455 4455

Asp Ser Asp Ser Tyr TyrPhe PheVal ValVal ValLys LysArgArg His His Thr Thr Phe Phe Ser Ser Asn Asn Tyr Tyr Gln Gln 4460 4460 4465 4465 4470 4470

His Glu His Glu Glu GluThr ThrIle IleTyr TyrAsn AsnLeuLeu Leu Leu Lys Lys Asp Asp Cys Cys Pro Pro Ala Ala Val Val 4475 4475 4480 4480 4485 4485

Ala Lys Ala Lys His HisAsp AspPhe PhePhe PheLys LysPhePhe Arg Arg Ile I l Asp Gly e Asp Gly Asp AspMet MetVal Val 4490 4490 4495 4495 4500 4500

Pro His Pro His Ile IleSer SerArg ArgGln GlnArg ArgLeuLeu Thr Thr Lys Lys Tyr Tyr Thr Thr MetMet AlaAla AspAsp 4505 4505 4510 4510 4515 4515

Leu Val Leu Val Tyr TyrAla AlaLeu LeuArg ArgHis HisPhePhe Asp Asp Glu Glu Gly Gly Asn Asn CysCys AspAsp ThrThr 4520 4520 4525 4525 4530

70

Leu Lys Leu Lys Glu GluIle IleLeu LeuVal ValThr ThrTyrTyr Asn Asn Cys Cys Cys Cys Asp Asp AspAsp AspAsp TyrTyr 4535 4535 4540 4540 4545 4545

Phe Phe Asn LysLys Asn Lys LysAsp AspTrp TrpTyr TyrAsp AspPhe PheVal ValGlu GluAsn AsnProPro AspAsp IleIle 4550 4550 4555 4555 4560 4560

Leu Arg Leu Arg Val ValTyr TyrAla AlaAsn AsnLeu LeuGlyGly Glu Glu Arg Arg Val Val Arg Arg GlnGln AlaAla LeuLeu 4565 4565 4570 4570 4575 4575

Leu Lys Leu Lys Thr ThrVal ValGln GlnPhe PheCys CysAspAsp Ala Ala Met Met Arg Arg Asn Asn AlaAla GlyGly IleIle 4580 4580 4585 4585 4590 4590

Val Gly Val Gly Val ValLeu LeuThr ThrLeu LeuAsp AspAsnAsn Gln Gln Asp Asp Leu Leu Asn Asn Gly Gly Asn Asn Trp Trp 4595 4595 4600 4600 4605 4605

Tyr Asp Tyr Asp Phe PheGly GlyAsp AspPhe PheIle IleGlnGln Thr Thr Thr Thr Pro Pro Gly Gly SerSer GlyGly ValVal 4610 4610 4615 4615 4620 4620

Pro Val Pro Val Val ValAsp AspSer SerTyr TyrTyr TyrSerSer Leu Leu Leu Leu Met Met Pro Pro IleIle LeuLeu ThrThr 4625 4625 4630 4630 4635 4635

Leu Thr Leu Thr Arg ArgAla AlaLeu LeuThr ThrAla AlaGluGlu Ser Ser His His Val Val Asp Asp ThrThr AspAsp LeuLeu 4640 4640 4645 4645 4650 4650

Thr Lys Thr Lys Pro ProTyr TyrIle IleLys LysTrp TrpAspAsp Leu Leu Leu Leu Lys Lys Tyr Tyr AspAsp PhePhe ThrThr 4655 4655 4660 4660 4665 4665

Glu Glu Glu Glu Arg ArgLeu LeuLys LysLeu LeuPhe PheAspAsp Arg Arg Tyr Tyr Phe Phe Lys Lys TyrTyr TrpTrp AspAsp 4670 4670 4675 4675 4680 4680

Gln Thr Gln Thr Tyr TyrHis HisPro ProAsn AsnCys CysValVal Asn Asn Cys Cys Leu Leu Asp Asp Asp Asp Arg Arg Cys Cys

71

4685 4685 4690 4690 4695 4695

Ile Ile Leu His Cys Leu His Cys Ala Ala Asn Asn Phe Phe Asn AsnVal ValLeu LeuPhe PheSer SerThrThr ValVal PhePhe 4700 4700 4705 4705 4710 4710

Pro Pro Pro Pro Thr ThrSer SerPhe PheGly GlyPro ProLeuLeu Val Val Arg Arg Lys Lys Ile Ile PhePhe ValVal AspAsp 4715 4715 4720 4720 4725 4725

Gly Val Gly Val Pro ProPhe PheVal ValVal ValSer SerThrThr Gly Gly Tyr Tyr His His Phe Phe ArgArg GluGlu LeuLeu 4730 4730 4735 4735 4740 4740

Gly Val Gly Val Val ValHis HisAsn AsnGln GlnAsp AspValVal Asn Asn Leu Leu His His Ser Ser SerSer ArgArg LeuLeu 4745 4745 4750 4750 4755 4755

Ser Phe Ser Phe Lys LysGlu GluLeu LeuLeu LeuVal ValTyrTyr Ala Ala Ala Ala Asp Asp Pro Pro AlaAla MetMet HisHis 4760 4760 4765 4765 4770 4770

Ala Ala Ala Ala Ser SerGly GlyAsn AsnLeu LeuLeu LeuLeuLeu Asp Asp Lys Lys Arg Arg Thr Thr Thr Thr Cys Cys Phe Phe 4775 4775 4780 4780 4785 4785

Ser Val Ser Val Ala AlaAla AlaLeu LeuThr ThrAsn AsnAsnAsn Val Val Ala Ala Phe Phe Gln Gln ThrThr ValVal LysLys 4790 4790 4795 4795 4800 4800

Pro Gly Pro Gly Asn AsnPhe PheAsn AsnLys LysAsp AspPhePhe Tyr Tyr Asp Asp Phe Phe Ala Ala ValVal SerSer LysLys 4805 4805 4810 4810 4815 4815

Gly Phe Gly Phe Phe PheLys LysGlu GluGly GlySer SerSerSer Val Val Glu Glu Leu Leu Lys Lys HisHis PhePhe PhePhe 4820 4820 4825 4825 4830 4830

Phe Ala Phe Ala Gln GlnAsp AspGly GlyAsn AsnAla AlaAlaAla Ile Ile Ser Ser Asp Asp Tyr Tyr AspAsp TyrTyr TyrTyr 4835 4835 4840 4840 4845

72

Arg Tyr Arg Tyr Asn AsnLeu LeuPro ProThr ThrMet MetCysCys Asp Asp IleArg I le ArgGln GlnLeuLeu Leu Leu Phe Phe 4850 4850 4855 4855 4860 4860

Val Val Val Val Glu GluVal ValVal ValAsp AspLys LysTyrTyr Phe Phe Asp Asp Cys Cys Tyr Tyr Asp Asp Gly Gly Gly Gly 4865 4865 4870 4870 4875 4875

Cys Ile Cys Ile Asn AsnAla AlaAsn AsnGln GlnVal ValIleIle Val Val Asn Asn Asn Asn Leu Leu AspAsp LysLys SerSer 4880 4880 4885 4885 4890 4890

Ala Gly Ala Gly Phe PhePro ProPhe PheAsn AsnLys LysTrpTrp Gly Gly Lys Lys Ala Ala Arg Arg Leu Leu Tyr Tyr Tyr Tyr 4895 4895 4900 4900 4905 4905

