Peptide vaccines against viral infections

Abstract

Immunogenic compositions against viral infections are provided, in particular immunogenic compositions having peptides capable of binding to major histocompatibility complex (MHC) molecules and inducing broad-spectrum immunity against coronaviruses.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 190,116, filed May 18, 2021, the disclosure of which is incorporated by reference in its entirety.

[0002] [Background of the invention] 1. Field of the Invention

[0002] The present invention relates to immunogenic compositions against viral infections, in particular immunogenic compositions having peptides capable of binding to major histocompatibility complex (MHC) molecules and inducing broad-spectrum immunity against coronaviruses.

[0003] 2. Description of the Prior Art Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19), a global pandemic infection since late 2019. As of April 3, 2022, SARS-CoV-2 has caused 489,779,062 confirmed cases of COVID-19, including 6,152,095 deaths, reported to the WHO (https: / / covid19.who.int). These numbers are still increasing rapidly.

[0004]

[0004] SARS-CoV-2 has persisted through its evolutionary evolution and its widespread distribution among people. To date, six major subtypes of the SARS-CoV-2 virus have been identified worldwide. Mutations at the P681H (B.1.1.207), N501Y / 69-70del / P681H (B.1.1.7, alpha variant), N501Y / K417N / E484K (B.1.351, beta variant), and N501Y / E484K / K417T (P.1, gamma variant) positions in the spike protein of SARS-CoV-2 have occurred, as well as 10 delta mutations (B.1.617.2) and 30 omicron mutations (B.1.1.529). The mutation of SARS-CoV-2 virus continues, and what's worse, hybrids of two different subtypes of COVID-19 virus have emerged, such as XD, XE, and XF. Developing a broad-spectrum COVID-19 vaccine to stop the COVID-19 pandemic is urgent due to the high mutation rate of SARS-CoV-2.

[0005] [Summary of the Invention] The present invention relates to peptides designed based on coronavirus spike proteins. The peptides may be used to diagnose, prevent, or treat coronavirus infections in humans. The inventors have designed several peptides that are conserved among different coronaviruses and can bind to molecules of the major histocompatibility complex (MHC). Inclusion of one or more such peptides in a vaccine composition may confer protection against one or more coronaviruses and / or the ability to treat an existing coronavirus infection. Each peptide may also be used to diagnose the presence or absence of a coronavirus infection, for example, by detecting the presence or absence in a sample of a molecule (e.g., a T cell receptor or antibody) that can bind to the peptide. The coronavirus may be, for example, a coronavirus implicated in a human epidemic or pandemic. The coronavirus may be, for example, a coronavirus of zoonotic origin. The coronavirus may be, for example, a member of the Betacoronavirus genus. The coronavirus may be, for example, a member of the Sarbecoronavirus subgenus. The coronavirus may be, for example, SARS-CoV or SARS-CoV 2.

[0006]

[0006] Thus, the present invention provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26 and variant sequences thereof that are at least 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 26, wherein the variant induces T cells that bind to MHC molecules and / or cross-react with the variant peptide.

[0007] The present invention also provides: a nucleic acid encoding the peptide of the present invention; an immunogenic composition comprising the peptide of the present invention or the nucleic acid of the present invention; an antibody that specifically recognizes the peptide of the present invention; A T cell receptor capable of binding to the peptide of the present invention. a recombinant host cell comprising one component selected from the group consisting of the peptide of the present invention, the nucleic acid of the present invention, the antibody of the present invention or a fragment thereof, and the T cell receptor of the present invention or a fragment thereof; an in vitro or ex vivo method for producing activated T lymphocytes, comprising contacting T cells in vitro or ex vivo with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide of the invention; Activated T lymphocytes produced by the above method, which selectively recognize cells presenting the peptide of the present invention. a pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide of the present invention, the nucleic acid of the present invention, the antibody of the present invention or a fragment thereof, the T cell receptor of the present invention or a fragment thereof, the recombinant host cell of the present invention, and the activated T lymphocyte of the present invention; A method for preventing or treating a pathogenic infection in a subject in need thereof, comprising administering to the subject an effective amount of the immunogenic or pharmaceutical composition of the present invention; an immunogenic or pharmaceutical composition of the invention for use in the prevention or treatment of a pathogenic infection in a subject in need thereof; Use of the immunogenic or pharmaceutical composition of the present invention for the manufacture of a medicament for the prevention or treatment of a pathogenic infection in a subject in need thereof; A method for producing anti-coronavirus antibodies, comprising administering to an animal or human subject an immunogenic composition of the invention; an immunogenic composition of the invention for use in generating anti-coronavirus antibodies in an animal or human subject; Use of the immunogenic composition of the present invention for the manufacture of a medicament for the generation of anti-coronavirus antibodies in an animal or human subject; a complex comprising a 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, the method comprising the steps of contacting a peptide or complex of the invention with a sample obtained from the individual, and determining the presence or absence of binding between the peptide or complex and a molecule contained in the sample; Use of a peptide or complex of the invention in a method for determining the presence or absence of a current or previous coronavirus infection in an individual; A method for identifying coronavirus-specific T cells, comprising contacting a peptide or complex of the present invention with a sample obtained from an individual and determining the presence or absence of binding between the peptide or complex and a T cell receptor contained in the sample; Use of a peptide or complex of the invention in a method for identifying coronavirus-specific T cells; a method for identifying a coronavirus-specific T cell receptor, the method comprising contacting a peptide or complex of the present invention with a T cell receptor and determining the presence or absence of binding between the peptide or complex and the T cell receptor; Use of a peptide or complex of the invention in a method for identifying coronavirus-specific T cell receptors.

[0008] These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings. The present invention may be as follows. Item 1 A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26, and variant sequences thereof that are at least 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 26, wherein the variants bind to molecules of the major histocompatibility complex (MHC) and / or induce T cells that cross-react with the variant peptide. Item 2 It has the ability to bind to MHC class I or II molecules and, when bound to MHC, binds to CD4 + and / or CD8 + 2. The peptide according to item 1, which can be recognized by T cells. Item 3 A nucleic acid encoding the peptide according to item 1 or 2. Item 4 An immunogenic composition comprising the peptide according to item 1 or 2 or the nucleic acid according to item 3. Item 5 5. The immunogenic composition according to item 4, further comprising a pharmaceutically acceptable carrier and / or adjuvant. Item 6 An antibody that specifically recognizes the peptide according to item 1 or 2. Item 7 A T cell receptor capable of binding to the peptide according to item 1 or 2. Item 8 8. The T cell receptor of item 7, wherein the peptide is bound to an MHC molecule. Item 9 A recombinant host cell comprising one component selected from the group consisting of the peptide according to Item 1 or 2, the nucleic acid according to Item 3, the antibody or fragment thereof according to Item 6, and the T cell receptor or fragment thereof according to Item 7 or 8. Item 10 10. The recombinant host cell of item 9, selected from a dendritic cell, a T cell, or a natural killer (NK) cell. Item 11 1. An in vitro or ex vivo method for producing activated T lymphocytes, comprising the step of contacting T cells in vitro or ex vivo with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is a peptide according to item 1 or 2. Item 12 12. An activated T lymphocyte produced by the method according to Item 11, which selectively recognizes cells presenting the peptide according to Item 1 or 2. Item 13 A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of the peptide according to Item 1 or 2, the nucleic acid according to Item 3, the antibody or fragment thereof according to Item 6, the T cell receptor or fragment thereof according to Item 7 or 8, the recombinant host cell according to Item 9 or 10, and the activated T lymphocyte according to Item 12. Item 14 14. The pharmaceutical composition according to item 13, further comprising a pharmaceutically acceptable carrier, and / or a pharmaceutically acceptable excipient and / or stabilizer. Item 15 15. The immunogenic composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for use in the prevention or treatment of a pathogenic infection in a subject in need thereof. Item 16 16. The immunogenic composition or pharmaceutical composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for the use according to item 15, wherein the pathogenic infection is induced by a coronavirus. Item 17 Item 16. The immunogenic composition or pharmaceutical composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for the use according to item 16, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Item 18 15. The immunogenic composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for use in generating anti-coronavirus antibodies in an animal or human subject. Item 19 Item 18. The immunogenic composition or pharmaceutical composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for the use according to item 18, wherein the anti-coronavirus antibody is characterized in that it has binding affinity for coronavirus. Item 20 Item 19. The immunogenic composition or pharmaceutical composition according to item 4 or 5 or the pharmaceutical composition according to item 13 or 14 for the use according to item 19, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Item 21 3. A complex comprising the peptide according to item 1 or 2 bound to an MHC molecule. Item 22 22. The complex according to item 21, comprising two or more peptides according to item 1 or 2 and two or more MHC molecules. Item 23 23. The complex according to item 21 or 22, wherein each of the two or more peptides is bound to one of the two or more MHC molecules. Item 24 24. The complex according to any one of items 21 to 23, wherein each of the two or more MHC molecules is linked to a dextran backbone. Item 25 25. The conjugate of any one of items 21 to 24, wherein the conjugate further comprises a fluorophore, optionally wherein the fluorophore is attached to the dextran backbone. Item 26 26. Use of the peptide according to item 1 or 2 or the conjugate according to any one of items 21 to 25 in a method for determining the presence or absence of a current or previous coronavirus infection in an individual, the method comprising the steps of contacting the peptide according to item 1 or 2 or the conjugate according to any one of items 21 to 25 with a sample obtained from the individual, and determining the presence or absence of binding between the peptide or conjugate and a molecule contained in the sample. Item 27 27. The use according to item 26, wherein the molecule is an antibody or a T cell receptor. Item 28 28. Use according to item 26 or 27, wherein the presence of binding indicates the presence of a current or previous coronavirus infection and / or the absence of binding indicates the absence of a current or previous coronavirus infection. Item 29 26. Use of the peptide according to item 1 or 2 or the conjugate according to any one of items 21 to 25 in a method for identifying coronavirus-specific T cells, the method comprising the steps of contacting the peptide according to item 1 or 2 or the conjugate according to any one of items 21 to 25 with a sample obtained from an individual, and determining the presence or absence of binding between the peptide or conjugate and a T cell receptor contained in the sample. Item 30 30. The use of item 29, wherein the individual is currently infected with the coronavirus. Item 31 31. The use according to item 29 or 30, wherein the individual was previously infected with the coronavirus but is not currently infected. Item 32 26. Use of the peptide according to item 1 or 2 or the complex according to any one of items 21 to 25 in a method for identifying a coronavirus-specific T cell receptor, the method comprising the steps of contacting the peptide according to item 1 or 2 or the complex according to any one of items 21 to 25 with a T cell receptor, and determining the presence or absence of binding between the peptide or complex and the T cell receptor. Item 33 33. The use of item 32, wherein the presence of binding indicates that the T cell receptor is a coronavirus-specific T cell receptor, and / or the absence of binding indicates that the T cell receptor is not a coronavirus-specific T cell receptor.

