Diagnosis, prevention, and treatment of coronavirus infection

Abstract

This invention provides coronavirus peptides, as well as the use of such peptides for the diagnosis, treatment, and prevention of coronavirus infection. [Solution] A peptide comprising a specific sequence or a variant thereof is disclosed. Including one or more such peptides in a vaccine composition may confer protective ability against one or more coronaviruses and / or the ability to treat existing coronavirus infections. Each of the peptides may also be used to diagnose the presence or absence of a coronavirus infection by, for example, detecting the presence or absence of molecules that can bind to the peptide (such as T cell receptors or antibodies) in a sample.

Description

[Technical Field]

[0001] This application is based on UK Patent Application Publication No. 2008250.9, filed on June 2, 2020. The present specification claims priority, and its contents and elements are referred to in this specification for any purpose. It will be incorporated into the book.

[0002] This invention relates to coronavirus peptides, and to the diagnosis, treatment, and prevention of coronavirus infection. Regarding the use of such peptides for prevention. [Background technology]

[0003] Coronaviruses are a group of related viruses that cause disease in mammals and birds. The symptoms of coronavirus infection vary by species. For example, the symptoms of coronavirus in chickens... While viral infections cause upper respiratory tract diseases, coronavirus in cattle and pigs Rus infections tend to cause diarrhea.

[0004] In humans, coronaviruses cause respiratory infections. Some coronaviruses The illness caused by this infection can be mild, such as a common cold. This refers to more severe and potentially fatal diseases such as SARS, MERS, and COVID-19. It causes. [Overview of the project] [Problems that the invention aims to solve]

[0005] Several studies have identified target sites for the development of a vaccine against coronavirus. It has been characterized. For example, He et al. identified the antigenic site of the SARS coronavirus as We conducted an experiment to test the nucleocapsid protein (1). The SARS nucleocapsid protein is a possible It has been broadly characterized to identify a vaccine candidate (2), dominant helper T cells An epitope has been identified in this protein (3). More recently, COVID-19 Potential vaccine targets for coronaviruses have been identified in previous SARS-CoV immunological studies. It has been identified based on (4). Nevertheless, human coronavirus infections No vaccine for prevention or treatment is currently available on the market. In the human population, and / or Previous tests to diagnose coronavirus infection are limited. Controlling infectious disease outbreaks involves strategies including accurate testing and / or effective vaccination. Because it depends on [something], the provision of vaccines and tests is highly desirable. [Means for solving the problem]

[0006] This invention is used to diagnose, prevent, or treat coronavirus infections in humans. This invention relates to a coronavirus-derived peptide that may be used. The inventors have identified different coronaviruses. These are stored between cells and presented by MHC molecules on cells infected with those viruses. Several peptides were identified. One or more such peptides should be included in the vaccine composition. This will provide the ability to defend against one or more coronaviruses and / or existing coronaviruses It may confer the ability to treat infectious diseases. Each of the peptides also, for example, The presence or absence of molecules that can bind to peptides (such as T cell receptors or antibodies) To diagnose the presence or absence of coronavirus infection by detecting it in a sample. It may be used for, for example, human epidemics or pandemics. It may be a coronavirus. The coronavirus may be, for example, a coronavirus derived from a zoonosis. It may be a coronavirus. The coronavirus may be, for example, a member of the genus Betacoronavirus (Be tacoronavirus). The coronavirus may be, for example , a member of the subgenus Sarbecoronavirus. It may also be, for example, SARS coronavirus or SARS coronavirus 2.

[0007] Therefore, the present invention provides a peptide containing any one of SEQ ID NOs: 1 to 34 or a variant thereof.

[0008] The present invention also provides - a complex containing the peptide of the present invention bound to an MHC molecule, - a method for determining the presence or absence of a current or previous coronavirus infection in an individual using the peptide or complex of the present invention, - use of the peptide or complex of the present invention in a method for identifying coronavirus-specific T cells - use of the peptide or complex of the present invention in a method for identifying a coronavirus-specific T cell receptor - a T cell containing a T cell receptor capable of binding to a peptide containing any one of SEQ ID NOs: 1 to 34 or a variant thereof, - a vaccine composition containing a peptide that can bind to the peptide of the present invention or a peptide containing any one of SEQ ID NOs: 1 to 34 or a variant thereof and can bind to a T cell receptor that can bind to the peptide, - a peptide encoding a peptide that can bind to the peptide of the present invention or a peptide containing any one of SEQ ID NOs: 1 to 34 or a variant thereof and can bind to a T cell receptor that can bind to the peptide A vaccine composition containing polynucleotides, - A method for preventing or treating coronavirus infection, which involves being infected with coronavirus The present invention includes administering the vaccine composition to individuals that are dolphins or at risk of infection. Methods and - The present invention for use in methods for preventing or treating coronavirus infection in individuals. vaccine composition To provide. [Brief explanation of the drawing]

[0009] [Figure 1(1)] This figure shows the absorbance spectrum of Example 1. [Figure 1(2)] This figure shows the absorbance spectrum of Example 1. [Figure 1(3)] This figure shows the absorbance spectrum of Example 1. [Figure 1(4)] This figure shows the absorbance spectrum of Example 1. [Figure 2(1)] This figure shows the DLS data for Example 1. [Figure 2(2)] This figure shows the DLS data for Example 1. [Figure 2(3)] This figure shows the DLS data for Example 1. [Figure 2(4)] This figure shows the DLS data for Example 1. [Figure 3A] This figure shows the HPLC method of Example 1. [Figure 3B(1)] This figure shows the HPLC results for Example 1. EM009-064-01 The GNP without any peptides is 3.4%. [Figure 3B(2)] This figure shows the HPLC results for Example 1. EM009-064-02: GNP without any peptides is 0%. [Figure 3B(3)] This figure shows the HPLC results for Example 1. EM009-064-03: The GNP without any peptides is 2.2%. [Figure 4(1)]This figure shows the LC-MS peptide quantification in Example 1. [Figure 4(2)] This figure shows the LC-MS peptide quantification in Example 1. [Figure 4(3)] This figure shows the LC-MS peptide quantification in Example 1. [Figure 5] This is a schematic diagram showing the alignment of the peptide used in Example 1. [Modes for carrying out the invention]

[0010] peptide The present invention provides a peptide comprising one of SEQ ID NOs: 1 to 34 or a variant thereof. The mutants are defined in detail below. Sequence IDs 1-34 are shown in Table 1. [Table 1]

[0011] To identify sequence numbers 1-34, the memory T cell response in SARS-CoV was investigated. Using long peptide sequences that have been shown to cause this, short duplication of 9-10 residues in length A series of peptides was prepared. Then, these sequences were processed using MHCFlurry. Therefore, we predicted their binding affinities to various HLA alleles. SARS-C For the protein in OV2 (NCBI accession number NC_045512) Sequences with affinity of 100 nM or less and 100% identity were selected.

[0012] Therefore, the peptide of the present invention can bind to MHC class I molecules. Tide originates from immunoproteasome processing of the viral proteome within infected cells. It may also be done by having the peptide on the surface of one or more coronaviruses, or one The above may also refer to peptides expressed intracellularly within coronaviruses. This could be, for example, a coronavirus involved in human epidemics or pandemics. The coronavirus could be, for example, a coronavirus originating from a zoonotic disease. Viruses include, for example, the betacoronavirus species. It may also be a coronavirus. For example, a salve coronavirus (Sarbe Coronavirus may also be a member of the subgenus. Coronaviruses, for example, It may be SARS coronavirus or SARS coronavirus 2. The peptide is Structural peptides or functional peptides, such as peptides involved in the metabolism or replication of the coronavirus. It may be present. Preferably, the peptide is an internal peptide. Preferably, the peptide is It is preserved between two or more different coronaviruses or coronavirus serotypes. Each of the above different coronaviruses or coronavirus serotypes contains peptides and 50% Above (for example, 60%, 70%, 75%, 80%, 90%, 95%, 98%, or 99%) If the peptide encodes homologous sequences, it may represent two or more different coronaviruses or coronaviruses. It is preserved among viral serotypes.

[0013] A peptide may contain any number of amino acids, that is, any length. Good. Typically, peptides are about 8 to about 30, 35, or 40 amino acids long, for example, about 9. Approximately 29, approximately 10-28, approximately 11-27, approximately 12-26, approximately 13-25, approximately 13- Approximately 24, approximately 14-23, approximately 15-22, approximately 16-21, approximately 17-20, or approximately 18 It is approximately 29 amino acids long. Peptides preferably have a length of 9 or 10 amino acids. The peptide may also be a polypeptide. The peptide is one of the sequence numbers 1 to 34. Peptides may consist of, or essentially consist of, a single amino acid sequence. From lipeptide coronavirus antigens, chemical induction, for example, by proteolytic cleavage. It may be done in a manner well known in the art. More typically, coronavirus peptides are used in a manner well known in the art. It may be used in synthesis.

[0014] The peptide may contain only one of SEQ ID NOs: 1-34 or a variant thereof. In other words, the peptides are any combination of two or more of SEQ ID NOs: 1-34 or their variants. For example, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 1 0 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more , 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 3 It may contain 0 or more, 31 or more, 32 or more, or 33 or more. The peptide is sequence number Peptides may include all of numbers 1-34 or their variants. For example, the peptide is SEQ ID NO: 21. It may contain one or more of 1, 19, 28, 2, 27, 16, and 14. This includes, for example, all of sequence numbers 21, 1, 19, 28, 2, 27, 16, and 14. That's good too.

[0015] In certain embodiments, the peptide comprises one or more of SEQ ID NOs: 9, 24, and 25. That's fine.

[0016] HLRIAGHHL (sequence number 9) is the sequence HLR in SARS-CoV-1 M. Based on MAGHSL (Sequence ID 34).

[0017] SMWALIISV (SEQ ID NO: 24) is associated with SARS-CoV-1 pp1ab. Based on the SMWALVISV (sequence number 31) sequence.

[0018] TKAYNVTQAF (SEQ ID NO: 25) is a TKQ in SARS-CoV-1 N. Based on the YNVTQAF (sequence number 32) sequence.

[0019] While we do not wish to be bound by any particular theory, the inventors believe that substitution is We believe this will provide an advantage in the improved specific detection of SARS-CoV-2.

[0020] A peptide is one or more copies of SEQ ID NOs: 1-34, for example, two or more copies. For example, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 The above copies may be included. This may include one or more copies of sequence numbers 1 through 34. Peptides, or peptides containing one or more of SEQ ID NOs: 1-34, are approximately 18-250 A number of amino acids, for example, about 20, about 30, about 40 or about 50 amino acids ~ about 200, about 1 It may have a length of 50 or about 100 amino acids. In such peptides, the sequence number Two or more copies of sequence numbers 1-34, or one or more copies of sequence numbers 1-34 are They may be directly linked to each other, or consist of 2 to about 20 amino acids or about 3 to about 10 amino acids. It may be linked by one or more amino acids, etc. In a peptide, linked amino Acids typically link two or more sequences from the natural sequence numbers 1-34. It does not contain a amino acid sequence.