Asp Ser Asp Ser Met MetSer SerTyr TyrGlu GluAsp AspGlnGln Asp Asp Ala Ala Leu Leu Phe Phe Ala Ala Tyr Tyr Thr Thr 4910 4910 4915 4915 4920 4920

Lys Arg Lys Arg Asn AsnVal ValIle IlePro ProThr ThrIleIle Thr Thr Gln Gln Met Met Asn Asn LeuLeu LysLys TyrTyr 4925 4925 4930 4930 4935 4935

Ala Ile Ala Ile Ser SerAla AlaLys LysAsn AsnArg ArgAlaAla Arg Arg Thr Thr Val Val Ala Ala Gly Gly Val Val Ser Ser 4940 4940 4945 4945 4950 4950

Ile Ile Cys Ser Thr Cys Ser Thr Met Met Thr Thr Asn Asn Arg ArgGln GlnPhe PheHis HisGln GlnLysLys LeuLeu LeuLeu 4955 4955 4960 4960 4965 4965

Lys Ser Lys Ser Ile IleAla AlaAla AlaThr ThrArg ArgGlyGly Ala Ala Thr Thr Val Val Val Val IleIle GlyGly ThrThr 4970 4970 4975 4975 4980 4980

Ser Lys Ser Lys Phe PheTyr TyrGly GlyGly GlyTrp TrpHisHis Asn Asn Met Met Leu Leu Lys Lys ThrThr ValVal TyrTyr 4985 4985 4990 4990 4995 4995

Ser Asp Ser Asp Val ValGlu GluAsn AsnPro ProHis HisLeuLeu Met Met Gly Gly Trp Trp Asp Asp TyrTyr ProPro LysLys 5000 5000 5005 5005 5010

73

Cys Asp Cys Asp Arg ArgAla AlaMet MetPro ProAsn AsnMetMet Leu Leu Arg Arg IleMet I le MetAlaAla Ser Ser Leu Leu 5015 5015 5020 5020 5025 5025

Val Leu Val Leu Ala AlaArg ArgLys LysHis HisThr ThrThrThr Cys Cys Cys Cys Ser Ser Leu Leu Ser Ser His His Arg Arg 5030 5030 5035 5035 5040 5040

Phe Phe Tyr ArgLeu Tyr Arg LeuAla AlaAsn AsnGlu GluCys CysAla AlaGln GlnVal ValLeu LeuSerSer GluGlu MetMet 5045 5045 5050 5050 5055 5055

Val Met Val Met Cys CysGly GlyGly GlySer SerLeu LeuTyrTyr Val Val Lys Lys Pro Pro Gly Gly Gly Gly Thr Thr Ser Ser 5060 5060 5065 5065 5070 5070

Ser Gly Ser Gly Asp AspAla AlaThr ThrThr ThrAla AlaTyrTyr Ala Ala Asn Asn Ser Ser Val Val PhePhe AsnAsn IleIle 5075 5075 5080 5080 5085 5085

Cys Gln Cys Gln Ala AlaVal ValThr ThrAla AlaAsn AsnValVal Asn Asn Ala Ala Leu Leu Leu Leu SerSer ThrThr AspAsp 5090 5090 5095 5095 5100 5100

Gly Asn Gly Asn Lys LysIle IleAla AlaAsp AspLys LysTyrTyr Val Val Arg Arg Asn Asn Leu Leu GlnGln HisHis ArgArg 5105 5105 5110 5110 5115 5115

Leu Tyr Leu Tyr Glu GluCys CysLeu LeuTyr TyrArg ArgAsnAsn Arg Arg Asp Asp Val Val Asp Asp Thr Thr Asp Asp Phe Phe 5120 5120 5125 5125 5130 5130

Val Asn Val Asn Glu GluPhe PheTyr TyrAla AlaTyr TyrLeuLeu Arg Arg Lys Lys His His Phe Phe Ser Ser Met Met Met Met 5135 5135 5140 5140 5145 5145

Ile Ile Leu Ser Asp Leu Ser Asp Asp Asp Ala Ala Val Val Val ValCys CysPhe PheAsn AsnSer SerThrThr TyrTyr AlaAla 5150 5150 5155 5155 5160 5160

Ser Gln Ser Gln Gly GlyLeu LeuVal ValAla AlaSer SerIleIle Lys Lys Asn Asn Phe Phe Lys Lys SerSer ValVal LeuLeu 5165 5165 5170 5170 5175

74

Tyr Tyr Tyr Tyr Gln GlnAsn AsnAsn AsnVal ValPhe PheMetMet Ser Ser Glu Glu Ala Ala Lys Lys CysCys TrpTrp ThrThr 5180 5180 5185 5185 5190 5190

Glu Thr Glu Thr Asp AspLeu LeuThr ThrLys LysGly GlyProPro His His Glu Glu Phe Phe Cys Cys SerSer GlnGln HisHis 5195 5195 5200 5200 5205 5205

Thr Met Thr Met Leu LeuVal ValLys LysGln GlnGly GlyAspAsp Asp Asp Tyr Tyr Val Val Tyr Tyr LeuLeu ProPro TyrTyr 5210 5210 5215 5215 5220 5220

Pro Asp Pro Asp Pro ProSer SerArg ArgIle IleLeu LeuGlyGly Ala Ala Gly Gly Cys Cys Phe Phe ValVal AspAsp AspAsp 5225 5225 5230 5230 5235 5235

Ile Ile Val Lys Thr Val Lys Thr Asp Asp Gly Gly Thr Thr Leu LeuMet MetI Ile Glu Arg le Glu Arg Phe PheVal ValSer Ser 5240 5240 5245 5245 5250 5250

Leu Ala Leu Ala Ile IleAsp AspAla AlaTyr TyrPro ProLeuLeu Thr Thr Lys Lys His His Pro Pro AsnAsn GlnGln GluGlu 5255 5255 5260 5260 5265 5265

Tyr Ala Tyr Ala Asp AspVal ValPhe PheHis HisLeu LeuTyrTyr Leu Leu Gln Gln Tyr Tyr Ile Ile ArgArg LysLys LeuLeu 5270 5270 5275 5275 5280 5280

His Asp His Asp Glu GluLeu LeuThr ThrGly GlyHis HisMetMet Leu Leu Asp Asp Met Met Tyr Tyr SerSer ValVal MetMet 5285 5285 5290 5290 5295 5295

Leu Thr Leu Thr Asn AsnAsp AspAsn AsnThr ThrSer SerArgArg Tyr Tyr Trp Trp Glu Glu Pro Pro GluGlu PhePhe TyrTyr 5300 5300 5305 5305 5310 5310

Glu Ala Glu Ala Met MetTyr TyrThr ThrPro ProHis HisThrThr Val Val Leu Leu Gln Gln Ala Ala ValVal GlyGly AlaAla 5315 5315 5320 5320 5325 5325

Cys Val Cys Val Leu LeuCys CysAsn AsnSer SerGln GlnThrThr Ser Ser Leu Leu Arg Arg Cys Cys GlyGly AlaAla CysCys

75

5330 5330 5335 5335 5340 5340

Ile Ile Arg Arg Pro Arg Arg Pro Phe Phe Leu Leu Cys Cys Cys CysLys LysCys CysCys CysTyr TyrAspAsp HisHis ValVal 5345 5345 5350 5350 5355 5355

Ile Ile Ser Thr Ser Ser Thr Ser His His Lys Lys Leu Leu Val ValLeu LeuSer SerVal ValAsn AsnProPro TyrTyr ValVal 5360 5360 5365 5365 5370 5370