[0009] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or like elements of an embodiment. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1 shows the HLA class I binding ability of peptide 1 (SEQ ID NO: 1), peptide 12 (SEQ ID NO: 12), and CMVpp65495-503 (SEQ ID NO: 27; positive control) of the present invention at concentrations of 1 nM, 3 nM, and 8.9 nM. Different concentrations of peptides were incubated with β2-microglobulin light chain subunit and biotin-labeled recombinant HLA-A201 to form peptide-HLA complexes. The peptide-HLA complexes were then captured by streptavidin-coated beads and detected by flow cytometry with anti-human β2-microglobulin antibody. Each data point represents the mean fluorescence intensity corresponding to the number of peptide-HLA complexes formed, and error bars represent the standard deviation (n = 2). [Figure 2] 1 shows the HLA class I binding ability of peptides 1 to 26 of the present invention (SEQ ID NOs: 1 to 26) at a concentration of 8.9 nM. MHC-peptide binding assays were performed as depicted in Figure 1. Each bar represents the mean fluorescence intensity corresponding to the number of peptide-HLA complexes formed, and the error bars represent the standard deviation (n=2). [Figure 3]

[0023] Figure 1 shows the results of an MHC class I tetramer assay of peptide 1 (SEQ ID NO: 1) and peptide 12 (SEQ ID NO: 12) of the present invention. Human PBMCs were treated with MHC class I tetramers that bind to peptide 1 (SEQ ID NO: 1) or peptide 12 (SEQ ID NO: 12) for 20 minutes, co-stained with anti-CD8 antibody, and then analyzed by flow cytometry. Results are shown as means with error bars representing standard error (n=3). *p<0.05. [Figure 4A] Figure 1 shows cytokine (IFN-γ and IL-4) release of CD4+ T cells in response to peptide 1 (SEQ ID NO: 1) of the present invention on day 12 of peptide treatment. Human PBMCs were treated with peptide 1 (SEQ ID NO: 1) at concentrations of 0.4, 2, 10, 50, and 250 nM for 12 days and then subjected to intracellular cytokine staining and multiparameter flow cytometry. Each data point represents the mean fluorescence intensity corresponding to the amount of cytokine, and error bars represent the standard error (n=3). [Figure 4B]Figure 1 shows cytokine (IFN-γ and IL-4) release of CD4+ T cells in response to peptide 12 (SEQ ID NO: 12) of the present invention on day 12 of peptide treatment. Human PBMCs were treated with peptide 12 (SEQ ID NO: 12) at concentrations of 0.4, 2, 10, 50, and 250 nM for 12 days and then subjected to intracellular cytokine staining and multiparameter flow cytometry. Each data point represents the mean fluorescence intensity corresponding to the amount of cytokine, and error bars represent the standard error (n=3). [Figure 5A] Figure 1 shows the total IgG amount in BALB / c mice two weeks after each immunization. BALB / c mice (n=10 / group) were intramuscularly injected with 45 μg of peptide 1 (SEQ ID NO: 1) of the present invention three times at two-week intervals (on days 0, 14, and 28). Antisera were collected one day before the first injection and two weeks after each injection (on days -1, 13, 27, and 41) and subjected to a total IgG ELISA assay. Each point represents the total IgG amount of an individual serum sample. Each boxplot shows the 25th to 75th percentile measurements. Error bars correspond to the 10th and 90th percentiles, and the horizontal bar of each box represents the mean. ***p<0.001. [Figure 5B] Figure 5 shows anti-spike IgG titers in BALB / c mice 2 weeks after the third immunization. Mice were immunized as depicted in Figure 5A (n = 10 per group), and antisera were collected 1 day before the first injection (day -1, pre-dose) and 2 weeks after the third injection (day 41, post-dose) and subjected to anti-spike IgG ELISA assays. Each point represents the anti-spike IgG titer of an individual serum sample. Each boxplot shows the 25th to 75th percentile measurements. Error bars correspond to the 10th and 90th percentiles, and the horizontal bar in each box represents the mean. ***p<0.001. [Figure 6A]Figure 1 shows the total IgG amount in BALB / c mice two weeks after the third immunization. BALB / c mice (n=3-4 / group) were intramuscularly injected with 400 μg of peptide 12 (SEQ ID NO: 12) of the present invention three times at two-week intervals (on days 0, 14, and 28). Antisera were collected one day before the first injection (day -1, pre-dose) and two weeks after the third injection (day 41, post-dose) and subjected to a total IgG ELISA assay. Each point represents the total IgG amount of an individual serum sample, and the horizontal bar represents the mean for each group. ***p<0.001. [Figure 6B] Figure 6 shows anti-spike IgG titers in BALB / c mice 2 weeks after the third immunization. Antisera were collected (n=4 / group) as depicted in Figure 6A and subjected to anti-spike IgG ELISA assay. Each point represents the anti-spike IgG titer of an individual serum sample, and the horizontal bar represents the mean for each group. *p<0.05, ***p<0.001. [Figure 7A] Figure 1 shows the total IgG amount in BALB / c mice two weeks after the third immunization. BALB / c mice (n=5 / group) were orally administered 200 μg of peptide 1 (SEQ ID NO: 1) of the present invention formulated with 1% (v / v) PLCL-PEG-PLCL three times at two-week intervals (days 0, 14, and 28). Antisera were collected one day before the first injection (day -1, pre-dose) and two weeks after the third injection (day 41, post-dose) and subjected to a total IgG ELISA assay. Each point represents the total IgG amount of an individual serum sample, and the horizontal bar represents the mean for each group. ***p<0.001. [Figure 7B] Figure 7 shows anti-spike IgG titers in BALB / c mice 2 weeks after the third immunization. Antisera were collected (n=5 / group) as depicted in Figure 7A and subjected to anti-spike IgG ELISA assay. Each point represents the anti-spike IgG titer of an individual serum sample, and the horizontal bar represents the mean for each group. **p<0.01.

[0011] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention relates to peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26 and variant sequences thereof that are at least 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 26, wherein the variants induce T cells that bind to molecules of the major histocompatibility complex (MHC) and / or cross-react with the variant peptide. SEQ ID NOs: 1 to 26 are shown in Table 1.