[0021] Like any one of sequence numbers 1-34 or its variants, the peptide contains one or more C D8+ T cell epitope, one or more CD4+ T cell epitopes and / or one or more B It may contain cellular epitopes. For example, the peptide may contain two or more, for example, three or more, four. In addition, 5 or more, 10 or more, 15 or more, or 20 or more CD8+ T cell epitopes It may contain two or more peptides, for example, three or more, four or more, five or more, or ten or fewer. The above may contain 15 or more or 20 or more CD4+ T cell epitopes. The peptide is , two or more, for example, three or more, four or more, five or more, ten or more, fifteen or twenty or twenty The above B cell epitopes may also be included.

[0022] The CD8+ T cell epitope is preferably one of SEQ ID NOs: 1 to 34 or the same This is a CD8+ T cell epitope that does not contain mutants. A CD8+ T cell epitope is, for example, If a coronavirus CD8+ epitope is present, that is, one or more coronaviruses It is expressed and (i) presented by class I MHC molecules and (ii) present on CD8+ T cells. It may also be a peptide that can be recognized by the T cell receptor (TCR). Alternatively, C The D8+ T cell epitope is a CD8+ T cell epitope that is not expressed by one or more coronaviruses. It can also be a cellular epitope.

[0023] CD4+ T cell epitopes are, for example, coronavirus CD4+ epitopes, i.e. , expressed by one or more coronaviruses, and (i) expressed by class II MHC molecules (ii) Recognizable by T cell receptors (TCRs) present on CD4+ T cells It may also be a peptide. Alternatively, the CD4+ T cell epitope may be one or more corona It may also be a CD4+ T cell epitope that is not expressed by the virus.

[0024] A B cell epitope is, for example, a coronavirus B cell epitope, i.e., one or more. It is expressed by the coronavirus and is activated by the B cell receptor (BCR) present on B cells. It may also be a recognizable peptide. Alternatively, the B cell epitope may be one or more colo It may also be a B cell epitope that is not expressed by Navirus.

[0025] Coronaviruses are, for example, coronaviruses that are involved in human epidemics or pandemics. It's acceptable. Coronaviruses, for example, are coronaviruses that originate from zoonotic diseases. It is fine. Coronaviruses include, for example, beta-coronaviruses. It may also be a member of the genus Irus. Coronaviruses are, for example, Salvecoronavirus. It may also be a member of the subgenus *Sarbecoronavirus*. Coronaviruses include, for example, SARS coronavirus and / or SARS coronavirus It may include 2.

[0026] The term "peptide" refers to a group of amino acid residues linked by peptide (-CO-NH-) bonds. This includes not only linked molecules but also molecules with reversed peptide bonds. Troinversopeptide mimics are made by methods known in the art, for example, Meziere e The method described in t al (1997) J.Immunol. 159, 3230-3237 It may be prepared using this method. This approach involves changes in the main chain rather than the orientation of the side chains. This includes the preparation of pseudopeptides containing [the specified substance]. Meziere et al. (1997) stated that at least These pseudo-peptides are useful for MHC class II and helper T cell responses. This indicates that it contains an NH-CO bond instead of a CO-NH peptide bond. Inverse peptides are far more resistant to protein degradation.

[0027] Similarly, peptide bonds are linked by appropriate linkers that maintain the spacing between carbon atoms in amino acid residues. The linker portion may be completely omitted as long as the portion is used. It is particularly preferable that they have the same charge distribution and substantially the same planarity. The peptide is To help reduce sensitivity to soprotein digestion, its N-terminus or C It should also be understood that it is acceptable for the terminal to be conveniently blocked. For example, the N-terminal a of a peptide. The mino group may be protected by reacting with a carboxylic acid, and the C-terminal group of the peptide The ruboxyl group may be protected by reacting with an amine. Other examples of modification include These include glycosylation and phosphorylation. Another possible modification is the R or K side chain amine. The hydrogen atoms on the NH group may be replaced by methylene groups (-NH2 is -NH(Me)). (Or it may be modified to -N(Me)2).

[0028] The term "peptide" also refers to a peptide that increases or decreases the half-life in vivo. Contains plutide variants. The half-life of the peptide used according to the present invention can be increased. Examples of similar analogs include peptoid analogs of peptides and D-amino acid derivatives of peptides. Examples include peptide-peptoid hybrids. Mutants used according to the present invention. Further embodiments of the polypeptide include the D-amino acid form of the polypeptide. The preparation of polypeptides using D-amino acids instead of ano acids is possible through normal metabolic processes. To significantly reduce the undesirable degradation of such drugs, and to reduce the need for administration along with the frequency of administration, Reduce the amount of a certain drug.

[0029] mutant As described above, the peptide may contain one of the variants of SEQ ID NOs: 1 to 34. The peptides include, for example, two or more of the sequence numbers 1-34, for example, three or more, four or more. 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more Top, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 It may contain one or more variants or 33 or more variants. The peptide is, for example, SEQ ID NOs: 1-34 It may include all variants of the peptide, for example, SEQ ID NOs: 21, 1, 19, 28. The peptide may include one or more variants of 2, 27, 16, and 14. This includes all variants of sequence numbers 21, 1, 19, 28, 2, 27, 16 and 14. That's good too.

[0030] Any one of the variants of sequence numbers 1-34 is one of the variants of sequence numbers 1-34 and five others. The following (for example, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer) amino acids in different sequences It may be present. Each of the five or fewer amino acid differences is selected from sequence numbers 1-34. This may involve amino acid substitutions, deletions, or insertions in the relevant sequence. Amino acid substitutions may include, for example, Alternatively, conservative amino acid substitutions may be used.

[0031] Preferably, one of the variants of SEQ ID NOs. 1 to 34 is any of SEQ ID NOs. 1 to 34 The sequence may also have one or fewer amino acids that are different. For example, sequence numbers 1-34 The selected sequence variants involve a single amino acid substitution, deletion, or insertion into the associated sequence. It may be included. The amino acid substitution may be, for example, a conservative amino acid substitution.

[0032] Conservative substitutions involve replacing an amino acid with another amino acid that has a similar chemical structure, similar chemical properties, or similar side chain volume. Replace with the amino acids. The amino acids introduced are similar to the amino acids they replace. It may have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge. Or, Conservative substitution is the substitution of an existing aromatic or aliphatic amino acid with another aromatic or aliphatic amino acid. Amino acids may be introduced. Conservative amino acid changes are well known in this art, as shown in Table 2 below. The 20 main amino acids may be selected according to their properties as defined. If present, this is determined by referring to the hydroxyl scale of amino acid side chains in Table 3. It is also possible. [Table 2] [Table 3]

[0033] complex The present invention provides a complex comprising the peptide of the present invention bound to an MHC molecule. The complex then binds one of the sequence numbers 1-34 or a variant thereof to the MHC molecule. Contains or comprises peptides.

[0034] Peptide:MHC linkage is well known in the art. Preferably, the complex contains peptides The bond between the plutide and the MHC molecule is non-covalent. The bond is formed by, for example, electrostatic interaction. It may also be mediated by hydrogen bonding, van der Waals forces and / or hydrophobic interactions. stomach.

[0035] The MHC molecule may be an MHC class 1 molecule or an MHC class II molecule. In other words, MHC molecules are MHC class I molecules. MHC class I molecules are any HLA class It may also be a supertype. For example, MHC class I molecules are supertype A 2, A203 / A2, A23, A24, A2403 / A2, A2403 / A24, A39 , A3, A11, A30, A31, A32, A68, A69, B7, B8, B35, B3 7, B44, B48, B53, B60, B61, B62, B63, B72, B75, CW It may be either type 1 or Cw6.

[0036] The complex may contain two or more peptides and two or more MHC molecules of the present invention. Example For example, a complex can consist of three or more, for instance, four or more, five or more, six or more, seven or more, eight or more. , 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more Above, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more of the Peptide of the present invention It may include "do". The complex may consist of three or more, for example, four or more, five or more, six or more, seven or more. Above, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or less Above, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more M The complex may contain HC molecules, for example, three or more, four or more, five or more, or six. 1 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more or 20 or more peptides of the present invention, and 3 or more, for example, 4 or more, 5 or more, 6 or more, 7 1 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 1 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more The complex may contain the same number of MHC molecules as the PEP of the present invention. It may contain cydops. The complex contains a number of the peptides of the present invention that is different from the number of MHC molecules. However, the complex may contain, for example, four MHC molecules. It may contain a body or consist of an MHC tetramer. The complex may, for example, consist of 12 The complex may contain MHC molecules. The complex may contain an MHC dodecamer, or an MHC 12 It may also consist of dimers.

[0037] If the complex contains two or more peptides of the present invention, each of the two or more peptides is They may be the same. Alternatively, each of the two or more peptides may be different. If the combination contains three or more peptides of the present invention, each of the three or more peptides is the same. It may be present. If the complex contains three or more peptides of the present invention, the three or more peptides Each may be different. If the complex contains three or more peptides of the present invention, then three Some of the above peptides may be the same, while some of three or more peptides may be different. This is also fine. The complex is, for example, sequence numbers 21, 1, 19, 28, 2, 27, 16 and 14. It may include two or more of these. The complex is, for example, sequence numbers 21, 1, 19, 28, 2 This may include all of 27, 16, and 14.

[0038] If the complex contains two or more MHC molecules, each of the two or more MHC molecules is the same. It may be present. Alternatively, each of the two or more MHC molecules may be different. If the body contains three or more peptides of the present invention, each of the three or more MHC molecules is the same. It may be present. If the complex contains three or more peptides of the present invention, then three or more MHC molecules Each of them may be different. If the complex contains three or more MHC molecules, then three or more Some of the MHC molecules may be the same, while some of the three or more MHC molecules may be different. That's good too.

[0039] If the complex contains two or more peptides and two or more MHC molecules of the present invention, each peptide The compound may be bound to one of two or more MHC molecules. That is, it may be included in the complex. Each peptide present may be bound to an MHC molecule contained in the complex. Preferably, Each peptide in the complex is bound to a different MHC molecule within the complex. In other words, each MHC molecule in the complex is bound to one or fewer peptides in the complex. It is preferable that the complex is not bound to an MHC molecule. The complex may contain one or more peptides not bound to the peptide of the present invention. It may contain more than one MHC molecule.