Cys Asn Cys Asn Ala AlaPro ProGly GlyCys CysAsp AspValVal Thr Thr Asp Asp Val Val Thr Thr GlnGln LeuLeu TyrTyr 5375 5375 5380 5380 5385 5385

Leu Gly Leu Gly Gly GlyMet MetSer SerTyr TyrTyr TyrCysCys Lys Lys Ser Ser His His Lys Lys ProPro ProPro IleIle 5390 5390 5395 5395 5400 5400

Ser Ser Phe ProLeu Phe Pro LeuCys CysAla AlaAsn AsnGly GlyGln GlnVal ValPhe PheGly GlyLeuLeu TyrTyr LysLys 5405 5405 5410 5410 5415 5415

Asn Thr Asn Thr Cys CysVal ValGly GlySer SerAsp AspAsnAsn Val Val Thr Thr Asp Asp Phe Phe Asn Asn Ala Ala Ile Ile 5420 5420 5425 5425 5430 5430

Ala Thr Ala Thr Cys CysAsp AspTrp TrpThr ThrAsn AsnAlaAla Gly Gly Asp Asp Tyr Tyr Ile Ile Leu Leu Ala Ala Asn Asn 5435 5435 5440 5440 5445 5445

Thr Cys Thr Cys Thr ThrGlu GluArg ArgLeu LeuLys LysLeuLeu Phe Phe Ala Ala Ala Ala Glu Glu Thr Thr Leu Leu Lys Lys 5450 5450 5455 5455 5460 5460

Ala Thr Ala Thr Glu GluGlu GluThr ThrPhe PheLys LysLeuLeu Ser Ser Tyr Tyr Gly Gly Ile Ile Ala Ala Thr Thr Val Val 5465 5465 5470 5470 5475 5475

Arg Glu Arg Glu Val ValLeu LeuSer SerAsp AspArg ArgGluGlu Leu Leu His His Leu Leu Ser Ser Trp Trp Glu Glu Val Val 5480 5480 5485 5485 5490

76

Gly Lys Gly Lys Pro ProArg ArgPro ProPro ProLeu LeuAsnAsn Arg Arg Asn Asn Tyr Tyr Val Val PhePhe ThrThr GlyGly 5495 5495 5500 5500 5505 5505

Tyr Arg Tyr Arg Val ValThr ThrLys LysAsn AsnSer SerLysLys Val Val Gln Gln Ile Ile Gly Gly GluGlu TyrTyr ThrThr 5510 5510 5515 5515 5520 5520

Phe Glu Phe Glu Lys LysGly GlyAsp AspTyr TyrGly GlyAspAsp Ala Ala Val Val Val Val Tyr Tyr ArgArg GlyGly ThrThr 5525 5525 5530 5530 5535 5535

Thr Thr Thr Thr Tyr TyrLys LysLeu LeuAsn AsnVal ValGlyGly Asp Asp Tyr Tyr Phe Phe Val Val LeuLeu ThrThr SerSer 5540 5540 5545 5545 5550 5550

His Thr His Thr Val ValMet MetPro ProLeu LeuSer SerAlaAla Pro Pro Thr Thr Leu Leu Val Val Pro Pro Gln Gln Glu Glu 5555 5555 5560 5560 5565 5565

His Tyr His Tyr Val ValArg ArgIle IleThr ThrGly GlyLeuLeu Tyr Tyr Pro Pro Thr Thr Leu Leu AsnAsn Ile I le Ser Ser 5570 5570 5575 5575 5580 5580

Asp Glu Asp Glu Phe PheSer SerSer SerAsn AsnVal ValAlaAla Asn Asn Tyr Tyr Gln Gln Lys Lys Val Val Gly Gly Met Met 5585 5585 5590 5590 5595 5595

Gln Lys Gln Lys Tyr TyrSer SerThr ThrLeu LeuGln GlnGlyGly Pro Pro Pro Pro Gly Gly Thr Thr GlyGly LysLys SerSer 5600 5600 5605 5605 5610 5610

His Phe His Phe Ala AlaIle IleGly GlyLeu LeuAla AlaLeuLeu Tyr Tyr Tyr Tyr Pro Pro Ser Ser Ala Ala Arg Arg Ile Ile 5615 5615 5620 5620 5625 5625

Val Tyr Val Tyr Thr ThrAla AlaCys CysSer SerHis HisAlaAla Ala Ala Val Val Asp Asp Ala Ala Leu Leu Cys Cys Glu Glu 5630 5630 5635 5635 5640 5640

Lys Ala Lys Ala LeuLeu LysLys TyrTyr Leu Leu Pro Pro Ile Lys I le Asp AspCys LysSer CysArgSer ArgIleIle I (le Ile 5645 5645 5650 5650 5655

77

Pro Ala Pro Ala Arg ArgAla AlaArg ArgVal ValGlu GluCysCys Phe Phe Asp Asp Lys Lys Phe Phe LysLys ValVal AsnAsn 5660 5660 5665 5665 5670 5670

Ser Thr Ser Thr Leu LeuGlu GluGln GlnTyr TyrVal ValPhePhe Cys Cys Thr Thr Val Val Asn Asn AlaAla LeuLeu ProPro 5675 5675 5680 5680 5685 5685

Glu Thr Glu Thr Thr ThrAla AlaAsp AspIIle Val Val le Val ValPhe PheAsp AspGlu GluIle IleSerSer Met Met Ala Ala 5690 5690 5695 5695 5700 5700

Thr Asn Thr Asn Tyr TyrAsp AspLeu LeuSer SerVal ValValVal Asn Asn Ala Ala Arg Arg Leu Leu Arg Arg Ala Ala Lys Lys 5705 5705 5710 5710 5715 5715

His Tyr His Tyr Val ValTyr TyrIIle Gly Asp le Gly Asp Pro ProAla AlaGln GlnLeu LeuPro ProAlaAla Pro Pro Arg Arg 5720 5720 5725 5725 5730 5730

Thr Leu Thr Leu Leu LeuThr ThrLys LysGly GlyThr ThrLeuLeu Glu Glu Pro Pro Glu Glu Tyr Tyr PhePhe AsnAsn SerSer 5735 5735 5740 5740 5745 5745

Val Cys Val Cys Arg ArgLeu LeuMet MetLys LysThr ThrIleIle Gly Gly Pro Pro Asp Asp Met Met Phe Phe Leu Leu Gly Gly 5750 5750 5755 5755 5760 5760

Thr Cys Thr Cys Arg ArgArg ArgCys CysPro ProAla AlaGluGlu Ile Ile Val Val Asp Asp Thr Thr Val Val Ser Ser Ala Ala 5765 5765 5770 5770 5775 5775

Leu Val Leu Val Tyr TyrAsp AspAsn AsnLys LysLeu LeuLysLys Ala Ala His His Lys Lys Asp Asp Lys Lys Ser Ser Ala Ala 5780 5780 5785 5785 5790 5790

Gln Cys Gln Cys Phe PheLys LysMet MetPhe PheTyr TyrLysLys Gly Gly Val Val Ile Ile Thr Thr HisHis AspAsp ValVal 5795 5795 5800 5800 5805 5805

Ser Ser Ser Ser Ala AlaIle IleAsn AsnArg ArgPro ProGlnGln Ile Ile Gly Gly Val Val Val Val ArgArg GluGlu PhePhe 5810 5810 5815 5815 5820

78

Leu Thr Leu Thr Arg ArgAsn AsnPro ProAla AlaTrp TrpArgArg Lys Lys Ala Ala Val Val Phe Phe IleIle SerSer ProPro 5825 5825 5830 5830 5835 5835