[0022]

[0012] [Table 1]

[0013]

[0023] To design SEQ ID NOS: 1-26, highly conserved regions of the human SARS-CoV-2 spike protein were selected, and short antigen peptides with a length of 9-10 residues were generated based on the molecular structure of human leukocyte antigens (HLA). These peptides were then subjected to HLA binding assays to confirm their binding activity with HLA molecules, and peptides with strong binding activity with HLA were selected from the peptides. Therefore, the peptides of the present invention can bind to MHC molecules, particularly MHC class I molecules.

[0014]

[0024] In some embodiments, the peptides of the present invention have the ability to bind to MHC class I or II molecules, and when the peptide binds to the MHC, the peptide binds to CD4 + and / or CD8 + It can be recognized by T cells.

[0015]

[0025] In some embodiments, the variant sequence is at least 80% homologous to SEQ ID NO: 1 through SEQ ID NO: 26. In some embodiments, the variant sequence is at least 80%, at least 85%, at least 88%, at least 90%, or at least 95% homologous to SEQ ID NO: 1 through SEQ ID NO: 26. Each possibility represents a separate embodiment of the present invention.

[0016]

[0026] As used herein, the term "coronavirus (CoV)" refers to a group of related RNA viruses in the Coronaviridae family that cause disease in mammals and birds. Seven human coronaviruses (HCoVs) have been identified to date: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and novel coronavirus (2019-nCoV, also known as SARS-CoV-2). Unlike the highly pathogenic SARS-CoV, MERS-CoV, and 2019-nCoV, the four so-called common HCoVs generally cause mild upper respiratory tract illness and account for 15%–30% of cold cases in adults, although severe and life-threatening lower respiratory tract infections can occasionally occur in infants, the elderly, or immunocompromised patients (Encyclopedia of Virology. 2021:428–440).

[0017]

[0027] As used herein, the term "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" refers to the coronavirus strain that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a positive-sense, single-stranded RNA virus with a genome size of 29,903 bases. Each SARS-CoV-2 virion is 50–200 nanometers in diameter and contains four structural proteins known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein houses the RNA genome, and the S, E, and M proteins together produce the viral envelope. The spike protein is responsible for allowing the virus to attach to and fuse with the membrane of a host cell; specifically, its S1 subunit catalyzes attachment, and the S2 subunit catalyzes fusion.

[0018]

[0028] As used herein, the term "major histocompatibility complex (MHC)" refers to a group of genes encoding proteins found on cell surfaces that help the immune system recognize foreign substances. MHC proteins are found in all higher vertebrates. In humans, the complex is also called the human leukocyte antigen (HLA) system. MHC exists primarily in a membrane-bound form and is responsible for regulating the immune system. MHC activates cytotoxic T cells by presenting peptide fragments of foreign or self proteins on the cell surface so that their T cell receptors (TCRs) can recognize and bind them. MHC can be classified into two main types: MHC class I is present on the surface of almost all cells, while MHC class II is present only on antigen-presenting cells (APCs) such as NK cells, macrophages, and dendritic cells. MHC class I, when loaded with specific peptide fragments, can selectively activate cytotoxic T cells expressing TCRs. Therefore, recombinant MHC class I with its membrane-bound domain removed is used for the treatment of cancer or infectious diseases. Unlike MHC class II, most MHC class I molecules are unstable when no peptide is loaded into the groove. Therefore, recombinant MHC class I molecules are prepared in a form loaded with a target synthetic peptide or fused with an α chain. In addition, because there is a risk that refolding may not be performed properly when expressed using microorganisms, recombinant MHC class I molecules are generally expressed using animal cells. In the art, HLA and MHC are used interchangeably with the same meaning, and HLA is used instead of MHC in humans. In this specification, the terms "HLA" and "MHC" can be used interchangeably with the same meaning.

[0019]

[0029] As used herein, the nomenclature used to describe the peptides of the present invention follows conventional practice, where the amino group (N-terminus) and / or 5' is designated on the left and the carboxyl group (C-terminus) and / or 3' is designated on the right.

[0020]

[0030] As used herein, the term "peptide" refers to a molecular chain of amino acids, including both L- and D-forms. The amino acids can be modified in vivo or in vitro, if necessary, by, for example, mannosylation, glycosylation, amidation (particularly C-terminal amides), carboxylation, or phosphorylation, provided that these modifications preserve the biological activity of the original molecule. In addition, peptides can be part of chimeric proteins.

[0021]

[0031] Functional derivatives of peptides are also included in the present invention. Functional derivatives are intended to include peptides that differ in one or more amino acids across the entire sequence, such as peptides with deletions, substitutions, inversions, or additions. Amino acid substitutions that are not expected to substantially alter biological and immunological activity are described. Amino acid exchanges between related amino acids or exchanges that have frequently occurred in evolution include, among others, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val.

[0022]

[0032] The peptides according to the invention can be produced synthetically or by recombinant DNA technology. Methods for producing synthetic peptides are well known in the art.

[0023]

[0033] Organic chemical methods for peptide synthesis are believed to involve coupling of the required amino acids by condensation reactions in a homogeneous phase or with the aid of so-called solid phase. Condensation reactions can be carried out as follows: condensation of a compound (amino acid, peptide) having a free carboxyl group and protected other reactive groups with a compound (amino acid, peptide) having a free amino group and protected other reactive groups in the presence of a condensing agent; condensation of a compound (amino acid, peptide) having an activated carboxyl group and free or protected other reactive groups with a compound (amino acid, peptide) having a free amino group and free or protected other reactive groups. Activation of the carboxyl group can be carried out, inter alia, by converting it to an acid halide, azide, anhydride, imidazolide, or activated ester, such as N-hydroxysuccinimide, N-hydroxybenzotriazole, or p-nitrophenyl ester.

[0024]

[0034] The most common methods for the above condensation reaction are: the carbodiimide method, the azide method, the mixed acid anhydride method, and the method using activated esters, such as those described in The Peptides, Analysis, Synthesis, Biology, Vol. 1-3 (Gross, E. and Meienhofer, J., eds.) 1979, 1980, 1981 (Academic Press, Inc.).

[0025]

[0035] By "variant" of a given amino acid sequence, we mean that e.g., one or two side chains of the amino acid residues have been altered (e.g., by exchanging them for the side chains of another naturally occurring amino acid residue or for some other side chain) so that the peptide is still able to bind to an MHC molecule in substantially the same way as a peptide consisting of the given amino acid sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 26. For example, the peptide may be modified to at least maintain, if not improve, its ability to interact with and bind to the binding groove of an appropriate MHC molecule, such as HLA-A*02.

[0026]

[0036] One skilled in the art would be able to assess whether T cells induced by a variant of a specific peptide are capable of cross-reacting with the peptide itself.

[0027]

[0037] The present invention also relates to nucleic acids encoding the peptides of the present invention, which thus encode a peptide comprising or consisting of any one of SEQ ID NOs: 1 to 26, or a variant thereof.

[0028]

[0038] As used herein, the term "nucleic acid encoding a peptide" refers to a nucleotide sequence that encodes a peptide. Nucleic acids encoding a particular peptide, oligopeptide, or polypeptide can be naturally occurring or synthetically constructed. A nucleic acid (e.g., a polynucleotide) can be, for example, a single- and / or double-stranded polynucleotide, or a native or stabilized form of deoxyribonucleic acid (DNA), complementary DNA (cDNA), peptide nucleic acid (PNA), ribonucleic acid (RNA), or combinations thereof, e.g., a polynucleotide having a phosphorothioate backbone, and may or may not contain introns, so long as the nucleic acid encodes a peptide.

[0029]

[0039] The present invention also relates to an immunogenic composition comprising a peptide of the present invention or a nucleic acid of the present invention. Thus, the immunogenic composition comprises a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof. Alternatively, the immunogenic composition comprises a nucleic acid encoding a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof.

[0030]

[0040] In some embodiments, the immunogenic composition further comprises a pharmaceutically acceptable carrier and / or an adjuvant.

[0031]

[0041] In some embodiments, the immunogenic composition comprises at least one peptide of the present invention. In some embodiments, the immunogenic composition comprises two or more peptides of the present invention. An immunogenic composition comprising a peptide of the present invention is also called a peptide vaccine.

[0032]

[0042] As used herein, the term "peptide vaccine" refers to a preparation composed of at least one peptide that improves immunity to a particular pathogen.

[0033]

[0043] In some embodiments, the immunogenic composition comprises a nucleic acid encoding at least one peptide of the invention. In some embodiments, the immunogenic composition comprises a nucleic acid encoding two or more peptides of the invention. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0034]

[0044] As used herein, the term "immunogenic composition" refers to a composition capable of producing an immune response.