[0040] One or more MHC molecules included in the complex may be linked to each other. For example. Each of the one or more MHC molecules in the complex is bound to a backbone molecule or nanoparticle. This is also acceptable. One or more MHC molecules in the complex are bound to the dextran skeleton. It is also possible that the complex contains an MHC dextramer, or an MHC dextramer. It may consist of xanthelmers. One or more MHC molecules are bound to the dextran skeleton. The mechanism is known in the art. Any number of MHC molecules bind to the dextran skeleton. You may do so. For example, one or more, two or more, three or more, for example, four or more, five or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more This includes 20 or more peptides of the present invention, and 3 or more, for example, 4 or more, 5 or more, 6 or more 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 The above MHC molecules may be bound to the dextran skeleton.

[0041] The complex may contain a fluorophore. Fluorophores are well known in the art. FITC (fluorescein isothiocyanate), PE (phycoerythrin), and AP One example is C (allophycocyanin). The complex contains any number of fluorophores. This is also acceptable. For example, a complex can consist of two or more, three or more, for example, four or more, five or more, six or more. 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 The peptides of the present invention described above, and three or more, for example, four or more, five or more, six or more, seven or more 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more , 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more full It may also contain an fluorophore. If the complex contains multiple fluorophores, the complex may contain The fluorophores may be the same or different. The complex has a dextran skeleton. In any case involving the skeleton, the fluorophore is preferably bonded to the dextran skeleton. The mechanism by which ruorophores are attached to the dextran skeleton is known in the art.

[0042] use The peptide or complex of the present invention can be used in several ways, including the uses described below. It's okay.

[0043] Determination of the presence or absence of current or past coronavirus infections This invention determines the presence or absence of current or previous coronavirus infection in an individual. The present invention provides the use of peptides or complexes in a method.

[0044] This method involves contacting a peptide or complex with a sample obtained from an organism, and the peptide This includes determining the presence or absence of a bond between the cydore or complex and the molecules contained in the sample. That's fine.

[0045] The sample may be, for example, a blood sample, serum sample, plasma sample, urine sample, saliva sample, or any substance present in an individual. The sample may be obtained by wiping the mucosal surface. Preferably, the sample is a blood sample. , a serum sample or a plasma sample.

[0046] The molecule may be a molecule that has immune function. For example, the molecule may be part of the innate immune system or adaptive system. It may be included in the immune system. Preferably, the molecule plays a role in adaptive immunity. The molecule is For example, it may be an antibody or an antibody fragment. The antibody or antibody fragment is B-type The antibody or antibody fragment may be on the surface of the cell or contained within the B cell. The molecule may be free in the sample. The molecule may be, for example, a T cell receptor. The receptor may be a CD4+ T cell receptor. The T cell receptor may be a CD8+ T cell receptor. It may be in the body. The T cell receptor may be on the surface of the T cell or contained within the T cell. It is also acceptable for the T cell to be a CD4+ T cell. The T cell may also be a CD8+ T cell. That's fine.

[0047] Preferably, the bond between the peptide or complex and the molecule is non-covalent. For example, electrostatic interactions, hydrogen bonds, van der Waals forces and / or hydrophobic interactions Therefore, it may be mediated. Detecting the binding between a peptide or peptide-containing complex and a molecule. Methods include, for example, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoabsorption spot assay. This is well known in the art, including ELISpot and flow cytometry.

[0048] The presence of a binding may indicate the presence of a current or previous coronavirus infection. Non-existence may indicate the absence of current or previous coronavirus infections.

[0049] In the current coronavirus infection, coronavirus particles or their components (e.g., peptides) In the current coronavirus infection, Antibodies specific to coronavirus particles or their components (e.g., peptides, proteins), B-type Cells, CD8+ T cells and / or CD4+ T cells may be present within the individual. Preferably In current coronavirus infections, (i) coronavirus particles or their components (for example) (ii) peptides, proteins, and (ii) coronavirus particles or their components (e.g., t Protein-specific antibodies, B cells, CD8+ T cells and / or CD4+ T cells within the individual It exists.

[0050] In previous coronavirus infections, coronavirus particles or their components (e.g., peptides) In some cases, the protein (or other substance) may not be present in the individual. In previous coronavirus infections, Antibodies specific to coronavirus particles or their components (e.g., peptides, proteins), B-type Cells, CD8+ T cells and / or CD4+ T cells may be present within the individual. Preferably In previous coronavirus infections, coronavirus particles or their components (e.g., phenylalanine) were found to be involved. The individual does not have cytoplasm or proteins, and coronavirus particles or their components (e.g., peptides) Antibodies specific to T cells (proteins), B cells, CD8+ T cells and / or CD4+ T cells It exists within the individual.

[0051] Coronaviruses are, for example, coronaviruses that are involved in human epidemics or pandemics. It's acceptable. Coronaviruses, for example, are coronaviruses that originate from zoonotic diseases. It is fine. Coronaviruses include, for example, beta-coronaviruses. It may also be a member of the genus Irus. Coronaviruses are, for example, Salvecoronavirus. It may also be a member of the subgenus Sarbecoronavirus. Rus could be, for example, SARS coronavirus or SARS coronavirus 2. .

[0052] Identification of coronavirus-specific T cells The present invention relates to a method for identifying coronavirus-specific T cells, and the peptide or The method provides for the use of a complex. This method involves contacting a peptide or complex with a sample obtained from an organism. Contact, and the existence of binding between the peptide or complex and the T cell receptor contained in the sample. This may include determining whether something is present or not.

[0053] The sample may be, for example, a blood sample, serum sample, plasma sample, urine sample, saliva sample, or any substance present in an individual. The sample may be obtained by wiping the mucosal surface. Preferably, the sample is a blood sample. be.

[0054] The T cell receptor may also be a CD4+ T cell receptor. It may be a cell receptor. Preferably, the T cell receptor is a CD8+ T cell receptor.

[0055] Preferably, the T cell receptor may be located on the surface of the T cell or contained within the T cell. That is also fine. The T cell may be a CD4+ T cell. The T cell may be a CD8+ T cell. This is also acceptable. Preferably, the T cells are CD8+ T cells.

[0056] Preferably, the binding between the peptide or complex and the T cell receptor is non-covalent. The bonding includes, for example, electrostatic interactions, hydrogen bonds, van der Waals forces and / or hydrophobic phases. The interaction may be mediated by the interaction between the peptide or peptide-containing complex and the T cell receptor. Methods for detecting the binding between them include, for example, enzyme-linked immunosorbent assay (ELISA). This technology includes immunosuppressive absorption spots (ELISpot) and flow cytometry. This is common knowledge.

[0057] The presence of binding may indicate the presence of one or more coronavirus-specific T cells. The absence of [specific T cells] may indicate the absence of coronavirus-specific T cells.

[0058] Individuals may currently be infected with the coronavirus. Coronavirus particles or their components (e.g., peptides, proteins) are present within an individual. In some cases, coronavirus particles or their components (e.g.) may be present. For example, antibodies specific to peptides, proteins, B cells, CD8+ T cells and / or CD 4+ T cells may be present within an individual. Preferably, in the current coronavirus infection. (i) coronavirus particles or their components (e.g., peptides, proteins) and ( ii) Antibodies specific to coronavirus particles or their components (e.g., peptides, proteins) The body contains B cells, CD8+ T cells, and / or CD4+ T cells.

[0059] An individual may have been previously infected with the coronavirus, but is currently showing symptoms of the coronavirus. Infection is not required. In previous coronavirus infections, coronavirus particles or In some cases, those components (e.g., peptides, proteins) may not be present in the individual. In viral infections, coronavirus particles or their components (e.g., peptides, proteins) Antibodies, B cells, CD8+ T cells, and / or CD4+ T cells specific to the substance are present in the individual. This may occur. Preferably, in previous coronavirus infections, coronavirus particles or The components (e.g., peptides, proteins) are not present in the organism, and the coronavirus particles or These include antibodies, B cells, and CD8+ T cells that are specific to their components (e.g., peptides, proteins). And / or CD4+ T cells are present in the individual. Therefore, previous coronavirus infection In this disease, coronavirus particles or their components (e.g., peptides, proteins) are present in the individual. Although they may not be present, coronavirus particles or their components (e.g., peptides, proteins) Antibodies, B cells, CD8+ T cells, and / or CD4+ T cells specific to the substance are present in the individual. It may happen.

[0060] Coronaviruses are, for example, coronaviruses that are involved in human epidemics or pandemics. It's acceptable. Coronaviruses, for example, are coronaviruses that originate from zoonotic diseases. It is fine. Coronaviruses include, for example, beta-coronaviruses. It may also be a member of the genus Irus. Coronaviruses are, for example, Salvecoronavirus. It may also be a member of the subgenus Sarbecoronavirus. Rus could be, for example, SARS coronavirus or SARS coronavirus 2. .

[0061] Identification of coronavirus-specific T cell receptors The present invention relates to a method for identifying coronavirus-specific T cell receptors. This method provides the use of a peptide or complex, which involves contacting the peptide or complex with a T cell receptor. To cause this to occur, and to determine the presence or absence of binding between the peptide or complex and the T cell receptor. This may include defining the definition.

[0062] The T cell receptor may also be a CD4+ T cell receptor. It may be a cell receptor. Preferably, the T cell receptor is a CD8+ T cell receptor.

[0063] T cell receptors may be located on the surface of T cells or may be contained within T cells. The cells may be CD4+ T cells. The T cells may be CD8+ T cells. More precisely, T refers to CD8+ T cells.

[0064] Preferably, the binding between the peptide or complex and the T cell receptor is non-covalent. The bonding includes, for example, electrostatic interactions, hydrogen bonds, van der Waals forces and / or hydrophobic phases. The interaction may be mediated by the interaction between the peptide or peptide-containing complex and the T cell receptor. Methods for detecting the binding between them include, for example, enzyme-linked immunosorbent assay (ELISA). This technology includes immunosuppressive absorption spots (ELISpot) and flow cytometry. This is common knowledge.

[0065] The presence of binding indicates that T cell receptors, upon contact with the peptide or complex, are affected by coronavirus-specific T It may indicate that it is a cell receptor. The absence of binding indicates contact with the peptide or complex. This may indicate that the T cell receptor is not a coronavirus-specific T cell receptor.

[0066] Coronaviruses are, for example, coronaviruses that are involved in human epidemics or pandemics. It's acceptable. Coronaviruses, for example, are coronaviruses that originate from zoonotic diseases. It is fine. Coronaviruses include, for example, beta-coronaviruses. It may also be a member of the genus Irus. Coronaviruses are, for example, Salvecoronavirus. It may also be a member of the subgenus Sarbecoronavirus. Rus could be, for example, SARS coronavirus or SARS coronavirus 2. .

[0067] T cells This invention relates to a peptide containing any one of SEQ ID NOs: 1 to 34 or a variant thereof, which binds to the peptide. The present invention provides T cells containing T cell receptors that can perform the following action: Methods for detecting the compound include, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunoassay. This is well known in the art, including absorption spot (ELISpot) and flow cytometry. The T cell receptor is identified using the method described above for identifying coronavirus-specific T cell receptors. It may be identified as such.