Tyr Asn Tyr Asn Ser SerGln GlnAsn AsnAla AlaVal ValAlaAla Ser Ser Lys Lys Ile Ile Leu Leu Gly Gly Leu Leu Pro Pro 5840 5840 5845 5845 5850 5850

Thr Gln Thr Gln Thr ThrVal ValAsp AspSer SerSer SerGlnGln Gly Gly Ser Ser Glu Glu Tyr Tyr AspAsp TyrTyr ValVal 5855 5855 5860 5860 5865 5865

Ile Ile Phe Thr Gln Phe Thr Gln Thr Thr Thr Thr Glu Glu Thr ThrAla AlaHis HisSer SerCys CysAsnAsn ValVal AsnAsn 5870 5870 5875 5875 5880 5880

Arg Phe Arg Phe Asn AsnVal ValAla AlaIle IleThr ThrArgArg Ala Ala Lys Lys Val Val Gly Gly Ile Ile Leu Leu Cys Cys 5885 5885 5890 5890 5895 5895

Ile Ile Met Ser Asp Met Ser Asp Arg Arg Asp Asp Leu Leu Tyr TyrAsp AspLys LysLeu LeuGln GlnPhePhe ThrThr SerSer 5900 5900 5905 5905 5910 5910

Leu Glu Leu Glu Ile IlePro ProArg ArgArg ArgAsn AsnValVal Ala Ala Thr Thr Leu Leu Gln Gln AlaAla GluGlu AsnAsn 5915 5915 5920 5920 5925 5925

Val Thr Val Thr Gly GlyLeu LeuPhe PheLys LysAsp AspCysCys Ser Ser Lys Lys Val Val Ile Ile Thr Thr Gly Gly Leu Leu 5930 5930 5935 5935 5940 5940

His Pro His Pro Thr ThrGln GlnAla AlaPro ProThr ThrHisHis Leu Leu Ser Ser Val Val Asp Asp Thr Thr Lys Lys Phe Phe 5945 5945 5950 5950 5955 5955

Lys Thr Lys Thr Glu GluGly GlyLeu LeuCys CysVal ValAspAsp Ile Pro I (le Pro Gly Gly Ile Ile Pro ProLys LysAsp Asp 5960 5960 5965 5965 5970 5970

Met Thr Met Thr Tyr TyrArg ArgArg ArgLeu LeuIle IleSerSer Met Met Met Met Gly Gly Phe Phe Lys Lys Met Met Asn Asn

79

5975 5975 5980 5980 5985 5985

Tyr Gln Tyr Gln Val ValAsn AsnGly GlyTyr TyrPro ProAsnAsn Met Met Phe Phe Ile Ile Thr Thr Arg Arg Glu Glu Glu Glu 5990 5990 5995 5995 6000 6000

Ala Ile Ala Ile Arg ArgHis HisVal ValArg ArgAla AlaTrpTrp IleGly I le GlyPhe PheAsp AspValVal Glu Glu Gly Gly 6005 6005 6010 6010 6015 6015

Cys His Cys His Ala AlaThr ThrArg ArgGlu GluAla AlaValVal Gly Gly Thr Thr Asn Asn Leu Leu ProPro LeuLeu GlnGln 6020 6020 6025 6025 6030 6030

Leu Gly Leu Gly Phe PheSer SerThr ThrGly GlyVal ValAsnAsn Leu Leu Val Val Ala Ala Val Val ProPro ThrThr GlyGly 6035 6035 6040 6040 6045 6045

Tyr Val Tyr Val Asp AspThr ThrPro ProAsn AsnAsn AsnThrThr Asp Asp Phe Phe Ser Ser Arg Arg Val Val Ser Ser Ala Ala 6050 6050 6055 6055 6060 6060

Lys Pro Lys Pro Pro ProPro ProGly GlyAsp AspGln GlnPhePhe Lys Lys His His Leu Leu Ile Ile ProPro LeuLeu MetMet 6065 6065 6070 6070 6075 6075

Tyr Lys Tyr Lys Gly GlyLeu LeuPro ProTrp TrpAsn AsnValVal Val Val Arg Arg Ile Lys I (le Lys Ile IleVal ValGln Gln 6080 6080 6085 6085 6090 6090

Met Leu Met Leu Ser SerAsp AspThr ThrLeu LeuLys LysAsnAsn Leu Leu Ser Ser Asp Asp Arg Arg Val Val Val Val Phe Phe 6095 6095 6100 6100 6105 6105

Val Leu Val Leu Trp TrpAla AlaHis HisGly GlyPhe PheGluGlu Leu Leu Thr Thr Ser Ser Met Met Lys Lys Tyr Tyr Phe Phe 6110 6110 6115 6115 6120 6120

Val Lys Val Lys I Ile Gly Pro le Gly Pro Glu Glu Arg Arg Thr ThrCys CysCys CysLeu LeuCys CysAspAsp Arg Arg Arg Arg 6125 6125 6130 6130 6135

80

Ala Thr Ala Thr Cys CysPhe PheSer SerThr ThrAla AlaSerSer Asp Asp Thr Thr Tyr Tyr Ala Ala Cys Cys Trp Trp His His 6140 6140 6145 6145 6150 6150

His Ser His Ser Ile IleGly GlyPhe PheAsp AspTyr TyrValVal Tyr Tyr Asn Asn Pro Pro Phe Phe MetMet I eIle AspAsp 6155 6155 6160 6160 6165 6165

Val Gln Val Gln Gln GlnTrp TrpGly GlyPhe PheThr ThrGlyGly Asn Asn Leu Leu Gln Gln Ser Ser Asn Asn His His Asp Asp 6170 6170 6175 6175 6180 6180

Leu Tyr Leu Tyr Cys CysGln GlnVal ValHis HisGly GlyAsnAsn Ala Ala His His Val Val Ala Ala SerSer CysCys AspAsp 6185 6185 6190 6190 6195 6195

Ala Ile Ala Ile Met MetThr ThrArg ArgCys CysLeu LeuAlaAla Val Val His His Glu Glu Cys Cys Phe Phe Val Val Lys Lys 6200 6200 6205 6205 6210 6210

Arg Val Arg Val Asp AspTrp TrpThr ThrIle IleGlu GluTyrTyr Pro Pro IleIle I le IleGly GlyAspAsp Glu Glu Leu Leu 6215 6215 6220 6220 6225 6225

Lys Lys Ile AsnAla Ile Asn AlaAla AlaCys CysArg ArgLys LysVal ValGln GlnHis HisMet MetValVal ValVal LysLys 6230 6230 6235 6235 6240 6240

Ala Ala Ala Ala Leu LeuLeu LeuAla AlaAsp AspLys LysPhePhe Pro Pro Val Val Leu Leu His His Asp Asp Ile Ile Gly Gly 6245 6245 6250 6250 6255 6255

Asn Pro Asn Pro Lys LysAla AlaIle IleLys LysCys CysValVal Pro Pro Gln Gln Ala Ala Asp Asp Val Val Glu Glu Trp Trp 6260 6260 6265 6265 6270 6270

Lys Phe Lys Phe Tyr TyrAsp AspAla AlaGln GlnPro ProCysCys Ser Ser Asp Asp Lys Lys Ala Ala TyrTyr LysLys IleIle 6275 6275 6280 6280 6285 6285

Glu Glu Glu Glu Leu LeuPhe PheTyr TyrSer SerTyr TyrAlaAla Thr Thr His His Ser Ser Asp Asp LysLys PhePhe ThrThr 6290 6290 6295 6295 6300