[0035]

[0045] In some embodiments, the immunogenic composition, upon administration, exhibits T cell activation. In some embodiments, the immunogenic composition, upon administration, exhibits CD4 + In some embodiments, the immunogenic composition, upon administration, induces activation of CD8 T cells. + In some embodiments, the immunogenic composition, upon administration, induces activation of CD4 T cells. + T cells and CD8 + Shows complex activation of T cells.

[0036]

[0046] In some embodiments, the immunogenic composition, upon administration, demonstrates the production of specific antibodies (of any immunoglobulin class) directed against epitopes within the peptides of the invention. Each possibility represents a separate embodiment.

[0037]

[0047] As used herein, the term "adjuvant" refers to any component of a pharmaceutical composition that is not the active agent.

[0038]

[0048] In some embodiments, the adjuvant is selected from the group comprising oil emulsions, cytokines, immune stimulating complexes (ISCOMs), saponin-type adjuvants, Montanide ISA 51VG, liposomes, aluminum hydroxide (alum), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), lipopolysaccharide (LPS) or derivatives such as monophosphoryl lipid A (MPL), CpG DNA, microbial DNA / RNA, nanoparticles (e.g., gold particles), bacterial ghosts, ligands or agonist antibodies against TNFα, TLR (Toll-like receptor)-based adjuvants (see, e.g., Heit et al., Eur. J. Immunol., 2007, 37:2063-2074), or combinations thereof.

[0039]

[0049] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible. In certain embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosal, or transdermal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0040]

[0050] In some embodiments, the peptide antigen is associated with a polymer, such as poly(lactide-co-caprolactone)-block-poly(ethylene glycol)-block-poly(lactide-co-caprolactone) (PLCL-PEG-PLCL), poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL), or other polymers known in the art for encapsulation and delivery of peptides, including, but not limited to, carboxymethylcellulose (CMC), chitosan, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0041]

[0051] In some embodiments, no adjuvant is added, in some embodiments, one adjuvant is added, in some embodiments, a combination of adjuvants is added.

[0042]

[0052] The present invention also relates to antibodies that specifically recognize the peptides of the present invention. Thus, the antibodies specifically recognize peptides comprising or consisting of any one of SEQ ID NOS: 1 to 26, or variants thereof.

[0043]

[0053] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides containing at least one binding domain formed by folding of polypeptide chains, resulting in a three-dimensional binding space with an internal shape and charge distribution complementary to the antigenic determinants of an antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and labeled antibodies in soluble or conjugated form, as well as fragments, variants, or derivatives thereof, including epitope-binding fragments, alone or in combination with other amino acid sequences. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single-chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or fragment thereof. Those skilled in the art will further recognize that other fusion products may be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0044]

[0054] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0045]

[0055] The term "antibody" or "antibodies" is used broadly herein and includes both polyclonal and monoclonal antibodies. The term "antibody" includes intact or "complete" immunoglobulin molecules as well as fragments of those immunoglobulin molecules (e.g., CDRs, Fv, Fab, and Fc fragments) or polymers and humanized versions of immunoglobulin molecules, so long as they exhibit any of the desired properties, i.e., specifically recognize a peptide according to the present invention or a variant thereof. Whenever possible, antibodies of the present invention can be purchased from commercial sources. Antibodies of the present invention may also be generated using well-known methods. Antibodies of the present invention can be used as therapeutic or diagnostic agents.

[0046]

[0056] The present invention also relates to a T cell receptor capable of binding to the peptide of the present invention, which is therefore capable of binding to a peptide comprising or consisting of any one of SEQ ID NOs: 1 to 26, or a variant thereof.

[0047]

[0057] In some embodiments, the peptide is conjugated to an MHC molecule.

[0048]

[0058] As used herein, the term "T cell receptor" (TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to peptide antigens presented by HLA molecules. The term also includes so-called gamma / delta TCRs.

[0049]

[0059] The present invention also relates to a recombinant host cell comprising one component selected from the group consisting of the peptide of the present invention, the nucleic acid of the present invention, the antibody of the present invention or a fragment thereof, and the T cell receptor of the present invention or a fragment thereof. Thus, the recombinant host cell comprises one component selected from the group consisting of a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, a nucleic acid encoding a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, an antibody or fragment thereof that specifically recognizes a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, and a T cell receptor capable of binding to a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof.

[0050]

[0060] In some embodiments, the recombinant host cell is selected from an antigen-presenting cell, such as a dendritic cell, a T cell, or a natural killer (NK) cell.

[0051]

[0061] The present invention also relates to an in vitro or ex vivo method for producing activated T lymphocytes, comprising contacting T cells in vitro or ex vivo with antigen-loaded human class I or II MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide of the invention.

[0052]

[0062] The present invention also relates to activated T lymphocytes produced by the above-mentioned in vitro or ex vivo method, wherein the activated T lymphocytes selectively recognize cells presenting a peptide of the present invention. Thus, the activated T lymphocytes selectively recognize cells presenting a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof.

[0053]

[0063] The present invention also relates to a pharmaceutical composition comprising at least one active ingredient selected from the group consisting of a peptide of the present invention, a nucleic acid of the present invention, an antibody or fragment thereof of the present invention, a T cell receptor or fragment thereof of the present invention, a recombinant host cell of the present invention, and an activated T lymphocyte of the present invention. Accordingly, the pharmaceutical composition comprises at least one active ingredient selected from the group consisting of a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, a nucleic acid encoding a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, an antibody or fragment thereof that specifically recognizes a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof, and a T cell receptor capable of binding to a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26 or a variant thereof.

[0054]

[0064] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and / or a pharmaceutically acceptable excipient and / or stabilizer.

[0055]

[0065] As used herein, the term "pharmaceutical composition" refers to a composition suitable for administration to humans in a medical setting. Preferably, pharmaceutical compositions are sterile and produced according to GMP guidelines.

[0056]

[0066] When preparing pharmaceutical compositions of the present invention, it may be desirable to modify peptide antigens or combine or conjugate peptides with other drugs to alter pharmacokinetics and biodistribution. Several methods for altering pharmacokinetics and biodistribution are known to those skilled in the art. Examples of such methods include protecting proteins, protein complexes, and polynucleotides in vesicles composed of other proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers. For example, the vaccine agents of the present invention can be incorporated into liposomes to enhance their pharmacokinetic and biodistribution characteristics. A wide variety of methods are available for preparing liposomes, as described, for example, in U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. For use with liposome delivery vehicles, peptides are typically encapsulated within liposomes or lipid vesicles or attached to the exterior of the vesicles.

[0057]

[0067] The present invention also relates to a method for preventing or treating a pathogenic infection in a subject in need thereof, comprising administering to the subject an effective amount of the immunogenic or pharmaceutical composition of the present invention.The present invention also relates to the immunogenic or pharmaceutical composition of the present invention for use in preventing or treating a pathogenic infection in a subject in need thereof.The present invention also relates to the use of the immunogenic or pharmaceutical composition of the present invention for the manufacture of a medicament for preventing or treating a pathogenic infection in a subject in need thereof.

[0058]

[0068] In some embodiments, the pathogenic infection is induced by a coronavirus, hi some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0059]

[0069] In some embodiments, the invention provides methods of treating or preventing a pathogenic infection, comprising administering to a subject in need thereof an enriched population of T cells, wherein the enriched population of T cells is obtained by administering an immunogenic composition to the T cell population in vitro.

[0060]

[0070] As used herein, an "effective amount" or "sufficient amount" of a substance is an amount sufficient to cause a beneficial or desired result, including a clinical result; therefore, the "effective amount" depends on the context in which the substance is applied. In the context of administering an immunogenic composition, an effective amount is an immunologically effective amount containing sufficient immunogenic composition of the invention to elicit an immune response. In the context of administering a pharmaceutical composition, an effective amount is a pharmaceutically effective amount containing sufficient pharmaceutical composition of the invention to maintain or produce a desired physiological result. An effective amount can be administered in one or more doses.

[0061]

[0071] As used herein, the term "immunologically effective amount" refers to amounts effective, in combination and at the necessary dosage and duration, to elicit a specific T lymphocyte-mediated immune response and / or humoral response, which can be determined by conventional assays for T cell activation, including, but not limited to, assays to detect antibody production, proliferation, specific cytokine activation, and / or cytotoxic activity, using, for example, antibody concentration / titer assays (e.g., via ELISA).