[0068] The T cells may be isolated T cells.

[0069] The T cell may also be a CD4+ T cell. The T cell receptor is the CD4+ T cell receptor. It's okay to have it.

[0070] The T cell may also be a CD8+ T cell. The T cell receptor is a CD8+ T cell receptor. It may be present. Preferably, the T cells are CD8+ T cells. Preferably, T cell receptor This is the CD8+ T cell receptor.

[0071] T cells may also be chimeric antigen receptor (CAR) expressing cells. T cell receptors are CA R is also acceptable.

[0072] Vaccine composition The present invention relates to the peptide of the present invention, or any one of SEQ ID NOs: 1 to 34, or a mutation thereof. Peptides that can bind to T cell receptors, including those that can bind to peptides in the body. The present invention provides a vaccine composition containing the following. The variants are defined above. The vaccine composition contains the following It has several advantages, as will become clear from the discussion below. However, the most important advantages are summarized here. ru.

[0073] Firstly, the vaccine composition can stimulate an immune response against coronavirus. Preferably, the immune response is a cellular immune response (e.g., a CD8+ T cell response). 8+ cytotoxic T lymphocytes (CTLs) exert their cytotoxic activity against infected cells. It mediates viral clearance. Therefore, stimulation of cellular immunity is necessary for coronavirus It may offer beneficial protection against infectious diseases.

[0074] Secondly, the peptides identified by the present inventors are found in different coronaviruses (e.g., SARS coronavirus and SARS coronavirus 2) are preserved, and It may be presented by MHC molecules on cells infected with one or more viruses. (i) related (ii) a type of virus, (ii) multiple species of coronavirus and / or (iii) a specific There is a possibility that the ability to defend against multiple strains or serotypes of a species, i.e., cross-defense, may be conferred. 100% homology between viruses is not necessary for cross-protection to be conferred. Furthermore, if specific residues are held in the correct position, cross-protection can, for example, prevent different viruses from crossing over. CD8+ T cell epitopes expressed in cells infected with the virus are expressed in approximately 50% or more (for example, 60%). Immunity with homologous sequences (percentages of %, 70%, 75%, 80%, 90%, 95%, 98%, or 99%) This may occur after conversion. Therefore, one of sequence numbers 1 to 34 or the same One or more peptides containing mutants, or one of SEQ ID NOs: 1-34 or a variant thereof. Peptides that can bind to T cell receptors that can bind to allopropyl groups Vaccine compositions containing or corresponding polynucleotides exceed those listed in Table 1. It may be possible to provide cross-protection against various existing coronaviruses. Including one or more conserved peptides in the vaccine composition also helps with coronavirus This may confer a protective ability against emerging coronavirus strains related to the evolution of the scogene. In this way, a single coronavirus vaccine composition can be used to treat various different coronaviruses. It can provide protection against the coronavirus. To control dispersion and provide a cost-effective means of doing so.

[0075] Thirdly, the different peptides identified by the inventors are different HLA supertypes. They can be bonded together. Each has a different HLA supertype (or corresponding poly By including multiple peptides that can bind to nucleotides, different HLs can be created. A vaccine composition effective in individuals having type A can be obtained. Using this coronavirus vaccine composition, it is possible to confer protection to the majority of the human population. Yes, it is possible. This also provides a cost-effective means of controlling the spread of coronavirus infection. do.

[0076] Fourth, the coronavirus peptide contained in the vaccine composition of the present invention is a nanoparticle, e.g. For example, it may bind to gold nanoparticles. As will be explained in more detail below, to the nanoparticles The binding of nanoparticles reduces or eliminates the need to include adjuvants in the vaccine composition. Binding to the offspring also reduces or eliminates the need to include the virus in the vaccine composition. Therefore, the vaccine composition of the present invention may cause adverse clinical effects when administered to an individual. The possibility is low.

[0077] Vaccine compositions, each selected from SEQ ID NOs: 1-34 or their variants The peptides may include two or more peptides according to claim 1, each of the peptides It may have any of the properties described in the "Peptide" section above. For example, each peptide D is sequence numbers 1-34 or their variants, and optionally one or more CD8+ T cells. Epitope, one or more CD4+ T cell epitopes and / or one or more B cell epitopes The vaccine composition may include multiple sequences selected from the following. In one embodiment, the vaccine composition is, Three or more different sequences selected from sequence numbers 1-34 or their variants 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more Top, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 The vaccine composition may contain one or more peptides or 33 or more peptides. Combinations may be included. For example, a vaccine composition in which each of the following is sequence numbers 1-34 or It may also contain 34 peptides, each containing a different sequence selected from the variants of the original.

[0078] Each vaccine composition contains one of SEQ ID NOs: 1-34 or a variant thereof. Two or more different T cell receptors that can bind to peptides that can bind to different T cell receptors It may also contain peptides, and each different T cell receptor may be SEQ ID NOs: 1-34 or a variant thereof. It can bind to different sequences selected from the above. Each of the peptides is a "peptide" It may have any of the properties described in the section on "Tide". For example, it may be contained in a vaccine composition. Each peptide contains one of the peptides from SEQ ID NOs: 1-34 or a variant thereof. It has the potential to bind to multiple different T cell receptors that can bind to it. The vaccine contains one or more CD8+ T cell epitopes and one or more CD4+ T cell epitopes. The vaccine may contain a tope and / or one or more B cell epitopes. In one embodiment, the vaccine compound Each product is linked to a peptide containing one of sequence numbers 1-34 or a variant thereof. Three or more, four or more, five different T cell receptors that can bind to each other. 1 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or less Top, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more The vaccine composition may contain the above or 33 or more peptides. It may also contain. Vaccine composition, for example, each of SEQ ID NOs: 1 to 34 It can bind to different T cell receptors, including peptides containing one or a variant thereof. It may contain 34 peptides that can perform certain functions.

[0079] Cross-defense Sequence IDs 1-34, identified by the present inventors, are caused by multiple coronaviruses. Therefore, the vaccine composition contains two or more coronaviruses, for example, SAR. Potential to induce a protective immune response against S coronavirus and SARS coronavirus 2. In other words, the vaccine composition of the present invention is effective against several different coronaviruses. This may trigger an immune response that is a form of cross-protection between different coronaviruses. This could be, for example, a coronavirus involved in a human epidemic or pandemic. Coronaviruses can be, for example, coronaviruses that originate from zoonotic diseases. Naviruses include, for example, those belonging to the genus Betacoronavirus. They can also be members. Coronaviruses are, for example, salvage coronaviruses (Sarb It may also be a member of the ecoronavirus subgenus.

[0080] Vaccination with a composition containing an epitope 100% homologous to a sequence from another virus. The immune response generated by this can protect against subsequent infection by the virus. It is possible. The sequence is approximately 50% or more (for example, 60%, 70%) of the sequence encoded by another virus. Compositions containing homologous epitopes (75%, 80%, 90%, 95%, 98%, or 99%) The immune response generated by vaccination protects against subsequent infections by that virus. It may offer protection. In some cases, the protective effect depends on the epitope and other viruses. This relates to the preservation of specific residues between the encoded sequence and the sequence. Therefore, the vaccine of the present invention Immunization with this composition can affect various viruses not listed in Table 1, such as other coronaviruses. It may induce a protective immune response against Rus.

[0081] Therefore, the vaccine composition of the present invention incorporates interspecies and / or inter-species efficacy. It may have the property of being a cross-protective vaccine composition. Therefore Therefore, the single coronavirus vaccine composition of the present invention is effective against various different coronaviruses. It may be used to provide protection against the spread of coronavirus infection. To control dispersion and provide a cost-effective means of doing so.

[0082] Including conserved peptides in the vaccine composition is related to the evolution of the coronavirus genome. This could provide protection against emerging coronavirus strains related to This may help in the long-term control of coronavirus infections.

[0083] Interaction with HLA supertypes The vaccine composition contains at least one that interacts with different HLA supertypes. It may contain two peptides. A vaccine composition may contain multiple such peptides. As a result, the vaccine composition provides immunity to a larger proportion of individuals to whom the vaccine composition is administered. This makes it possible to induce a response (such as a CD8+ T cell response). However, one of the peptides contained in the vaccine composition interacts with an HLA supertype. This is because it must be possible to induce an immune response in all individuals. Chido is A2, A203 / A2, A23, A24, A2403 / A2, A2403 / A2 4, A39, A3, A11, A30, A31, A32, A68, A69, B7, B8, B 35, B37, B44, B48, B53, B60, B61, B62, B63, B72, B 75, Cw1 or Cw6, or any other HLA supertype known in the art It may interact with [something]. Any combination of peptides is possible.

[0084] The vaccine composition interacts with at least two different HLA supertypes. It may contain at least one peptide. Here again, this means that the vaccine composition is a vaccine A larger proportion of individuals administered the tin composition showed an immune response (such as a CD8+ T cell response). This makes it possible to induce it. Each vaccine composition contains at least two different HLs. At least two, at least three, at least four, and at least two that interact with subtype A. Five of each, at least 10, at least 15, at least 20, at least 25 Alternatively, it may contain at least 30 peptides. Each peptide may contain, for example, at least 2 at least 3, at least 4, at least 5, at least 6, at least 7 At least 8, at least 9, at least 10, at least 11, and at least 12, at least 13, at least 14, or at least 15 different HLAs It may interact with supertypes. Each peptide may interact with A2, A2 in any combination. , A203 / A2, A23, A24, A2403 / A2, A2403 / A24, A39, A3, A11, A30, A31, A32, A68, A69, B7, B8, B35, B37 , B44, B48, B53, B60, B61, B62, B63, B72, B75, Cw1 and Cw6, or any two or more other HLA supertypes known in the art, It may have an effect.

[0085] Preferably, the vaccine composition contains peptides that interact with A3, A11, and A31. In this case, the vaccine composition may include, for example, a peptide containing SEQ ID NO: 1 and / or 11. It may also contain. For example, the vaccine composition may contain a peptide including SEQ ID NO: 1 and SEQ ID NO: 11 It may also contain peptides that include [the specified substance].

[0086] Preferably, the vaccine composition contains peptides that interact with B7 and B35. In this case, the vaccine composition may include, for example, a peptide containing SEQ ID NO: 2.

[0087] Preferably, the vaccine composition contains peptides that interact with B72, A2, and A203 / A2. Contains tide. In this case, the vaccine composition contains, for example, a peptide including SEQ ID NO: 3. That's good too.

[0088] Preferably, the vaccine composition contains peptides that interact with B72, B62, and B75. Includes. In this case, the vaccine composition includes, for example, peptides containing SEQ ID NOs: 4 and / or 15. The vaccine composition may include, for example, a peptide containing SEQ ID NO: 4 and SEQ ID NO: 1 It may also contain a peptide that includes 5.