81

Asp Gly Asp Gly Val ValCys CysLeu LeuPhe PheTrp TrpAsnAsn Cys Cys Asn Asn Val Val Asp Asp Arg Arg Tyr Tyr Pro Pro 6305 6305 6310 6310 6315 6315

Ala Asn Ala Asn Ser SerIle IleVal ValCys CysArg ArgPhePhe Asp Asp Thr Thr Arg Arg Val Val Leu Leu Ser Ser Asn Asn 6320 6320 6325 6325 6330 6330

Leu Asn Leu Asn Leu LeuPro ProGly GlyCys CysAsp AspGlyGly Gly Gly Ser Ser Leu Leu Tyr Tyr ValVal AsnAsn LysLys 6335 6335 6340 6340 6345 6345

His Ala His Ala Phe PheHis HisThr ThrPro ProAla AlaPhePhe Asp Asp Lys Lys Ser Ser Ala Ala PhePhe ValVal AsnAsn 6350 6350 6355 6355 6360 6360

Leu Lys Leu Lys Gln GlnLeu LeuPro ProPhe PhePhe PheTyrTyr Tyr Tyr Ser Ser Asp Asp Ser Ser ProPro CysCys GluGlu 6365 6365 6370 6370 6375 6375

Ser His Ser His Gly GlyLys LysGln GlnVal ValVal ValSerSer Asp Asp Ile Ile Asp Asp Tyr Tyr ValVal ProPro LeuLeu 6380 6380 6385 6385 6390 6390

Lys Ser Lys Ser Ala AlaThr ThrCys CysIle IleThr ThrArgArg Cys Cys Asn Asn Leu Leu Gly Gly GlyGly Ala AI a Val Val 6395 6395 6400 6400 6405 6405

Cys Arg Cys Arg His HisHis HisAla AlaAsn AsnGlu GluTyrTyr Arg Arg Leu Leu Tyr Tyr Leu Leu AspAsp AlaAla TyrTyr 6410 6410 6415 6415 6420 6420

Asn Met Asn Met Met MetIle IleSer SerAla AlaGly GlyPhePhe Ser Ser Leu Leu Trp Trp Val Val Tyr Tyr Lys Lys Gln Gln 6425 6425 6430 6430 6435 6435

Phe Asp Phe Asp Thr ThrTyr TyrAsn AsnLeu LeuTrp TrpAsnAsn Thr Thr Phe Phe Thr Thr Arg Arg LeuLeu GlnGln SerSer 6440 6440 6445 6445 6450 6450

Leu Glu Leu Glu Asn AsnVal ValAla AlaPhe PheAsn AsnValVal Val Val Asn Asn Lys Lys Gly Gly HisHis PhePhe AspAsp 6455 6455 6460 6460 6465

82

Gly Gln Gly Gln Gln GlnGly GlyGlu GluVal ValPro ProValVal Ser Ser IleIle I le IleAsn AsnAsnAsn Thr Thr Val Val 6470 6470 6475 6475 6480 6480

Tyr Thr Tyr Thr Lys LysVal ValAsp AspGly GlyVal ValAspAsp Val Val Glu Glu Leu Leu Phe Phe GluGlu AsnAsn LysLys 6485 6485 6490 6490 6495 6495

Thr Thr Thr Thr Leu LeuPro ProVal ValAsn AsnVal ValAlaAla Phe Phe Glu Glu Leu Leu Trp Trp Ala Ala Lys Lys Arg Arg 6500 6500 6505 6505 6510 6510

Asn Ile Asn Ile Lys LysPro ProVal ValPro ProGlu GluValVal Lys Lys IleLeu I le LeuAsn AsnAsnAsn Leu Leu Gly Gly 6515 6515 6520 6520 6525 6525

Val Asp Val Asp Ile IleAla AlaAla AlaAsn AsnThr ThrValVal Ile Ile Trp Trp Asp Asp Tyr Tyr Lys Lys Arg Arg Asp Asp 6530 6530 6535 6535 6540 6540

Ala Pro Ala Pro Ala AlaHis HisIle IleSer SerThr ThrIleIle Gly Gly Val Val Cys Cys Ser Ser Met Met Thr Thr Asp Asp 6545 6545 6550 6550 6555 6555

Ile Ile Ala Lys Lys Ala Lys Lys Pro Pro Thr Thr Glu Glu Thr ThrIle IleCys CysAla AlaPro ProLeuLeu ThrThr ValVal 6560 6560 6565 6565 6570 6570

Phe Phe Phe Phe Asp AspGly GlyArg ArgVal ValAsp AspGlyGly Gln Gln Val Val Asp Asp Leu Leu PhePhe ArgArg AsnAsn 6575 6575 6580 6580 6585 6585

Ala Arg Ala Arg Asn AsnGly GlyVal ValLeu LeuIle IleThrThr Glu Glu Gly Gly Ser Ser Val Val Lys Lys Gly Gly Leu Leu 6590 6590 6595 6595 6600 6600

Gln Pro Gln Pro Ser SerVal ValGly GlyPro ProLys LysGlnGln Ala Ala Ser Ser Leu Leu Asn Asn GlyGly ValVal ThrThr 6605 6605 6610 6610 6615 6615

Leu Ile Leu Ile Gly GlyGlu GluAla AlaVal ValLys LysThrThr Gln Gln Phe Phe Asn Asn Tyr Tyr TyrTyr LysLys LysLys

83

6620 6620 6625 6625 6630 6630

Val Asp Val Asp Gly GlyVal ValVal ValGln GlnGln GlnLeuLeu Pro Pro Glu Glu Thr Thr Tyr Tyr Phe Phe Thr Thr Gln Gln 6635 6635 6640 6640 6645 6645

Ser Ser Arg AsnLeu Arg Asn LeuGln GlnGlu GluPhe PheLys LysPro ProArg ArgSer SerGln GlnMetMet GluGlu IleIle 6650 6650 6655 6655 6660 6660

Asp Phe Asp Phe Leu LeuGlu GluLeu LeuAla AlaMet MetAspAsp Glu Glu Phe Phe IleGlu I le GluArgArg Tyr Tyr Lys Lys 6665 6665 6670 6670 6675 6675

Leu Glu Leu Glu Gly GlyTyr TyrAla AlaPhe PheGlu GluHisHis Ile Ile Val Val Tyr Tyr Gly Gly AspAsp PhePhe SerSer 6680 6680 6685 6685 6690 6690

His Ser His Ser Gln GlnLeu LeuGly GlyGly GlyLeu LeuHisHis Leu Leu Leu Leu Ile Ile Gly Gly Leu Leu Ala Ala Lys Lys 6695 6695 6700 6700 6705 6705

Arg Phe Arg Phe Lys LysGlu GluSer SerPro ProPhe PheGluGlu Leu Leu Glu Glu Asp Asp Phe Phe Ile Ile Pro Pro Met Met 6710 6710 6715 6715 6720 6720

Asp Ser Asp Ser Thr ThrVal ValLys LysAsn AsnTyr TyrPhePhe Ile Ile Thr Thr Asp Asp Ala Ala Gln Gln Thr Thr Gly Gly 6725 6725 6730 6730 6735 6735

Ser Ser Ser Ser Lys LysCys CysVal ValCys CysSer SerValVal Ile Ile Asp Asp Leu Leu Leu Leu LeuLeu AspAsp AspAsp 6740 6740 6745 6745 6750 6750