[0062]

[0072] As used herein, the term "pharmaceutically effective amount" refers to an amount capable of maintaining or producing a desired physiological result, including, but not limited to, treating, reducing, eliminating, substantially preventing, or preventing the onset of a disease, disorder, or combination thereof, or a combination thereof. A pharmaceutically effective amount may include one or more doses administered sequentially or simultaneously. One of ordinary skill in the art will know how to adjust the doses of the present invention to account for various types of formulations, including, but not limited to, sustained release formulations. As used herein, the term "prophylactic" refers to a composition that can substantially prevent or prevent the onset of any aspect of a disease, disorder, or combination thereof. As used herein, the term "therapeutic" refers to a composition that can treat, reduce, halt the progression of, slow the progression of, favorably alter, eliminate, or a combination thereof, any aspect of a disease, disorder, or combination thereof.

[0063]

[0073] The term "dose" as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken (administered or received) by a subject at one time.

[0064]

[0074] As used herein, the term "immunization" refers to the process of increasing the response of a mammalian subject to an antigen, thereby improving the subject's ability to resist or overcome infection.

[0065]

[0075] As used herein, the term "vaccination" refers to the introduction of a vaccine into the body of a mammalian subject.

[0066]

[0076] As used herein, the term "subject" refers to animals, and more particularly to non-human mammals and human organisms. Non-human animal subjects may also include prenatal forms of animals, such as embryos or fetuses. Non-limiting examples of non-human animals include horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In some embodiments, the subject is a human. Human subjects may also include fetuses.

[0067]

[0077] As used herein, the term "subject" refers to any subject for whom treatment is desired, particularly mammalian subjects, such as humans.

[0068]

[0078] In some embodiments, the subject in need thereof is suffering from a pathogenic infection. In some embodiments, the subject in need thereof is susceptible to a pathogenic infection. In some embodiments, the subject in need thereof is potentially susceptible to a pathogenic infection.

[0069]

[0079] As used herein, the terms "treat," "treating," or "treatment" include alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition has been completely cured. To be an effective therapy, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or result in an improvement in the patient's or subject's quality of life.

[0070]

[0080] In some embodiments, the peptide vaccines of the present invention reduce transfection or transmission to other subjects.

[0071]

[0081] In some embodiments, the peptide vaccines of the present invention are administered in an effective amount, with or without a costimulatory molecule, drug, or adjuvant. By the methods of the present invention, the peptide vaccines can be administered to a subject in need of such treatment for a time and under conditions sufficient to prevent and / or ameliorate pathogen infection.

[0072]

[0082] In some embodiments, the immunogenic composition may be administered to a subject by a variety of modes of administration, including intradermal, intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, and transdermal delivery, or by topical application to the eye, ear, skin, or mucous membrane. Alternatively, the antigen may be administered ex vivo by direct exposure to cells, tissues, or organs from the subject (autologous) or another subject (allogeneic), optionally in a biologically suitable liquid or solid carrier.

[0073]

[0083] The peptide vaccine can be administered to a subject via injection, either alone or in combination with a suitable auxiliary or adjuvant. Alternatively, the peptide vaccine can be administered transdermally via the mucous membrane, for example, by spraying a solution. The unit dose of the peptide typically ranges from about 0.001 mg to 100 mg, more typically from about 1 μg to about 1,000 μg, and can be administered to a patient once or repeatedly.

[0074]

[0084] Examples of auxiliary agents or adjuvants that can be formulated with or conjugated to vectors for expressing peptide or protein antigens and / or costimulatory molecules to enhance the immunogenicity of peptide or protein antigens for use within the present invention include cytokines (e.g., GM-CSF), bacterial cell components such as BCG bacterial cell components, immunostimulating complexes extracted from tree bark called Quill A (ISCOMs), QS-21, saponin-type adjuvants, Montanide ISA 51VG, liposomes, aluminum hydroxide (alum), bovine serum albumin (BSA), tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), and TLR (Toll-like receptor)-based adjuvants (see, e.g., Heit et al., Eur. J. Immunol. (2007) 37:2063-2074).

[0075]

[0085] The present invention also relates to a method for producing anti-coronavirus antibodies, comprising administering an immunogenic composition of the present invention to an animal or human subject.The present invention also relates to the immunogenic composition of the present invention for use in producing anti-coronavirus antibodies in an animal or human subject.The present invention also relates to the use of the immunogenic composition of the present invention for the manufacture of a medicament for producing anti-coronavirus antibodies in an animal or human subject.

[0076]

[0086] In some embodiments, the anti-coronavirus antibody is characterized by having binding affinity to a coronavirus. In some embodiments, the anti-coronavirus antibody is an antibody against an alphacoronavirus peptide. In some embodiments, the coronavirus is 229E or NL63. In some embodiments, the anti-coronavirus antibody is an antibody against a betacoronavirus peptide. In some embodiments, the coronavirus is OC43, HKU1, MERS-CoV, SARS-CoV, or SARS-CoV-2.

[0077]

[0087] In some embodiments, the animal is a horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, or pig. Animals can also include prenatal forms of animals, such as an embryo or fetus.

[0078]

[0088] The present invention also relates to a complex comprising a peptide of the present invention bound to an MHC molecule, such as a peptide comprising or consisting of any one of SEQ ID NOS: 1 to 26, or a variant thereof, bound to an MHC molecule.

[0079]

[0089] In some embodiments, the MHC molecule is an MHC class 1 molecule. In some embodiments, the MHC molecule is an MHC class II molecule. Preferably, the MHC molecule is an MHC class I molecule. The MHC class I molecule can be of any HLA supertype. For example, the MHC class I molecule can be of supertype A2.

[0080]

[0090] In some embodiments, a complex comprises two or more peptides of the present invention and two or more MHC molecules. For example, a complex may comprise three or more peptides of the present invention, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. A complex may comprise three or more MHC molecules, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. A complex may comprise three or more peptides of the present invention and three or more MHC molecules, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. A complex may comprise the same number of peptides of the present invention as MHC molecules. A complex may comprise a different number of peptides than the number of MHC molecules. A complex may comprise, for example, four MHC molecules. The complex may comprise or consist of an MHC tetramer. The complex may, for example, comprise 12 MHC molecules. The complex may comprise or consist of an MHC dodecamer.

[0081]

[0091] When a complex comprises two or more peptides of the present invention, each of the two or more peptides may be the same. Alternatively, each of the two or more peptides may be different. When a complex comprises three or more peptides of the present invention, each of the three or more peptides may be the same. When a complex comprises three or more peptides of the present invention, each of the three or more peptides may be different. When a complex comprises three or more peptides of the present invention, some of the three or more peptides may be the same, and some of the three or more peptides may be different.

[0082]

[0092] When a complex contains two or more MHC molecules, each of the two or more MHC molecules may be the same. Alternatively, each of the two or more MHC molecules may be different. When a complex contains three or more peptides of the present invention, each of the three or more MHC molecules may be the same. When a complex contains three or more peptides of the present invention, each of the three or more MHC molecules may be different. When a complex contains three or more MHC molecules, some of the three or more MHC molecules may be the same, and some of the three or more MHC molecules may be different.

[0083]

[0093] In some embodiments, a complex may comprise two or more peptides of the present invention and two or more MHC molecules, and each peptide may be bound to one of two or more MHC molecules. That is, each peptide in the complex may be bound to an MHC molecule in the complex. Preferably, each peptide in the complex is bound to another MHC molecule in the complex. That is, each MHC molecule in the complex is preferably bound to no more than one peptide in the complex. However, a complex may comprise one or more peptides of the present invention that are not bound to an MHC molecule. A complex may comprise one or more MHC molecules that are not bound to a peptide of the present invention.

[0084]

[0094] One or more MHC molecules included in a complex may be linked to each other. For example, each of one or more MHC molecules of a complex may be linked to a scaffold molecule or nanoparticle. In some embodiments, each of two or more MHC molecules is linked to a dextran scaffold. That is, a complex may comprise or consist of an MHC dextramer. Mechanisms for linking one or more MHC molecules to a dextran scaffold are known in the art. Any number of MHC molecules may be linked to a dextran scaffold. For example, one or more, two or more, three or more peptides of the invention and three or more MHC molecules may be linked to a dextran scaffold.