[0089] Preferably, the vaccine composition contains peptides that interact with A68, A11, and A31. Includes. In this case, the vaccine composition includes, for example, a peptide containing SEQ ID NOs. 5 and / or 11. The vaccine composition may include, for example, a peptide containing SEQ ID NO: 5 and SEQ ID NO: 1 It may also contain a peptide that includes 1.

[0090] Preferably, the vaccine composition contains A203 / A2 and peptides that interact with A2. In this case, the vaccine composition is, for example, SEQ ID NOs: 3, 8, 13, 14, 21, 24 and The vaccine composition may also contain a peptide containing bi / or 26. For example, the vaccine composition may contain SEQ ID NO: 3 Peptides containing, peptides containing SEQ ID NO: 8, peptides containing SEQ ID NO: 13, SEQ ID NO: 14 Peptides containing sequence number 21, peptides containing sequence number 24 and sequence number It may also contain peptides including number 26.

[0091] Preferably, the vaccine composition includes peptides that interact with A2403 / A2 and A23. This includes. In this case, the vaccine composition may also include, for example, the peptide containing SEQ ID NO: 10. good.

[0092] Preferably, the vaccine composition interacts with A11, A30, A3, A68 and A31. It contains a peptide. In this case, the vaccine composition contains, for example, a peptide including SEQ ID NO: 11. It may include "do".

[0093] Preferably, the vaccine composition contains peptides that interact with A11, A30, A3, and A68. Contains cido. In this case, the vaccine composition contains, for example, a peptide containing SEQ ID NO: 12. But that's fine.

[0094] Preferably, the vaccine composition contains peptides that interact with B60, B48, and B44. Includes. In this case, the vaccine composition may include, for example, a peptide containing SEQ ID NO: 17. stomach.

[0095] Preferably, the vaccine composition contains compounds that interact with A68, B63 and A203 / A2. Contains a peptide. In this case, the vaccine composition contains, for example, a peptide containing SEQ ID NO: 22. That's fine.

[0096] Preferably, the vaccine composition comprises a peptide that interacts with A2, A203 / A2, A69, and A32. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 24 or SEQ ID NO: 31.

[0097] Preferably, the vaccine composition comprises a peptide that interacts with A2, A203 / A2, and A68. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 26.

[0098] Preferably, the vaccine composition comprises a peptide that interacts with B35, B53, and A29. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 27.

[0099] Preferably, the vaccine composition comprises a peptide that interacts with B37, B60, B61, B44, and B48. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 29.

[0100] Preferably, the vaccine composition comprises a peptide that interacts with Cw6 and Cw1. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 30.

[0101] Preferably, the vaccine composition comprises a peptide that interacts with A30, B7, B8, B62, and B72. In this case, the vaccine composition may comprise a peptide comprising, for example, SEQ ID NO: 34.

[0102] Nanoparticles The peptide or one or more peptides may be conjugated, for example, to the nanoparticles in the vaccine composition of the present invention. They may be combined. Any other peptides further contained in the vaccine composition may also be nanoparticles. It may be bound to the nanoparticles. Binding to nanoparticles, such as gold nanoparticles, is beneficial.

[0103] As described above, the binding of peptides to nanoparticles (such as gold nanoparticles) in the vaccine composition Reduces or eliminates the need to include viruses or adjuvants. Nanoparticles are peptides. Potentially contains immune "danger signals" that help effectively induce an immune response to [the disease]. There is a reason why nanoparticles induce the activation and maturation of dendritic cells (DCs), which are necessary for a robust immune response. It is possible that nanoparticles will be produced by cells such as antigen-presenting cells, and therefore by It may contain non-self components that improve the uptake of butyl. Therefore, to nanoparticles The binding of the peptide allows antigen-presenting cells to stimulate virus-specific T cells and / or B cells. It may enhance capabilities. Binding to nanoparticles can also be done subcutaneously, intradermally, transdermally, and orally / orally. This facilitates the delivery of vaccine compositions via pathways and provides flexibility in administration.

[0104] Nanoparticles can be used as a substrate for immobilizing ligands, ranging from 1 to 100 nanoparticles. These are particles with a size of nanometers (nm). In the vaccine composition of the present invention, nanoparticles The average diameter is 1-100, 20-90, 30-80, 40-70, or 50-60 nm. The average core diameter may be 5 to 40 nm, for example, 10 to It has an average diameter or average core diameter of 30 nm or 20-32 nm. Preferably, nanoparticles. The child has an average diameter or average core diameter of 5 nm. The diameter promotes the uptake of nanoparticles into the cytosol. The average diameter or average core diameter is Measurements can be taken using techniques well known in this field, such as transmission electron microscopy.

[0105] Nanoparticles suitable for delivering antigens such as peptides according to the present invention are known in the art. Methods for producing nanoparticles like the one described are also publicly known.

[0106] Examples of nanoparticles include polymer nanoparticles, inorganic nanoparticles, liposomes, and immunostimulatory complexes. (ISCOM), virus-like particles (VLPs), or self-assembling proteins may also be used. The nanoparticles are preferably calcium phosphate nanoparticles, silicon nanoparticles, or gold nanoparticles. It seems so.

[0107] The nanoparticles may also be polymer nanoparticles. Polymer nanoparticles are poly(d,l- Lactide-coglycolide (PLG), poly(d,l-lactic acid-coglycolic acid) (PL GA), poly(g-glutamic acid)(g-PGA)m poly(ethylene glycol)(PE G) May contain one or more synthetic polymers such as polystyrene. Polymer nanoparticles It is a polysaccharide, such as one or more natural polysaccharides, such as pullulan, arginate, inulin, and chitosan. The polymer may be included. The use of polymer nanoparticles may include polymers that may be included in the nanoparticles. - may be advantageous due to its properties. For example, the above natural and synthetic polymers are good Desirable biocompatibility and biodegradability, non-toxic properties, and / or the ability to be manipulated into desired shapes and sizes. Polymer nanoparticles may have the ability to form hydrogel nanoparticles. It is possible. Hydrogel nanoparticles are a nano-sized hydrophilic three-dimensional polymer network. It is one type. Hydrogel nanoparticles have a flexible mesh size and multivalent conjugation. having favorable properties including a large surface area, a high water content, and a high loading capacity for antigens Polymers such as poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are particularly suitable for forming hydrogel nanoparticles.

[0108] The nanoparticles may be inorganic nanoparticles. Typically, inorganic nanoparticles have a rigid structure and are non-biodegradable. However, inorganic nanoparticles may be biodegradable. Inorganic nanoparticles may include a shell in which an antigen may be encapsulated. Inorganic nanoparticles may include a core to which an antigen may be covalently bonded. The core may include a metal. For example, the core may include gold (Au), silver (Ag), or copper (Cu) atoms. The core may be formed from two or more types of atoms For example, the core may include an alloy such as an alloy of Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd, or Au / Ag / Cu / Pd. The core may include calcium phosphate (CaPO4). The core may include a semiconductor material such as cadmium selenide.

[0109] Other exemplary inorganic nanoparticles include carbon nanoparticles and silica-based nanoparticles. Carbon nanoparticles have good biocompatibility and can be synthesized into nanotubes and mesoporous spheres. Silica-based nanoparticles (SiNP) are biocompatible and can be prepared using structural parameters that can be adjusted to be suitable for their therapeutic applications.

[0110] The nanoparticles may be silicon nanoparticles such as elemental silicon nanoparticles. The nanoparticles may be mesoporous or may have a honeycomb pore structure. Preferably Nanoparticles are elemental silicon particles that have a honeycomb pore structure. This is known in the art and can be adjusted to virtually any load, route of administration, target, or release profile. It provides adjustable and controlled drug loading, targeting, and release that can be regulated. For example, such nanoparticles increase the bioavailability of their loads. And / or may improve the intestinal permeability and absorption of orally administered active substances. Nanoparticles The children may have a very high load capacity due to their porous structure and large surface area. It has properties. Nanoparticles last for several days, weeks, or months, depending on their physical properties. This has the potential to release those burdens. Silicon is an element that naturally exists in the human body. Therefore, nanoparticles may not elicit a response from the immune system. It is advantageous in terms of safety.

[0111] Any of the above SiNPs may be biodegradable or non-biodegradable. Biodegradable SiN P is orthosilicic acid, which is a biologically available form of silicon. It may dissolve in acid. (orthosilicic acid) It has been shown to be beneficial for the health of bones, connective tissue, hair, and skin.

[0112] Nanoparticles may also be liposomes. Liposomes are typically biodegradable and non-toxic. It contains a self-assembling phospholipid bilayer shell formed from aqueous phospholipids and having an aqueous core. Posomes are monolayer vesicles containing a single phospholipid bilayer, or separated by a layer of water. It may be a multilayer vesicle containing several concentric phospholipid shells. As a result, lipos The system incorporates hydrophilic molecules into the aqueous core or hydrophobic molecules into the phospholipid bilayer. It can be adjusted. Liposomes may encapsulate the antigen within the core for delivery. Posomes incorporate viral envelope glycoproteins into their shells to form virosomes. It may be done. Several liposome-based products have been established in this technology field and are used in humans. It has been approved for use.

[0113] The nanoparticles may also be immunostimulatory complexes (ISCOMs). ISCOMs are typically ISCOM is a cage-like particle formed from colloidal saponin-containing micelles. Resterol, phospholipids (phosphatidylethanolamine or phosphatidylcholine) (and saponins such as Quil A derived from the tree Quillia saponaria) ) may include. ISCOM has traditionally included herpes simplex virus type 1, hepatitis B, or 1. Viral envelope proteins such as envelope proteins derived from the flu virus It has been used to capture quality.

[0114] Nanoparticles may also be virus-like particles (VLPs). VLPs are biocompatible capsules. These are self-assembling nanoparticles that lack infectious nucleic acids and are formed by the self-assembly of proteins. VLPs typically have a diameter of about 20 to about 150 nm, for example, about 20 to about 40 nm, 30~140nm, 40~130nm, 50~120nm, 60~110 The wavelength is approximately 70-100 nm or 80-90 nm. VLPs interact with the immune system. It takes advantage of the power of the evolved viral structure that is naturally optimized for its action. The optimized nanoparticle size and repeating structural order are such that VLPs can be used even in the absence of adjuvants. This means inducing a strong immune response.

[0115] Nanoparticles may also be self-assembling proteins. For example, nanoparticles may be ferritin It may be included. Ferritin is a protein that can self-assemble into a nearly spherical 10nm structure. Quality. Nanoparticles may contain major vault protein (MVP). 96 units The MVP consists of barrel-shaped vaulted nanoparticles with a width of approximately 40 nm and a length of 70 nm. The offspring can self-assemble.