Phe Val Phe Val Glu GluIle IleIle IleLys LysSer SerGlnGln Asp Asp Leu Leu Ser Ser Val Val ValVal SerSer LysLys 6755 6755 6760 6760 6765 6765

Val Val Val Val Lys LysVal ValThr ThrI Ile Asp Tyr le Asp TyrThr ThrGlu GluI Ile Ser Phe le Ser PheMet MetLeu Leu 6770 6770 6775 6775 6780

84

Trp Cys Trp Cys Lys LysAsp AspGly GlyHis HisVal ValGluGlu Thr Thr Phe Phe Tyr Tyr Pro Pro Lys Lys Leu Leu Gln Gln 6785 6785 6790 6790 6795 6795

Ser Ser Ser Ser Gln GlnAla AlaTrp TrpGln GlnPro ProGlyGly Val Val Ala Ala Met Met Pro Pro AsnAsn LeuLeu TyrTyr 6800 6800 6805 6805 6810 6810

Lys Met Lys Met Gln GlnArg ArgMet MetLeu LeuLeu LeuGluGlu Lys Lys Cys Cys Asp Asp Leu Leu GlnGln AsnAsn TyrTyr 6815 6815 6820 6820 6825 6825

Gly Asp Gly Asp Ser SerAla AlaThr ThrLeu LeuPro ProLysLys Gly Gly Ile Ile Met Met Met Met AsnAsn ValVal AlaAla 6830 6830 6835 6835 6840 6840

Lys Tyr Lys Tyr Thr ThrGln GlnLeu LeuCys CysGln GlnTyrTyr Leu Leu Asn Asn Thr Thr Leu Leu ThrThr LeuLeu AlaAla 6845 6845 6850 6850 6855 6855

Val Pro Val Pro Tyr TyrAsn AsnMet MetArg ArgVal ValIleIle His His Phe Phe Gly Gly Ala Ala Gly Gly Ser Ser Asp Asp 6860 6860 6865 6865 6870 6870

Lys Gly Lys Gly Val ValAla AlaPro ProGly GlyThr ThrAlaAla Val Val Leu Leu Arg Arg Gln Gln TrpTrp LeuLeu ProPro 6875 6875 6880 6880 6885 6885

Thr Gly Thr Gly Thr ThrLeu LeuLeu LeuVal ValAsp AspSerSer Asp Asp Leu Leu Asn Asn Asp Asp PhePhe ValVal SerSer 6890 6890 6895 6895 6900 6900

Asp Ala Asp Ala Asp AspSer SerThr ThrLeu LeuIle IleGlyGly Asp Asp Cys Cys Ala Ala Thr Thr Val Val His His Thr Thr 6905 6905 6910 6910 6915 6915

Ala Asn Ala Asn Lys LysTrp TrpAsp AspLeu LeuIle IleIleIle Ser Ser Asp Asp Met Met Tyr Tyr Asp Asp Pro Pro Lys Lys 6920 6920 6925 6925 6930 6930

Thr Lys Thr Lys Asn AsnVal ValThr ThrLys LysGlu GluAsnAsn Asp Asp Ser Ser Lys Lys Glu Glu GlyGly PhePhe PhePhe 6935 6935 6940 6940 6945

85

Thr Tyr Thr Tyr Ile IleCys CysGly GlyPhe PheIle IleGlnGln Gln Gln Lys Lys Leu Leu Ala Ala LeuLeu GlyGly GlyGly 6950 6950 6955 6955 6960 6960

Ser Ser Val AlaIle Val Ala IleLys LysIIle le Thr GluHis Thr Glu HisSer SerTrp TrpAsn AsnAla AlaAsp AspLeu Leu 6965 6965 6970 6970 6975 6975

Tyr Lys Tyr Lys Leu LeuMet MetGly GlyHis HisPhe PheAlaAla Trp Trp Trp Trp Thr Thr Ala Ala PhePhe ValVal ThrThr 6980 6980 6985 6985 6990 6990

Asn Val Asn Val Asn AsnAla AlaSer SerSer SerSer SerGluGlu Ala Ala Phe Phe Leu Leu Ile Ile Gly Gly Cys Cys Asn Asn 6995 6995 7000 7000 7005 7005

Tyr Leu Tyr Leu Gly GlyLys LysPro ProArg ArgGlu GluGlnGln Ile Ile Asp Asp Gly Gly Tyr Tyr ValVal MetMet HisHis 7010 7010 7015 7015 7020 7020

Ala Asn Ala Asn Tyr TyrIle IlePhe PheTrp TrpArg ArgAsnAsn Thr Thr Asn Asn Pro Pro Ile Ile Gln Gln Leu Leu Ser Ser 7025 7025 7030 7030 7035 7035

Ser Ser Tyr SerLeu Tyr Ser LeuPhe PheAsp AspMet MetSer SerLys LysPhe PhePro ProLeu LeuLysLys LeuLeu ArgArg 7040 7040 7045 7045 7050 7050

Gly Thr Gly Thr Ala AlaVal ValMet MetSer SerLeu LeuLysLys Glu Glu Gly Gly Gln Gln Ile Ile AsnAsn AspAsp MetMet 7055 7055 7060 7060 7065 7065

Ile Ile Leu Ser Leu Leu Ser Leu Leu Leu Ser Ser Lys Lys Gly GlyArg ArgLeu LeuIle IleIle IleArgArg GluGlu AsnAsn 7070 7070 7075 7075 7080 7080

Asn Arg Asn Arg Val ValVal ValIle IleSer SerSer SerAspAsp Val Val Leu Leu Val Val Asn Asn Asn Asn 7085 7085 7090 7090 7095 7095

<210> <210> 22 22 <211> <211> 275

86

<212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 22 22

Met Asp Met Asp Leu LeuPhe PheMet Met ArgArg IlePhePhe I le ThrThr Ile Ile Gly Gly Thr Thr Val Leu Val Thr ThrLys Leu Lys 1 1 5 5 10 10 15 15

Gln Gly Gln Gly Glu GluI Ile LysAsp le Lys AspAla AlaThrThr ProPro SerSer Asp Asp Phe Phe Val Ala Val Arg ArgThr Ala Thr 20 20 25 25 30 30

Ala Thr Ala Thr Ile IlePro ProIle Ile GlnGln AlaAla Ser Ser Leu Leu Pro Gly Pro Phe Phe Trp GlyLeu TrpIle Leu ValIle Val 35 35 40 40 45 45

Gly Val Gly Val Ala AlaLeu LeuLeu Leu AlaAla ValVal Phe Phe Gln Gln Ser Ser Ser Ala Ala Lys SerIle LysIle Ile ThrIle Thr 50 50 55 55 60 60

Leu Lys Leu Lys Lys LysArg ArgTrp Trp GlnGln LeuLeu Ala Ala Leu Leu Ser Gly Ser Lys Lys Val GlyHis ValPhe His ValPhe Val

70 70 75 75 80 80

Cys Asn Leu Cys Asn LeuLeu LeuLeu LeuLeuLeu PhePhe Val Val Thr Thr Val Val Tyr His Tyr Ser SerLeu HisLeu LeuLeuLeu Leu 85 85 90 90 95 95

Val Ala Val Ala Ala AlaGly GlyLeu Leu GluGlu AlaAla Pro Pro Phe Phe Leu Leu Leu Tyr Tyr Tyr LeuAla TyrLeu Ala ValLeu Val 100 100 105 105 110 110