[0085]

[0095] In some embodiments, the conjugate further comprises a fluorophore, optionally linked to the dextran backbone. Fluorophores are well known in the art and include FITC (fluorescein isothiocyanate), PE (phycoerythrin), and APC (allophycocyanin). The conjugate can comprise any number of fluorophores. For example, the conjugate can comprise two or more, three or more, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more peptides of the present invention and three or more, e.g., four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more fluorophores. When the conjugate comprises multiple fluorophores, the fluorophores included in the conjugate can be the same or different. When the conjugate comprises a backbone such as a dextran backbone, the fluorophores are preferably linked to the dextran backbone. Mechanisms for attaching fluorophores to a dextran backbone are known in the art.

[0086]

[0096] The present invention also relates to a method for determining the presence or absence of a current or previous coronavirus infection in an individual, the method comprising the steps of contacting a peptide or complex of the present invention with a sample obtained from the individual and determining the presence or absence of binding between the peptide or complex and a molecule contained in the sample.The present invention also relates to the use of a peptide or complex of the present invention in a method for determining the presence or absence of a current or previous coronavirus infection in an individual, the method comprising the steps of contacting a peptide or complex of the present invention with a sample obtained from the individual and determining the presence or absence of binding between the peptide or complex and a molecule contained in the sample.

[0087]

[0097] The sample can be, for example, a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, or a sample obtained by swabbing a mucosal surface present in an individual. Preferably, the sample is a blood sample, a serum sample, or a plasma sample.

[0088]

[0098] In some embodiments, the molecule is an antibody or a T cell receptor. The molecule can be, for example, an antibody or an antibody fragment. The antibody or antibody fragment can be on the surface of a B cell or contained in a B cell. The antibody or antibody fragment can be free in a sample. The molecule can be, for example, a T cell receptor. The T cell receptor can be CD4 + The T cell receptor may be a CD8 + It can be a T cell receptor. T cell receptors can be on the surface of T cells or contained within T cells. T cells can be CD4 + T cells can be CD8 + It may be a T cell.

[0089]

[0099] Methods for detecting binding of a peptide or peptide-containing complex to a molecule are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), and flow cytometry.

[0090]

[0100] In some embodiments, the presence of binding indicates the presence of a current or previous coronavirus infection, and / or the absence of binding indicates the absence of a current or previous coronavirus infection.

[0091]

[0101] In current coronavirus infections, coronavirus particles or their components (e.g., peptides, proteins) may be present in individuals. In current coronavirus infections, antibodies, B cells, CD8 + T cells and / or CD4 + T cells may be present in an individual. Preferably, in a current coronavirus infection, (i) coronavirus particles or components thereof (e.g., peptides, proteins) and (ii) antibodies, B cells, CD8 + T cells and / or CD4 + T cells are present within an individual.

[0092]

[0102] In a previous coronavirus infection, coronavirus particles or their components (e.g., peptides, proteins) may not be present in the individual. In a previous coronavirus infection, antibodies, B cells, CD8 + T cells and / or CD4 + T cells may be present in the individual. Preferably, coronavirus particles or components thereof (e.g., peptides, proteins) have not been present in the individual in a previous coronavirus infection, and antibodies, B cells, CD8 + T cells and / or CD4 + T cells are present within an individual.

[0093]

[0103] The present invention also relates to a method for identifying coronavirus-specific T cells, the method comprising the steps of contacting a peptide or complex of the invention with a sample obtained from an individual and determining the presence or absence of binding between the peptide or complex and a T cell receptor present in the sample.The present invention also relates to the use of a peptide or complex of the invention in a method for identifying coronavirus-specific T cells, the method comprising the steps of contacting a peptide or complex of the invention with a sample obtained from an individual and determining the presence or absence of binding between the peptide or complex and a T cell receptor present in the sample.

[0094]

[0104] The sample can be, for example, a blood sample, a serum sample, a plasma sample, a urine sample, a saliva sample, or a sample obtained by swabbing a mucosal surface present on an individual. Preferably, the sample is a blood sample.

[0095]

[0105] T cell receptor is CD4 + It can be a T cell receptor. T cell receptors are CD8 + It may be a T cell receptor. Preferably, the T cell receptor is CD8 + It is a T cell receptor.

[0096]

[0106] T cell receptors may be located on the surface of T cells or may be contained within T cells. T cells are CD4 + T cells can be CD8 + The T cells may be CD8 T cells. Preferably, the T cells are CD8 + T cells.

[0097]

[0107] Methods for detecting binding of a peptide or peptide-containing complex to a T cell receptor are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), and flow cytometry.

[0098]

[0108] The presence of binding can indicate the presence of one or more coronavirus-specific T cells. The absence of binding can indicate the absence of coronavirus-specific T cells.

[0099]

[0109] In some embodiments, the individual is currently infected with a coronavirus. In a current coronavirus infection, coronavirus particles or components thereof (e.g., peptides, proteins) may be present in the individual. In a current coronavirus infection, antibodies, B cells, CD8 + T cells and / or CD4 + T cells may be present in an individual. Preferably, in a current coronavirus infection, (i) coronavirus particles or components thereof (e.g., peptides, proteins) and (ii) antibodies, B cells, CD8 + T cells and / or CD4 + T cells are present within an individual.

[0100]

[0110] In some embodiments, the individual has previously been infected with a coronavirus but is not currently infected. In a previous coronavirus infection, coronavirus particles or components thereof (e.g., peptides, proteins) may not be present in the individual. In a previous coronavirus infection, antibodies, B cells, CD8 + T cells and / or CD4 + T cells may be present in the individual. Preferably, coronavirus particles or components thereof (e.g., peptides, proteins) have not been present in the individual in a previous coronavirus infection, and antibodies, B cells, CD8 + T cells and / or CD4 +T cells are present in an individual. Thus, although coronavirus particles or their components (e.g., peptides, proteins) may not be present in an individual, antibodies, B cells, CD8 + T cells and / or CD4 + The T cells may be present in an individual.

[0101]

[0111] The present invention also relates to a method for identifying a coronavirus-specific T cell receptor, the method comprising the steps of contacting a peptide or complex of the present invention with a T cell receptor and determining the presence or absence of binding between the peptide or complex and the T cell receptor.The present invention also relates to the use of a peptide or complex of the present invention in a method for identifying a coronavirus-specific T cell receptor, the method comprising the steps of contacting a peptide or complex of the present invention with a T cell receptor and determining the presence or absence of binding between the peptide or complex and the T cell receptor.

[0102]

[0112] In some embodiments, the presence of binding indicates that the T cell receptor is a coronavirus-specific T cell receptor, and / or the absence of binding indicates that the T cell receptor is not a coronavirus-specific T cell receptor.

[0103]

[0113] T cell receptor is CD4 + It can be a T cell receptor. T cell receptors are CD8 + It may be a T cell receptor. Preferably, the T cell receptor is CD8 + It is a T cell receptor.

[0104]

[0114] T cell receptors may be located on the surface of T cells or may be contained within T cells. T cells are CD4 + T cells can be CD8 + The T cells may be CD8 T cells. Preferably, the T cells are CD8 + T cells.

[0105]

[0115] Methods for detecting binding of a peptide or peptide-containing complex to a T cell receptor are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), and flow cytometry.

[0106]

[0116] The meanings of technical and scientific terms used herein can be clearly understood by those skilled in the art.

[0107]

[0117] As used herein, the terms "about," "approximately," or "nearly" when combined with a value refer to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ±100 nm.

[0108]

[0118] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides; a reference to a "polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those skilled in the art; and so forth. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate for use of exclusive terminology such as "only," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0109]

[0119] When a convention similar to "such as at least one of A, B, and C" is used, such configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0110]

[0120] This invention is further illustrated by the following examples, which are provided for purposes of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0111]

[0121] [Example]

[0122] Example 1 Assay of antigen peptide binding ability to MHC class I

[0123] Materials and Methods

[0124] MHC-Peptide Binding Assay. An MHC-peptide binding assay was used to determine the ability of each antigen peptide of the present invention to bind to HLA-A201, the most common human MHC class I allele in the population (Wills, MR et al., J. Virol. 1996, 70:7569-7579; Weekes, MP et al., J. Virol. 1999, 73:2099-2108; Reiser, JB et al., Acta Cryst. Sect. F Struct. Biol. Cryst. Commun. 2009, 65:1157-1161). MHC class I refolding assays (easYmer, immunAware Aps, Copenhagen, Denmark) were preformed according to the manufacturer's instructions. Briefly, each antigen peptide of the present invention (SEQ ID NOS: 1 to 26) was incubated with β2-microglobulin light chain subunit and biotin-labeled recombinant HLA-A201 at 18°C ​​for 48 hours to form a peptide-HLA complex. The biotin-labeled peptide-HLA complex was then captured by streptozocin-coated beads and detected by flow cytometry with PE-conjugated anti-human β2-microglobulin. The peptides showed a binding affinity (IC) of 45 nM for HLA-A201. 50 ) is a known peptide strongly binding to HLA-A201, derived from the cytomegalovirus (CMV) pp65 protein ( 495 NLVPMVATV 503 ;SEQ ID NO: 27; hereafter referred to as CMVpp65 495-503 ) (Hassan C. et al., J. Biol. Chem. 2015, 290:2593-2603) was used as a positive control. No peptide was used as a negative control.