[0116] The nanoparticles may also be calcium phosphate (CaPO4) nanoparticles. No particles are one or more (for example, two or more, ten or more, twenty or more, fifty or more, 100 or more) The core may contain (1 or more, 200 or more, or 500 or more) CaPO4 molecules. CaPO4 nanoparticles and methods for producing them are known in the art. For example, CAP A stable nano-suspension of nanoparticles is obtained by mixing an inorganic salt solution of calcium and phosphate under constant conditions. It may also be produced by mixing in a fixed ratio.

[0117] CaPO4 nanoparticles are approximately 80-100 nm in size, for example, approximately 82-98 nm, approximately 84- It may have an average particle size of approximately 96 nm, approximately 86-94 nm, or approximately 88-92 nm. This particle size is better than other larger particle sizes in terms of immune cell uptake and immune response. It can potentially bring about performance. For example, 1 month, 2 months, 3 months, 6 months, 12 months, 1 When measured over periods of 8 months, 24 months, 36 months, or 48 months, the particle size is stable. There is a possibility that (i.e., no significant change) will occur.

[0118] CaPO4 nanoparticles are adsorbed onto the surface of the nanoparticles during particle synthesis, or CaPO4 It can be co-formulated with one or more antigens co-precipitated with the present invention. Peptides such as D are dissolved in DMSO (for example, at a concentration of about 10 mg / ml). N-acetyl-glucosamine (GlcNAc) (e.g., 0.093 mol / L and ultra- Add the CaPO4 nanoparticle suspension to pure water and let it steep at room temperature for approximately 4 hours (for example, 1 hour, 2 hours). By mixing for 3, 5, 6, 7, 8, 9, or 10 hours It can also be bonded to CaPO4 nanoparticles.

[0119] The vaccine composition is approximately 0.15 to 0.8%, for example, 0.2 to 0.75%, 0.25 ~approximately 0.7%, 0.3~approximately 0.6%, 0.35~approximately 0.65%, 0.4~approximately 0.6%, or It may contain 0.45 to approximately 0.55% CaPO4 nanoparticles. Preferably, the vaccine compound The product contains approximately 0.3% CaPO4 nanoparticles.

[0120] CaPO4 nanoparticles have a high degree of bioavailability due to their chemical similarity to human hard tissues such as bone and teeth. It is biocompatible. Therefore, advantageously, CaPO4 nanoparticles can be used in therapeutic applications. In that case, it is nontoxic. CaPO4 nanoparticles are delivered via intramuscular, subcutaneous, oral, or inhalation routes. It is safe to administer. CaPO4 nanoparticles are also easy to synthesize commercially. Furthermore, CaPO4 nanoparticles may be involved in the sustained release of antigens, which is related to nanoparticles. It may enhance the induction of an immune response to the bound peptide. CaPO4 nanoparticles The child may be used as both an adjuvant and a drug delivery vehicle.

[0121] The nanoparticles may be gold nanoparticles. Gold nanoparticles are well known in the art, in particular International Publication No. 2002 / 32404, International Publication No. 2006 / 037979 Brochure, International Publication No. 2007 / 122388 Brochure, International Publication No. 2007 In pamphlet No. / 015105 and international publication No. 2013 / 034726 It is described. Gold nanoparticles bound to each peptide are described in International Publication No. 2002 / 32404. Pamphlet No. 2006 / 037979, International Publication No. 200 Pamphlet No. 7 / 122388, International Publication No. 2007 / 015105 and The gold nanoparticles are as described in either of the international publications, No. 2013 / 034726. That's fine.

[0122] Gold nanoparticles contain a core containing gold (Au) atoms. The core contains one or more Fe, Cu or It may further contain Gd atoms. The core can be Au / Fe, Au / Cu, Au / Gd, Au / Formed from gold alloys such as Fe / Cu, Au / Fe / Gd, or Au / Fe / Cu / Gd. That's also fine. The total number of atoms in the core is 100 to 500 atoms, for example, 150 to 450 atoms, 2 The number of atoms may be 00-400 or 250-350. Gold nanoparticles may be 1-100 They may have an average diameter of 20-90, 30-80, 40-70, or 50-60 nm. Preferably, the gold nanoparticles have an average diameter of 20 to 40 nm.

[0123] The nanoparticles are coated with α-galactose and / or β-GlcNAc on a surface. It may also contain α-galactose and / or β-GlcNAc. The nanoparticles may include a passivated surface. In this case, the nanoparticles may be, for example, metal and / or semiconductor. The nanoparticles may also include a core containing conductive atoms. For example, the nanoparticles may be gold nanoparticles. It is also possible. β-GlcNAc can activate bacterial pathogens that can trigger antigen-presenting cells. This is a PAMP (Particle-Amplified Pattern) pattern. It is then coated with β-GlcNAc. Alternatively, nanoparticles containing a passivated surface may nonspecifically stimulate the immune response. Therefore, one or more of the CD8+ T cell epitopes shown in Sequence IDs 1-23 or The binding of flavivirus peptides, including variants of the present invention, to such nanoparticles is The administration of the tin composition to an individual may improve the immune response induced by it.

[0124] One or more ligands other than peptides may be linked to the nanoparticles, as described above. Any type of nanoparticle may be used. The ligand partially or completely coats the surface of the core. A "corona" may form a covering, layer, or coating. The corona is It is possible that this is an organic layer surrounding or partially surrounding the nanoparticle core. Corona may cause or be involved in the passivation of the nanoparticle core. Therefore, in certain cases, the corona is not complete enough to stabilize the core. It may also be a coating layer. Corona promotes the solubility of the nanoparticles of the present invention, such as water solubility. It's possible.

[0125] The nanoparticles consist of at least 10, at least 20, at least 30, and at least 4 It may contain 0 or at least 50 ligands. A ligand is one or more peptides. , protein domain, nucleic acid molecule, lipid group, carbohydrate group, anionic group, or cationic It may also contain a carbohydrate group, glycolipids and / or glycoproteins. The carbohydrate group may be a polysaccharide, oligosaccharide or It may also be a monosaccharide group (e.g., glucose). One or more ligands are non-self components. This may also be the case, and as a result, due to the similarity with pathogenic components, nanoparticles are detected by antigen-presenting cells. The likelihood of incorporation increases. For example, one or more ligands can be incorporated into the carbohydrate portion (bacterial char). It may also contain a hydrated portion, a surfactant portion, and / or a glutathione portion. (Example) Suitable ligands include thiolated glucose and N-acetylglucosamine (GlcNAc). ), glutathione, 2'-thioethyl-β-D-glucopyranoside and 2'-thioethyl -D-glucopyranosides are one example. Preferred ligands include those that form sugar nanoparticles. These include complex carbohydrates.

[0126] The linkage of ligands to the core may be facilitated by linkers. Linkers are, It may contain thiol groups, alkyl groups, glycol groups, or peptide groups. For example, Linka - may contain C2-C15 alkyl and / or C2-C15 glycol. Linker - A sulfur-containing group, amino-containing group, phosphate-containing group or that can be covalently bonded to the core. It may contain an oxygen-containing group. Alternatively, the ligand may contain, for example, a sulfur-containing group. It is directly linked to the core via a group, an amino acid-containing group, a phosphate-containing group, or an oxygen-containing group. That's good too.

[0127] Binding to nanoparticles The peptide may be bound to a nanoparticle at its N-terminus. Typically, The molecule is bound to the core of the nanoparticle, but it may also be able to bind to the corona or ligand. be.

[0128] Peptides, for example, contain sulfur-containing groups, amino-containing groups, phosphate-containing groups, or acids. The atoms in the element-containing group and the atoms in the nanoparticle or its core are directly bonded together. They may be in contact with each other.

[0129] A linker may be used to link the peptide to the nanoparticles. The linker is the original component in the core. A sulfur-containing group, amino-containing group, phosphate-containing group, or oxygen-containing group that can be covalently bonded to the child. It may contain groups. For example, the linker may contain a thiol group, an alkyl group, a glycol group, or a phenyl group. It may contain a butyl group.

[0130] The linker may include a peptide portion and a non-peptide portion. The peptide portion is sequence It may contain X1X2Z1, where X1 is an amino acid selected from A and G, and X2 is , is an amino acid selected from A and G, and Z1 is an amino acid selected from Y and F. Yes. The peptide portion may include the sequence AAY or FLAAY (SEQ ID NO: 44). The peptide portion of the linker may be linked to the N-terminus of the peptide. The thiomethyl group consists of C2-C15 alkyl and / or C2-C15 glycols, for example, thioethyl It may contain a thiopropyl group or a thiopropyl group.

[0131] The linker is (i) HS-(CH2)2-CONH-AAY, (ii) HS-(CH2 )2-CONH-LAAY (SEQ ID NO: 43), (iii)HS-(CH2)3-CONH -AAY, (iv)HS-(CH2)3-CONH-FLAAY (SEQ ID NO: 44), (v )HS-(CH2) 10 -(CH2OCH2)7-CONH-AAY, and (vi)HS -(CH2) 10-(CH2OCH2)7-CONH-FLAAY (sequence number 44) This is also acceptable. In this case, the thiol group of the non-peptide portion of the linker connects the linker to the core. To tie.

[0132] Other suitable linkers for binding peptides to nanoparticles are known in the art. This may be easily identified and implemented by those skilled in the art.

[0133] If the vaccine composition contains two or more peptides, then two or more (for example, three or more, four or more) The above (5 or more, 10 or more, or 20 or more) peptides are bound to the same nanoparticle. This is also acceptable. Two or more (for example, three or more, four or more, five or more, ten or more, or twenty or more) The peptides in ) may each be bound to different nanoparticles. However, if the peptides are bound The combined nanoparticles may be of the same type. For example, each peptide may be Each peptide may be bound to gold nanoparticles. Good. The nanoparticles to which the peptide is bound may be of different types. Example For example, one peptide may be bound to a gold nanoparticle, and another peptide may be bound to a CaPO4 nanoparticle. It may be bound to particles.

[0134] Polynucleotide vaccine The present invention relates to the peptide described in claim 1, or any one of SEQ ID NOs 1 to 34 or It can bind to T cell receptors that can bind to peptides containing that variant. The present invention provides a vaccine composition containing a polynucleotide encoding a peptide.

[0135] Vaccine compositions, each selected from SEQ ID NOs: 1-34 or their variants The polynucleotides may include sequences and may encode two or more peptides of the present invention. .

[0136] Each vaccine composition contains one of SEQ ID NOs: 1-34 or a variant thereof. Two or more different T cell receptors that can bind to peptides that can bind to different T cell receptors It may also contain a polynucleotide encoding a peptide, and each different T cell receptor has a sequence It can bind to different sequences selected from numbers 1 to 34 or their variants.

[0137] The vaccine composition comprises two or more polynucleotides, each encoding the peptide of the present invention. It may contain tides, and each peptide is selected from SEQ ID NOs: 1-34 or their variants. Includes the following array.