Tyr Phe Tyr Phe Leu LeuGln GlnSer Ser IleIle AsnAsn Phe Phe Val Val Arg Ile Arg Ile Ile Met IleArg MetLeu Arg TrpLeu Trp 115 115 120 120 125 125

Leu Cys Leu Cys Trp TrpLys LysCys Cys ArgArg SerSer Lys Lys Asn Asn Pro Leu Pro Leu Leu Tyr LeuAsp TyrAla Asp AsnAla Asn 130 130 135 135 140 140

Tyr Phe Tyr Phe Leu LeuCys CysTrp Trp HisHis ThrThr Asn Asn Cys Cys Tyr Tyr Tyr Asp Asp Cys TyrIle CysPro Ile TyrPro Tyr 145 145 150 150 155 155 160

87

Asn Ser Asn Ser Val ValThr ThrSer Ser SerSer IleIle Val Val Ile Ile Thr Gly Thr Ser Ser Asp GlyGly AspThr Gly ThrThr Thr 165 165 170 170 175 175

Ser Pro Ser Pro Ile IleSer SerGlu Glu HisHis AspAsp Tyr Tyr Gln Gln Ile Gly Ile Gly Gly Tyr GlyThr TyrGlu Thr LysGlu Lys 180 180 185 185 190 190

Trp Glu Trp Glu Ser SerGly GlyVal Val LysLys AspAsp Cys Cys Val Val Val His Val Leu Leu Ser HisTyr SerPhe Tyr ThrPhe Thr 195 195 200 200 205 205

Ser Asp Ser Asp Tyr TyrTyr TyrGln Gln LeuLeu TyrTyr Ser Ser Thr Thr Gln Ser Gln Leu Leu Thr SerAsp ThrThr Asp GlyThr Gly 210 210 215 215 220 220

Val Glu Val Glu His HisVal ValThr Thr PhePhe PhePhe I leIle TyrTyr Asn Asn Lys Lys Ile Ile Val Glu Val Asp AspPro Glu Pro 225 225 230 230 235 235 240 240

Glu Glu Glu Glu His HisVal ValGln Gln IleIle HisHis Thr Thr Ile Ile Asp Ser Asp Gly Gly Ser SerGly SerVal Gly ValVal Val 245 245 250 250 255 255

Asn Pro Asn Pro Val ValMet MetGlu Glu ProPro IleIle Tyr Tyr Asp Asp Glu Thr Glu Pro Pro Thr ThrThr ThrThr Thr SerThr Ser 260 260 265 265 270 270

Val Pro Val Pro Leu Leu 275 275

<210> <210> 23 23 <211> <211> 61 61

<212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 23 23

Met Phe Met Phe His HisLeu LeuVal Val AspAsp PhePhe Gln Gln Val Val Thr Ala Thr Ile Ile Glu AlaIle GluLeu Ile LeuLeu Leu 1 1 55 10 10 15

88

Ile Ile Met Ile Ile MetArg ArgThr ThrPhePhe LysLys ValVal Ser Ser Ile Ile Trp Leu Trp Asn AsnAsp LeuTyr AspIleTyr Ile 20 20 25 25 30 30

Ile Asn Leu Ile Asn LeuIle IleIle IleLysLys AsnAsn LeuLeu Ser Ser Lys Lys Ser Thr Ser Leu LeuGlu ThrAsn GluLysAsn Lys 35 35 40 40 45 45

Tyr Ser Tyr Ser Gln GlnLeu LeuAsp Asp GluGlu GluGlu Gln Gln Pro Pro Met Ile Met Glu Glu Asp Ile Asp 50 50 55 55 60 60

<210> <210> 24 24 <211> <211> 121 121

<212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 24 24

Met Lys Met Lys Ile IleIle IleLeu Leu PhePhe LeuLeu Ala Ala Leu Leu Ile Leu Ile Thr Thr Ala LeuThr AlaCys Thr GluCys Glu 1 1 55 10 10 15 15

Leu Tyr Leu Tyr His HisTyr TyrGln Gln GluGlu CysCys Val Val Arg Arg Gly Thr Gly Thr Thr Val ThrLeu ValLeu Leu LysLeu Lys 20 20 25 25 30 30

Glu Pro Glu Pro Cys CysSer SerSer Ser GlyGly ThrThr Tyr Tyr Glu Glu Gly Ser Gly Asn Asn Pro SerPhe ProHis Phe ProHis Pro 35 35 40 40 45 45

Leu Ala Leu Ala Asp AspAsn AsnLys Lys PhePhe AlaAla Leu Leu Thr Thr Cys Ser Cys Phe Phe Thr SerGln ThrPhe Gln AlaPhe Ala 50 50 55 55 60 60

Phe Ala Phe Ala Cys CysPro ProAsp Asp GlyGly ValVal Lys Lys His His Val Gln Val Tyr Tyr Leu GlnArg LeuAla Arg ArgAla Arg

70 70 75 75 80 80

Ser Val Ser Val Ser SerPro ProLys Lys LeuLeu PhePhe Ile Ile Arg Arg Gln Glu Gln Glu Glu Val GluGln ValGlu Gln LeuGlu Leu 85 85 90 90 95

89

Tyr Ser Tyr Ser Pro ProIle IlePhe Phe LeuLeu IleIle Val Val Ala Ala Ala Val Ala Ile Ile Phe ValIle PheThr Ile LeuThr Leu 100 100 105 105 110 110

Cys Phe Cys Phe Thr ThrLeu LeuLys Lys ArgArg LysLys Thr Thr Glu Glu 115 115 120 120

<210> <210> 25 25 <211> <211> 121 121

<212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 25 25

Met Lys Met Lys Phe PheLeu LeuVal Val PhePhe LeuLeu Gly Gly Ile Ile Ile Thr Ile Thr Thr Val ThrAla ValAla Ala PheAla Phe 1 1 55 10 10 15 15

His Gln His Gln Glu GluCys CysSer Ser LeuLeu GlnGln Ser Ser Cys Cys Thr His Thr Gln Gln Gln HisPro GlnTyr Pro ValTyr Val 20 20 25 25 30 30

Val Asp Val Asp Asp AspPro ProCys Cys ProPro IleIle His His Phe Phe Tyr Lys Tyr Ser Ser Trp LysTyr TrpIle Tyr ArgIle Arg 35 35 40 40 45 45

Val Gly Val Gly Ala AlaArg ArgLys Lys SerSer AlaAla Pro Pro Leu Leu Ile Leu Ile Glu Glu Cys LeuVal CysAsp Val GluAsp Glu 50 50 55 55 60 60

Ala Gly Ala Gly Ser SerLys LysSer Ser ProPro IleIle Gln Gln Tyr Tyr Ile Ile Ile Asp Asp Gly IleAsn GlyTyr Asn ThrTyr Thr

70 70 75 75 80 80

Val Ser Val Ser Cys CysLeu LeuPro Pro PhePhe ThrThr Ile Ile Asn Asn Cys Glu Cys Gln Gln Pro GluLys ProLeu Lys GlyLeu Gly 85 85 90 90 95 95

Ser Leu Val Ser Leu ValVal ValArg ArgCysCys SerSer Phe Phe Tyr Tyr Glu Glu Asp Leu Asp Phe PheGlu LeuTyr GluHisTyr His 100 100 105 105 110

90

Asp Val Asp Val Arg Arg Val Val Val Val Leu Leu Asp Asp Phe Phe Ile Ile 115 115 120 120

<210> <210> 26 26 <211> <211> 38 38 <212> <212> PRT PRT <213> <213> Severe acute Severe acuterespiratory respiratory syndrome syndrome coronavirus coronavirus 2 2