[0112]

[0125] result

[0126] The peptides of the present invention have the ability to bind to human MHC class I. As shown in Figure 1, both peptide 1 (SEQ ID NO: 1) and peptide 12 (SEQ ID NO: 12) of the present invention bind to CMVpp65 at concentrations of 3 nM and 8.9 nM against HLA-A201. 495-503(SEQ ID NO: 27; positive control). In particular, the binding affinity of peptide 1 (SEQ ID NO: 1) and peptide 12 (SEQ ID NO: 12) to HLA-A201 was 8.9 nM, which is higher than that of CMVpp65. 495-503 (SEQ ID NO: 27; positive control) are 1.8 and 2.1 times higher, respectively.

[0113]

[0127] In addition, as shown in FIG. 2, all of the antigen peptides of the present invention (SEQ ID NOs: 1 to 26) exhibit strong binding affinity to HLA-A201 at a concentration of 8.9 nM.

[0114]

[0128] The results of Example 1 demonstrate that the peptides of the present invention (SEQ ID NOS: 1-26) have strong binding affinity to HLA-A201, the most common human MHC class I allele in the population. Thus, when one of the peptides of the present invention is administered to a population of human subjects, the peptide binds to MHC class I and elicits a subsequent immune response in the majority of the subjects.

[0115]

[0129] Example 2 MHC Class I Tetramer Assay

[0130] MHC tetramer reagents enable rapid and simple detection of antigen-specific T cells. MHC tetramer technology is based on the ability of MHC-peptide complexes to recognize antigen-specific T cells at the single-cell level. This technology allows researchers to accurately measure the responsible T cell responses involved in infectious diseases, cancer, and autoimmune diseases. The presence of an antigen-specific T cell immune response is considered to be the most important and meaningful outcome of anti-tumor or anti-viral responses for the development of vaccines and therapeutics. Due to the importance and significance of the presence of an antigen-specific T cell immune response for vaccine development, an MHC tetramer assay was used in this example to detect antigen-specific T cells.

[0116]

[0131] Materials and Methods

[0132] MHC Class I Tetramer Assay. The MHC Class I tetramer assay (Easimer®, immunAware Aps, Copenhagen, Denmark) was performed according to the manufacturer's instructions. Briefly, 3 μL of 75 μM peptide 1 (SEQ ID NO: 1) or peptide 12 (SEQ ID NO: 12) of the present invention was mixed with 45 μL of ddHO, 12 μL of folding buffer (x6), and 12 μL of Aisimer® and incubated at 18°C ​​for 48 hours to obtain approximately 500 nM folded monomer. 50 μL of the resulting folded monomer was then mixed with the equivalent of 2.1 μL of 0.2 mg / mL streptozocin-fluorophore and incubated at 4°C in the dark for at least 1 hour to tetramerize the monomer. For staining of human T cells, the tetramer was diluted to 30 nM in FACS buffer (PBS with 1 v / v% BSA and 0.1 v / v% NaN3).

[0117]

[0133] Next, approximately 1-2 x 10 5 Human peripheral blood mononuclear cells (PBMCs, HLA-A201 + ) (STEMCELL Technologies Inc, Vancouver, British Columbia, Canada) was plated in a 96-well microplate. Cells were centrifuged at 700 × g for 3 minutes, and the supernatant was removed. Cells were resuspended in 40 μL of diluted tetramer and incubated at room temperature (RT) in the dark for 20 minutes. Cells were washed once with cold FACS buffer and centrifuged at 700 × g for 3 minutes. After removing the supernatant, cells were co-stained with anti-CD8 antibody and incubated at 4°C in the dark for 30 minutes. Cells were washed twice with cold FACS buffer, resuspended in FACS buffer, and analyzed using a flow cytometer (BD LSRFortessa™ X20, Franklin Lakes, NJ, US).

[0118]

[0134] Statistical analysis. Microsoft Excel was used for statistical analysis. Student's t-test was used to calculate significance. *p<0.05, **p<0.01, ***p<0.001.

[0119]

[0135] result

[0136] The peptides of the present invention induce antigen-specific T cell immune responses. As shown in Figure 3, antigen-specific cytotoxic T cells (CTLs) of peptide 1 (SEQ ID NO: 1) and peptide 12 (SEQ ID NO: 12) of the present invention were expressed in human PBMC (HLA-A201 + The results show that the antigen peptides of the present invention can induce at least antigen-specific CTLs (i.e., CD8 + These peptide-tetramer complexes have been shown to induce immune responses (e.g., T cell responses). The peptide-tetramer complexes can detect corresponding specific CTLs, and these responding CTLs can recognize exogenous organisms or endogenous cells bearing the peptides. CTL recognition of the peptides can result in the elimination of the organisms or infected cells.

[0120]

[0137] In addition, the results showed that HLA class I tetramer binding to peptide 1 (SEQ ID NO: 1) or peptide 12 (SEQ ID NO: 12) stimulated approximately 2.2-3.3% of CD8+ cells in human PBMCs. + It has been shown to associate with T cells. Approximately 4 x 10 10 CD8 + Since it is estimated that there are approximately 0.9 to 1.3 × 10 T cells in the adult human body (Alanio, C. et al., Blood, 2010, 115(18):3718-3725), the peptides of the present invention can be used to treat approximately 0.9 to 1.3 × 10 T cells in the adult human body. 9 CD8 + T cells can be engaged to recognize virus-infected cells and induce apoptosis in the cells.

[0121]

[0138] Example 3 Intracellular cytokine assay

[0139] To further verify the ability of the antigenic peptides to induce T cell responses, intracellular cytokine staining was used to measure the CD4 T cells in response to the peptides of the present invention. + Cytokine production of helper T cells was detected.

[0122]

[0140] Materials and Methods

[0141] Intracellular cytokine staining and multiparameter flow cytometry. Approximately 1-2 x 10 5 Human PBMC (HLA-A201 + ) (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) were plated in X-VIVO™ 15 medium in a 96-well microplate. PBMCs were treated with peptide 1 (SEQ ID NO: 1) or peptide 12 (SEQ ID NO: 12) of the present invention, diluted 5-fold from 250 nM to 0.4 nM. The culture medium was refreshed every 5 days. On day 12 after peptide treatment, cells were harvested and stained with anti-CD4-PerCP-Cy5.5-conjugated antibodies, IFN-γ-FITC chrome-conjugated antibodies, and IL4-PE chrome-conjugated antibodies according to the manufacturer's instructions. Cells were then washed twice with cold FACS buffer, resuspended in FACS buffer, and analyzed using a flow cytometer (BD LSR Fortessa™ X20, Franklin Lakes, NJ, USA).

[0123]

[0142] result

[0143] The peptides of the present invention stimulate immune cells to secrete cytokines that enhance both cellular and humoral immunity. As shown in Figure 4A, peptide 1 (SEQ ID NO: 1) of the present invention stimulates helper T cells (CD4 + As shown in Figure 4B, peptide 12 of the present invention (SEQ ID NO: 12) stimulated CD4 T cells to secrete higher levels of IFN-γ at concentrations of 10 nM and 250 nM and IL-4 at a concentration of 10 nM. + The cells were stimulated.

[0124]

[0144] The results show that the peptides of the present invention can stimulate helper T cells to secrete the cytokines IFN-γ and IL-4, which represent cellular and humoral immunity, respectively. In addition, by stimulating helper T cells to secrete IFN-γ, the peptides of the present invention enhance CTL immune responses, which corresponds to the results of Example 2.

[0125]

[0145] Example 4 Immunogenicity Assay of Peptide 1 by Intramuscular Injection

[0146] Materials and Methods

[0147] Immunization of Mice. To determine the immunogenicity of the antigenic peptides of the present invention, peptide 1 (SEQ ID NO: 1) of the present invention was used as an example mouse model. Ten female BALB / c mice (7-9 weeks old) provided by Envigo (Indianapolis, IN, USA) were intramuscularly injected with 45 μg of peptide 1 (SEQ ID NO: 1) three times at 2-week intervals (days 0, 14, and 28). Serum was collected before each immunization day (days -1, 13, and 27). A final blood draw was performed on day 41. Isolated serum was stored at -80°C until serological analysis. Serum samples collected one day before the first injection (day -1, pre-dose group) and two weeks after the third injection (day 41, post-dose group) were used for anti-spike IgG ELISA assays.