[0138] Each vaccine composition contains one of SEQ ID NOs: 1-34 or a variant thereof. Encoding peptides that can bind to T cell receptors that can bind to peptides. It may contain two or more polynucleotides, and each peptide is one of the sequence numbers 1 to 34. Different T cell receptors that can bind to peptides containing either one or a variant thereof. Each different T cell receptor can bind, and each is selected from SEQ ID NOs: 1-34 or its variants. It can be combined with different selected sequences.

[0139] Polynucleotides may be DNA. Polynucleotides may be RNA. For example, the polynucleotide may be mRNA.

[0140] Pharmaceuticals, treatment methods and therapeutic uses The present invention relates to a method for preventing or treating coronavirus infection, wherein the coronavirus The vaccine composition of the present invention is administered to individuals who are infected or at risk of infection. The present invention also provides a method including the prevention of coronavirus infection in individuals or The present invention provides a vaccine composition for use in methods of treatment.

[0141] Coronavirus infection is caused by, for example, a coronavirus that is involved in human epidemics or pandemics. It may also be a coronavirus infection. Coronavirus infections, for example, are caused by zoonotic diseases. It could also be a coronavirus infection. Coronavirus infections include, for example, beta-coronavirus infections. It may also be an infection caused by a member of the genus Betacoronavirus. Coronaviruses include, for example, salvecoronaviruses. ) It may also be an infectious disease caused by a member of the subgenus. Coronavirus infection is, for example, SA It may be RSV infection or SARS-CoV-2 infection.

[0142] The vaccine composition may be provided as a pharmaceutical composition. The pharmaceutical composition is preferably a drug The pharmaceutical composition contains a scientifically acceptable carrier or diluent. The pharmaceutical composition is formulated using any preferred method. Cells may be prepared using standard pharmaceutically acceptable carriers and / or excipients. Formulation may be carried out using routine methods in the field of pharmacy. The precise properties of the formulation are The method depends on several factors, including the cells to be administered and the desired administration route. The formulation was described in Remington's Pharmaceutical Sciences. 19th Edition, Mack Publishing Company, Eas It is fully documented in tern Pennsylvania, USA.

[0143] The vaccine composition or pharmaceutical composition may be administered by any route. Preferred route This includes intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, transdermal, and oral / oral routes, however These are not the only options.

[0144] The composition may be prepared with a physiologically acceptable carrier or diluent. Typically Such compositions may be prepared as liquid suspensions of peptides and / or peptide-linked nanoparticles. The peptides and / or peptide-linked nanoparticles are manufactured. The peptides and / or peptide-linked nanoparticles are pharmaceutically acceptable and contain active ingredients. It may be mixed with a suitable excipient. Suitable excipients include, for example, water, saline solution, and These include chistrose, glycerol, and combinations thereof.

[0145] Furthermore, if desired, the pharmaceutical composition may contain a humectant or emulsifier and / or a pH buffer. It may contain any small amount of auxiliary substances.

[0146] Peptides or peptide-linked nanoparticles are administered in a manner compatible with the administered formulation, and so It is therapeutically effective in small amounts. The amount administered depends on the target being treated, the disease being treated, and the target. It depends on the capacity of the immune system. The exact amount of nanoparticles that need to be administered is at the discretion of the physician. They may exist, and may be specific to each object.

[0147] Any suitable number of peptides or peptide-linked nanoparticles may be administered to the target. For example , at least or about 0.2 × 10 per kg of patient 6 , 0.25 × 10 6 , 0.5 × 10 6 , 1.5×10 6 , 4.0×10 6 or 5.0 × 10 6 peptide or peptide linkage It may be possible to administer nono-particles. For example, at least or about 10 5 、10 6 、10 7 、10 8 、 10 9 peptide or peptide-conjugated nanoparticles may be administered. As a guide, the number of peptides or peptide-conjugated nanoparticles administered is 10 ~10 5 ~10 9 、preferably 10 6 ~10 8 and may be.

[0148] Example 1 Introduction As described below, the coronavirus peptide was conjugated to gold nanoparticles.

[0149] As shown in Table 4, eight peptides: P77, P81, P83, P86, P92, P9 6, P99 and P100 were selected.

Table 4

[0150] The purpose of this experiment was to produce a 100 mg Au scale batch for toxicity testing based on test GNP EM009-062-01 at the 4 mg Au scale 。The total peptide loading started from 5 equivalents per NP (estimated as 100 Au atoms / NP) for ligand exchange.

[0151] Method ]

Table 5

[0152] Calculation Table 5 relates to the preparation of DMSO solutions of eight peptides. Table 5 contains 90% peptide Assuming quantity / purity, the weighted amount of DMSO added to produce a 1 mM stock was The quantities and volumes are listed. Quantitative analysis of peptides is problematic, and for these peptides... Peptide purity is cited, but "peptide content" is not given, and these are time Since the actual rate can be as low as 50%, the inventors used 90% as an estimate, and then In-laboratory HPLC quantification was performed. [Table 6]

[0153] Since the weight of 1 mL of base GNP is 1.003 g, Au = 1.00 mg 3 * (100 / 3.193) = 31.4 g, and the base particles are weighted for accuracy. The actual volume obtained is based on the following calculation: 100 mg of Au = 505 μl Mole Au = 5 times excess peptide / NP, resulting in 5.05 μmoles of NP = 25.25 μmoles total Peptides, but since there are 8 peptides, = 3.16 μmol of each peptide.

[0154] The peptide was weighed, dissolved in a laminar flow hood (LAF), and placed in a newly sealed bottle of DMSO. The above peptide stock was used for solubilization. The peptide stock was assayed by HPLC. For example, Regarding P77, the inventors predict that the expected 70 AUC (Tyr at 278 nm) Instead of the area defined by the absorbance of the Tryp residue, an area of ​​50.1 was obtained. 1 mM Instead, the peptide was determined to be 0.716 mM, so 3.16 μmol To obtain P77, it was estimated that 4.43 ml would be required, as shown in the table below. Several peptide stocks contained disulfide peptides. Thiol peptide region Since only the region was quantified, these were ignored.

[0155] The actual volumes of the DMSO solutions containing the eight peptides collected for ligand exchange are shown in the table below. This is shown in 6. [Table 7]

[0156] procedure Weigh 31.4 g of ChemCon-based GNP into a 50 mL sterile Falcon tube. Then, all eight peptide DMSO solutions were added together in a 250 mL glass round-bottom flask. Next, ChemCon Tox-based GNP was added, mixed briefly, and the container was sterilized. The mixture was flushed and sealed. This ligand exchange solution mixture was heated in a water bath at 300 rpm for 30 minutes. The mixture was stirred continuously at ℃ for 3 hours.

[0157] After 3 hours, the dark brown GNP solution was placed in 15 mL of 10 kDa Amicon Tubes. Concentrated using ×8), then washed with sterile "water for injection," and all additions were carried out in LAF. ×5, 4000G per centrifuge for 8 minutes, DMSO in Amicon device (Keep the concentration below 15%). 12 1.5 mL tubes of GNP solution were dispensed from Amicon tubes. The aggregates were collected in a Pendorf tube and then centrifuged at 17G for 2 minutes to remove all aggregates. The residue was removed. The supernatant from each Eppendorf tube was combined and two 0.2 μm sterile sodium phosphate solutions were added. Filtered through an Igene syringe filter (approximately 5 mL of GNP solution per filter). The final sterile GNP solution (EM009-064-01) is 10 mL, and then this is used for 4 It was kept at °C. 200 μL of this final GNP solution was removed and kept separately for analysis.

[0158] I noticed that there is some GNP material on the Amicon tube membrane, and 17k After a final hard spin of 2 minutes at G, pellets were observed at the bottom of each Eppendorf tube. The subsequent investigation involved using 0.2M carbonate buffer (CB pH 10) to analyze the precipitated GNP. It was shown that it can be resuspended in .22). It was shown to be sedimented from Amicon and Eppendorf tubes. All the collected GNP was resuspended in 0.2M carbonate buffer (pH 10.22) and the same process was carried out from the previous day. Concentrate on 8 Amicon tubes, then wash again with 0.2M CB, and continue Washed with water (5 times, 8 minutes at 4000G per centrifuge). Amicon tube GNP was hardly observed in the sample. After centrifugation at 17kG for 2 minutes (precipitation was observed). (Undetected), this GNP solution (EM009-064-02) is 2.7 mL, The samples were kept at 4°C for further analysis. Below, the Au yield of the main sterile preparation was 62.6%. This indicates that 24.9% was lost as insoluble aggregates. To further increase yield, use 0.2M carbonate buffer (pH 10.22) before washing with water. ) Cleaning can be used.

[0159] EM009-064-01: Supernatant GNP solution derived from a large toxic batch. EM009-064-02: 0.2M CB (pH 10.22) derived from the primary toxic batch. ) Precipitated GNP after treatment. EM009-064-03:EM0090-64-01 (50 μL) and 02 (13.5 2 μL of the test mixture.

[0160] analysis Gold assay The results of the gold assay are shown in Table 7. [Table 8]

[0161] Absorbance spectrum ChenconTox base GNP, EM009-064-01, EM009-064 The absorbance spectra of -02 and EM009-064-03 are shown in Figure 1. ChemCon Tox-based GNP, EM009-064-01, 02 and 03 No plasmon band at 520nm was observed in the CT scan.

[0162] DLS The results of the DLS are shown in Figure 2 and summarized below. For ChemConTox-based GNP, size = 3.77nm (n=3), SD = ±1.22nm. In the case of EM009-064-01, size = 6.08nm (n=3). SD = ±2.62nm. In the case of EM009-064-02, size = 4.77nm (n = 3) SD = ±1.21 nm. For EM009-064-03, size = 4.75 nm (n=3), SD=±1.05nm.

[0163] HPLC of 400nm nanoparticles This method is summarized in Figure 3. Sample preparation is shown in Table 8 below. [Table 9]

[0164] For each batch, 16 μg of Au was added to 40 μL of water. HPLC was performed on 10 μL. This GNP solution was injected into the column, yielding 4 μg of Au per injection in each batch.

[0165] HPLC results showed that all three samples had excellent peptide uptake (>96%). This was shown. EM009-064-01 GNP without any peptides was 3.4%. Yes. EM009-064-02 GNP without any peptides is 0%. E M009-064-03: The GNP without any of the peptides is 2.2%.

[0166] LC-MS peptide quantification The sample preparation is shown in Table 9 below. [Table 10]

[0167] For each batch, 25 μg of Au was mixed with 0.1 M TCEP and incubated at 40°C for 4 hours. It was evaporated, and then supplemented with DMSO up to 100 μL. LC-MS showed that 32 μL of this The GNP solution was injected into the column, yielding 8 μg of Au per injection in each batch.