<400> <400> 26 26

Met Gly Met Gly Tyr Tyr Ile Ile Asn Asn Val Val Phe Phe Ala Ala Phe Phe Pro Pro Phe Phe Thr Thr Ile Ile Tyr Tyr Ser Ser Leu Leu 11 5 5 10 10 15 15

Leu Leu Leu Leu Cys CysArg ArgMet Met AsnAsn SerSer Arg Arg Asn Asn Tyr Ala Tyr Ile Ile Gln AlaVal GlnAsp Val ValAsp Val 20 20 25 25 30 30

Val Asn Val Asn Phe PheAsn AsnLeu Leu ThrThr 35 35

<210> <210> 27 27 <211> <211> 4 4 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> <223> aalinker linkerpeptide peptide

<400> <400> 27 27

Asn Lys Asn Lys Arg ArgLys Lys 11

<210> <210> 28 28 <211> <211> 22 22 <212> <212> DNA DNA <213> <213> Artificial Sequence Artificial Sequence

91

<220> <220> <223> aaPCR <223> PCRprimer primerfor forthe theTCR TCRanalysis analysis

<400> <400> 28 28 gtctaccaggcattcgcttc gtctaccagg cattcgcttcat at 22 22

<210> <210> 29 29 <211> <211> 24 24 <212> <212> DNA DNA <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> <223> aaPCR PCRprimer primerfor forthe theTCR TCRanalysis analysis

<400> <400> 2929 tcagctggac cacagccgca tcagctggac cacagccgca gcgt gcgt 24 24

<210> <210> 30 30 <211> 21 <211> 21 <212> DNA <212> DNA <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> <223> aaPCR PCRprimer primerfor forthe theTCR TCRanalysis analysis

<400> <400> 30 30 tcagaaatcctttctcttga tcagaaatcc tttctcttgaC c 21 21

<210> <210> 31 31

<211> <211> 24 24 <212> <212> DNA DNA <213> ArtificialSequence <213> Artificial Sequence

<220> <220> <223> <223> aaPCR PCRprimer primerfor forthe theTCR TCRanalysis analysis

<400> <400> 31 31

ctagcctctggaatcctttc ctagcctctg gaatcctttc tctt tctt 24

Claims (24)

[CLAIMS]

1. A peptide of less than 15 amino acids having cytotoxic T cell (CTL)-inducing ability, which comprises the amino acid sequence selected from the group below: (a) the 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;and (b) the amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added to the 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. The peptide of claim 1, which has either or both of features below to the amino acid sequence selected from the group consisting of 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15: (a) the second amino acid from the N terminus is substituted with an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine and tryptophan; and (b) the C-terminal amino acid is substituted with an amino acid selected from the group consisting of phenylalanine, leucine, isoleucine, tryptophan and methionine.

3. The peptide of claim 1, which has either or both of features below to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 7, 10, 12 and 13: (a) the second amino acid from the N terminus is substituted with an amino acid selected from the group consisting of leucine and methionine; and (b) the C-terminal amino acid is substituted with an amino acid selected from the group consisting of valine and leucine.

4. The peptide of claim 1, which consists of the 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.

5. A polynucleotide, which encodes the peptide of any one of claims 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) below: (a) one or more types of peptides of any one of claims I to 4; (b) one or more types of polynucleotides encoding the peptide(s) of any one of claims 1 to 4 in an expressible form;

(c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; and (e) a CTL that targets the peptide of any one of claims I to 4.

7. The composition of claim 5, which is a composition for inducing a CTL(s), wherein the active ingredient is at least one ingredient selected from the group consisting of (a) to (d) below: (a) one or more types of peptides of any one of claims I to 4; (b) one or more types of polynucleotides encoding the peptide(s) of any one of claims 1 to 4 in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; and (d) an exosome that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen.

8. The composition of claim 6, which is a pharmaceutical composition.

9. The composition of claim 8, which is for one or more uses selected from the group consisting of (i) treatment of coronavirus infectious disease, (ii) prevention (prophylaxis) of coronavirus infectious disease and (iii) suppression of aggravation of coronavirus infectious disease.

10. The composition of claim 8, which is for inducing an immune response against coronavirus infection.

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

12. The composition of any one of claims 6 to 11, which is formulated for administration to a subject positive for HLA-A24 or HLA-A02.

13. A method of inducing an APC(s) having CTL-inducing ability, which comprises a step selected from the group consisting of (a) and (b) below: (a) contacting an APC(s) with the peptide of any one of claims 1 to 4 in vitro, ex vivo or in vivo; and

(b) introducing a polynucleotide encoding the peptide of any one of claims 1 to 4 into an APC(s).

14. A method of inducing a CTL(s), which comprises a step selected from the group consisting of (a) to (c) below: (a) co-culturing a CD8-positive T cell(s) with an APC(s) that presents on its surface a complex of an HLA antigen and the peptide of any one of claims I to 4; (b) co-culturing a CD8-positive T cell(s) with an exosome(s) that presents on its surface a complex of an HLA antigen and the peptide of any one of claims I to 4; and (c) introducing into a CD8-positive T cell(s) a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of any one of claims I to 4 presented by an HLA antigen on a cell surface.

15. An APC that presents on its surface a complex of an HLA antigen and the peptide of any one of claims I to 4.

16. The APC of claim 15, which is induced by the method of claim 13.

17. A CTL that targets the peptide of any one of claims I to 4.

18. The CTL of claim 17, which is induced by the method of claim 14.

19. A method of inducing an immune response against coronavirus infection, which comprises administering to a subject at least one ingredient selected from the group consisting of (a) to (e) below: (a) one or more types of peptides of any one of claims I to 4; (b) one or more types of polynucleotides encoding the peptide(s) of any one of claims 1 to 4 in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of claims I to 4 and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; and (e) a CTL that targets the peptide of any one of claims 1 to 4.

20. A method of treating and/or preventing coronavirus infectious disease, and/or suppressing aggravation of coronavirus infectious disease, which comprises administering to a subject a composition comprising at least one active ingredient selected from the group consisting of (a) to (e) below: (a) one or more types of peptides of any one of claims I to 4; (b) one or more types of polynucleotides encoding the peptide(s) of any one of claims 1 to 4 in an expressible form; (c) an antigen-presenting cell (APC) that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; (d) an exosome that presents on its cell surface a complex of the peptide of any one of claims 1 to 4 and an HLA antigen; and (e) a CTL that targets the peptide of any one of claims I to 4.

21. An antibody that binds to the peptide of any one of claims I to 4.

22. A method of screening for a peptide having CTL-inducing ability, which comprises the steps of: (a) generating candidate sequences consisting of an amino acid sequence in which one, two or several amino acid residues are substituted, deleted, inserted and/or added to an original amino acid sequence consisting of the amino acid sequence selected from among SEQ ID NOs: 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13 and 15; (b) selecting from among the candidate sequences generated in (a), a candidate sequence that does not have significant homology (sequence identity) with any known human gene product; (c) contacting an APC(s) with a peptide consisting of the candidate sequence selected in (b); (d) contacting the APC(s) of (c) with a CD8-positive T cell(s); and (e) selecting a peptide having an equal to or higher CTL-inducing ability than that of a peptide consisting of the original amino acid sequence.

23. An emulsion comprising one or more types of peptides of any one of claims I to 4, a water soluble carrier and an oil adjuvant.

24. A kit comprising a container that houses the composition of any one of claims 6 to 12 and a container that houses an adjuvant.