[0168] The results are shown in Figure 4.

[0169] Table 10 shows the individual peptide loadings from all three batches. [Table 11]

[0170] conclusion The particle size of both batches is good. However, batch EM009-064-01 ( The supernatant GNP solution is slightly less than EM009-064-02 (precipitated GNP treated with CB). It has a large size. The 520nm plasmon band is in any of the three batches. It was not observed even when left unattended.

[0171] HPLC analysis of the total GNP products showed a minimum GNP <4% with no peptides present. A large single peak from the M009-064-02HPLC chromatogram indicates this batch This indicates that it has a higher loading of hydrophobic peptides (P92 and / or P100). That is probably why this batch precipitated from the aqueous solution during Amicon washing.

[0172] EM009-064-01: The total peptide load is 4.2 equivalents (starting from 5 equivalents). ). LC-MS has a slightly higher load for P81 and P77, compared to P86 and P83 showed a slightly lower load than expected.

[0173] EM009-064-02: The total peptide load is 9.4 equivalents (starting from 5 equivalents). ). LC-MS has slightly lower loads for P77 and P83, compared to P100. It showed a very high loading, almost twice as high as other peptides. P100 is highly hydrophobic, and such high loads mean that this batch is EM009-0 64-01 This explains the reason for precipitation during water washing in purification. However, 0.2M After washing with CB, this precipitated GNP can be resuspended in aqueous solution. The solution can be redissolved under non-sterile conditions and mixed with the main EM009-064-01 product. It is not intended to do so.

[0174] EM009-064-03: 50 μL of EM009-064-01 to 13.52 μL Mixed with EM009-064-02. The final peptide load was 4.89. P83 The load is still a little low, and the P100 load is still high, but this mixed batch is EM Compared to batches 009-064-01 and 02, this batch showed better overall peptide loading. The results were shown.

[0175] EM009-064-01 contains approximately 62.6 mg of Au, while EM009-064-02 It contains approximately 24.9 mg of Au. When both batches are combined, 87.5% Gold recovery yield is obtained. In the future, the final gold yield will be increased during the final GNP refining at Amicon. To improve the peptide ratio / level, 0.2 is added to maintain the GNP in the solution. M CB should be used.

[0176] Regarding the amount of product, 10 ml of material and 1.34 μmol / ml of Au. The total peptide content of ml, i.e., 13.4 μmoles of total peptides, is approximately 1 n mole of each peptide. We provide materials in doses exceeding 1600.

[0177] The following are examples of aspects of the present invention. Item 1 A peptide containing one of sequence numbers 9, 1-8, 10-34, or a variant thereof. Section 2 A complex containing the peptide described in item 1, bound to an MHC molecule. Section 3 The complex described in item 2, comprising two or more peptides described in item 1 and two or more MHC molecules. Section 4 The complex according to item 3, wherein each peptide is bound to one of the two or more MHC molecules. Section 5 The complex according to claim 3 or 4, wherein each of the two or more MHC molecules is bound to a dextran skeleton. Section 6 The complex according to claim 5, wherein the complex further comprises a fluorophore, which optionally binds to the dextran skeleton. Section 7 The complex according to any one of claims 3 to 7, wherein the complex comprises two or more of sequence numbers 21, 1, 19, 28, 2, 27, 16, and 14 or their variants, and optionally the complex comprises sequence numbers 21, 1, 19, 28, 2, 27, 16, and 14 or their variants. Section 8 Use of the peptide described in item 1 or the complex described in any one of items 2 to 7 in a method for determining the presence or absence of a current or previous coronavirus infection in an individual. Section 9 The use according to claim 8, wherein the method includes contacting the peptide or complex with a sample obtained from the individual, and determining the presence or absence of binding between the peptide or complex and molecules contained in the sample. Section 10 The use according to item 9, wherein the molecule is an antibody or a T cell receptor. Section 11 The use according to paragraph 9 or 10, wherein the presence of the binding indicates the presence of a current or previous coronavirus infection, and / or the absence of the binding indicates the absence of a current or previous coronavirus infection. Section 12 Use of the peptide described in item 1 or the complex described in any one of items 2 to 7 in a method for identifying coronavirus-specific T cells. Section 13 The use according to paragraph 12, wherein the method includes contacting the peptide or complex with a sample obtained from an organism, and determining the presence or absence of binding between the peptide or complex and T cell receptors contained in the sample. Section 14 The use described in paragraph 13, wherein the individual is currently infected with the coronavirus. Item 15: The use described in Item 14, wherein the individual was previously infected with the coronavirus but is not currently infected. Item 16 Use of the peptide described in item 1 or the complex described in any one of items 2 to 7 in a method for identifying coronavirus-specific T cell receptors. Item 17 The use according to paragraph 16, wherein the method includes contacting the peptide or complex with a T cell receptor and determining the presence or absence of binding between the peptide or complex and the T cell receptor. Section 18 The use according to paragraph 17, wherein the presence of the binding indicates that the T cell receptor is a coronavirus-specific T cell receptor, and / or the absence of the binding indicates that the T cell receptor is not a coronavirus-specific T cell receptor. Section 19 A T cell containing a T cell receptor capable of binding to a peptide containing one of the following: SEQ ID NOs: 9, 1-8, 10-34, or a variant thereof. Section 20 A vaccine composition comprising the peptide described in item 1, or a peptide capable of binding to the T cell receptor described in item 19. Section 21 A vaccine composition as described in item 20, (a) Two or more peptides as described in item 1, each containing a different sequence selected from SEQ ID NOs. 9, 1-8, 10-34 or their variants, or (b) Two or more peptides, each capable of binding to a different T cell receptor as described in item 19, wherein each different T cell receptor can bind to a different sequence selected from SEQ ID NOs. 9, 1-8, 10-34 or their variants. A vaccine composition containing the following: Section 22 The vaccine composition according to item 21, comprising two or more peptides, each containing a different sequence selected from SEQ ID NOs: 21, 1, 19, 28, 2, 27, 16, and 14 or their variants. Section 23 The vaccine composition according to item 21, wherein each of the two or more peptides interacts with a different HLA supertype. Section 24 A product comprising at least one peptide as described in item 20, wherein the peptide interacts with at least two different HLA supertypes, as described in any one of items 20 to 23. The vaccine composition. Section 25 The aforementioned at least two different HLA supertypes (i)A2, A203 / A2, A23, A24, A2403 / A2, A2403 / A24, A39, A3, A11, A30, A31, A32, A 68, A69, B7, B8, B35, B37, B44, B48, B53, B60, B61, B62, B63, B72, B75, Cw1 and Cw6, (ii) A3, A11 and A31, (iii) B7 and B35, (iv) B72, A2 and A203 / A2, (v) B72, B62 and B75, (vi) A68, A11 and A31, (vii) A203 / A2 and A2, (viii) A2403 / A2 and A23, (ix) A11, A30, A3, A68 and A31, (x) A11, A30, A3 and A68, (xi) B60, B48 and B44, (xii) A68, B63 and A203 / A2, (xiii) A2, A203 / A2, A69 and A32, (xiv) A2, A203 / A2 and A68, (xv)B35, B53, A29, (xvi) B37, B60, B61, B44 and B48, (xvii) Cw6 and Cw1, and (xviii) A30, B7, B8, B62 and B72 A vaccine composition selected from, according to item 23 or 24. Section 26 The vaccine composition according to any one of claims 20 to 25, wherein the peptide is bound to nanoparticles. Section 27 The vaccine composition according to any one of claims 21 to 26, wherein each of the two or more peptides is bound to a nanoparticle. Section 28 The vaccine composition according to claim 26 or 27, wherein the nanoparticles are gold nanoparticles, calcium phosphate nanoparticles, or silicon nanoparticles, and optionally the gold nanoparticles are coated with α-galactose and / or β-GlcNAc. Section 29 The vaccine composition according to any one of claims 26 to 28, wherein the peptide is bound to the nanoparticles via a linker. Item 30 A vaccine composition comprising a polynucleotide encoding a peptide that can bind to the T cell receptor described in item 1 or item 19. Section 31 A vaccine composition as described in item 30, (a) Two or more peptides as described in item 1, each containing a different sequence selected from SEQ ID NOs. 9, 1-8, 10-34 or their variants, or (b) Two or more peptides, each capable of binding to a different T cell receptor as described in item 19, wherein each different T cell receptor can bind to a different sequence selected from SEQ ID NOs. 1 to 34 or its variants. A vaccine composition containing a polynucleotide encoding [a specific character]. Section 32 A vaccine composition as described in item 30, (a) Two or more polynucleotides, each encoding a peptide as described in item 1, wherein each peptide contains a different sequence selected from SEQ ID NOs: 1 to 34 or its variants, or (b) Two or more polynucleotides, each encoding a peptide that can bind to a T cell receptor as described in item 18, wherein each peptide can bind to a different T cell receptor as described in item 18, and each different T cell receptor can bind to a different sequence selected from SEQ ID NOs: 1-34 or its variants. A vaccine composition containing the following: Section 33 A method for preventing or treating coronavirus infection, comprising administering a vaccine composition described in any one of items 20 to 32 to an individual who is infected with or at risk of becoming infected with coronavirus. Section 34 A vaccine composition according to any one of sub-sub Section 35 The use according to any one of claims 8 to 18, the T cell receptor according to claim 19, the vaccine composition according to any one of claims 20 to 32, the method for preventing or treating coronavirus infection according to claim 33, or the vaccine composition for use according to claim 34, wherein the coronavirus is SARS coronavirus or SARS coronavirus 2.

[0178] References 1.He Y, Zhou Y, Wu H, Kou Z, Liu S, Jiang S. Mapping of antigenic sites on the nucleo capsid protein of the severe acute respi ratory syndrome coronavirus.J Clin Micro biol.2004 Nov;42(11):5309-14.doi:10.1128 / JCM.42.11.5309-5314.2004. 2.Liu SJ, Leng CH, Lien SP, et al.Immunolo physical characterizations of the nucleocap sid protein based SARS vaccine candidate s.Vaccine.2006;24(16):3100-3108.doi:10.1 016 / j.vaccine.2006.01.058. 3.Zhao J,Huang Q,Wang W,Zhang Y,Lv P,Ga o XM.Identification and characterization of dominant helper T-cell epitopes in t he nucleocapsid protein of severe acute respiratory syndrome coronavirus.J Virol .2007;81(11):6079-6088.doi:10.1128 / JVI.0 2568-06 4.Ahmed SF,Quadeer AA,McKay MR.Prelimin ary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus(SAR S-CoV-2)Based on SARS-CoV Immunological Studies.Viruses.2020;12(3):254.https: / / d oi.org / 10.3390 / v12030254.

Claims

[Claim 1] A peptide containing one of sequence numbers 9, 1-8, 10-34, or a variant thereof.

Images