[0126]

[0148] Total IgG ELISA. An IgG (Total) Mouse Uncoated ELISA Kit (Cat. No. 88-50400, Thermo Fisher Scientific, MA, USA) was used for the Total IgG ELISA according to the manufacturer's instructions. Briefly, Nunc™ MaxiSorp™ 9018 ELISA plates were coated with 100 μL / well of capture antibody in coating buffer and incubated overnight at 4°C. The plates were washed twice with 400 μL / well of wash buffer. The wells were blocked with 250 μL of blocking buffer and incubated at room temperature for 2 hours, after which the plates were washed twice. A standard curve was generated by serially diluting the standards two-fold in Assay Buffer A. Then, 100 μL / well of Assay Buffer A was added to blank wells, and 90 μL / well of Assay Buffer A was added to sample wells. Serum samples were diluted at least 10,000-fold in assay buffer A, and then 10 μL / well of the prediluted serum sample was added to the appropriate wells. 50 μL / well of diluted detection antibody was added to all wells. The plate was then covered and incubated at room temperature for 2 hours, after which it was washed four times. 100 μL / well of substrate solution was added to each well, and the plate was incubated at room temperature for 15 minutes. 100 μL of stop solution was then added to each well, and the plate was read at OD 450 nm using a microplate reader.

[0127]

[0149] Anti-spike IgG ELISA. A human SARS-CoV-2 spike (trimer) IgG ELISA kit (catalog number BMS2325, Thermo Fisher Scientific, MA, USA) was used for anti-spike IgG ELISA according to the manufacturer's instructions. Briefly, a human SARS-CoV-2 spike (trimer)-coated plate (96 wells) was washed with wash buffer, and then 90 μL of assay buffer and 10 μL of assay buffer-diluted sample were added to the wells of the plate. The plate was covered with a plate cover and incubated at 37°C for 30 minutes. After incubation, the plate was washed with wash buffer, and 100 μL of anti-mouse horseradish peroxidase (HRP)-conjugated detection antibody (A90-131P, Bethyl, TX, USA) was added to the wells of the plate. The plate was covered with a plate cover and incubated at 37°C for an additional 30 minutes. After incubation, the plate was washed with wash buffer, and 100 μL of substrate solution (tetramethylbenzidine, TMB) was added to the wells of the plate. The plate was incubated at room temperature for 15 minutes. After incubation, 100 μL of stop solution was added to the wells of the plate. The plate was then read at OD 450 nm using a microplate reader.

[0128]

[0150] Statistical analysis. Prism5 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Significance was calculated using the t-test. *p<0.05, **p<0.01, ***p<0.001.

[0129]

[0151] result

[0152] Intramuscular administration of the peptide of the present invention induces immunogenicity against the SARS-CoV-2 spike protein. As shown in Figure 5A, intramuscular administration of peptide 1 (SEQ ID NO: 1) of the present invention to mice induces total IgG antibodies, and the efficacy reaches a plateau after the second administration. The results demonstrate that the peptide of the present invention has immunogenicity.

[0130]

[0153] As shown in Figure 5B, intramuscular administration of peptide 1 (SEQ ID NO: 1) of the present invention to mice induced anti-spike IgG antibodies. The results further demonstrate that the peptides of the present invention induce mice to produce specific antibodies against the human SARS-CoV-2 spike protein.

[0131]

[0154] Example 5 Immunogenicity Assay of Peptide 12 by Intramuscular Injection

[0155] Materials and Methods

[0156] Immunization of Mice. To determine the immunogenicity of the antigenic peptide of the present invention, peptide 12 (SEQ ID NO: 12) of the present invention was used as an example mouse model. Four female BALB / c mice (7-9 weeks old) supplied by BioLASCO (Taiwan Co. Ltd.) were intramuscularly injected with 400 μg of peptide 12 (SEQ ID NO: 12) three times at two-week intervals (on days 0, 14, and 28). Serum was collected one day before the first injection (day -1, pre-dosing group) and two weeks after the third injection (day 41, post-dosing group). The isolated serum was stored at -80°C until serum analysis.

[0132]

[0157] Total IgG ELISA. Total IgG ELISA assays were performed as described in Example 4.

[0133]

[0158] Anti-spike IgG ELISA. Anti-spike IgG ELISA assays were performed as described in Example 4.

[0134]

[0159] Statistical Analysis. Statistical analysis was performed as described in Example 4.

[0160] result

[0161] Intramuscular administration of the peptide of the present invention induces immunogenicity against the SARS-CoV-2 spike protein. As shown in Figure 6A, intramuscular administration of peptide 12 (SEQ ID NO: 12) of the present invention to mice induced total IgG antibodies. The results indicate that the peptide of the present invention has immunogenicity. The results of this example correspond to those of Example 2, in which the peptide of the present invention stimulates helper T cells to secrete IL-4, which activates B cells to produce antibodies.

[0135]

[0162] As shown in Figure 6B, intramuscular administration of peptide 12 (SEQ ID NO: 12) of the present invention to mice induced anti-spike IgG antibodies. The results further demonstrate that the peptides of the present invention induce mice to produce specific antibodies against the human SARS-CoV-2 spike protein.

[0136]

[0163] Example 6 Immunogenicity Assay of Peptide 1 by Oral Administration

[0164] Materials and Methods

[0165] Immunization of Mice. To determine the immunogenicity of the antigenic peptides of the present invention, Peptide 1 (SEQ ID NO: 1) of the present invention was used as an example mouse model. Five female BALB / c mice (7-9 weeks old) provided by BioLASCO (Taiwan Co. Ltd.) were orally administered 200 μL of 1 μg / μL Peptide 1 (SEQ ID NO: 1; 200 μg / dose) formulated with 1% (v / v) 77 mg / mL PLCL-PEG-PLCL (Sigma-Aldrich, St. Louis, MO, USA) three times at 2-week intervals (days 0, 14, and 28). Serum was collected one day before the first dose (day -1, pre-dose group) and two weeks after the third dose (day 41, post-dose group). The isolated serum was stored at -80°C until serological analysis.

[0137]

[0166] Total IgG ELISA. Total IgG ELISA assays were performed as described in Example 4.

[0138]

[0167] Anti-spike IgG ELISA. Anti-spike IgG ELISA assays were performed as described in Example 4.

[0139]

[0168] Statistical Analysis. Statistical analysis was performed as described in Example 4.

[0169] result

[0170] Oral administration of the peptide of the present invention induces immunogenicity against the SARS-CoV-2 spike protein. As shown in Figure 7A, oral administration of peptide 1 (SEQ ID NO: 1) of the present invention to mice induced total IgG antibodies. The results indicate that the peptide of the present invention has immunogenicity.

[0140]

[0171] As shown in Figure 7B, oral administration of peptide 1 (SEQ ID NO: 1) of the present invention to mice induced anti-spike IgG antibodies. The results further demonstrate that the peptide of the present invention induces mice to produce specific antibodies against the human SARS-CoV-2 spike protein.

[0141]

[0172] The peptides of the present invention are specifically designed for binding to human MHC molecules, and therefore, these peptides have high affinity for HLA-A201 (as shown in Figure 2). However, the peptides of the present invention also have lower affinity for mouse MHC molecules. For example, peptide 1 (SEQ ID NO: 1) of the present invention exhibited half-maximal inhibitory concentrations (IC) of 2.2, 78.1, and 82.5 μM for mouse MHC class H2-Kd, H2-Ld, and H-2-Dd molecules, respectively. 50 ) (https: / / www.iedb.org). The peptides of the present invention have lower affinity for mouse MHC molecules, but can still induce immune responses in mice (shown in Figures 5A to 7B). These results indicate that the peptides of the present invention can induce not only immune responses with different subtypes of MHC molecules, but also higher immune responses in human subjects.

[0142]

[0173] In conclusion, the peptides of the present invention exhibit strong binding affinity to MHC molecules, especially MHC class I molecules, and bind to CD4 + T cell responses and CD8 + The peptides of the present invention induce both T cell responses, participate in CTL responses, and stimulate the production of specific antibodies against the human SARS-CoV-2 spike protein via both intramuscular and oral routes. Because the peptides of the present invention are designed based on a well-conserved region of the human SARS-CoV-2 spike protein and induce both cellular and humoral immune responses, the peptides are important candidates for the development of broad-spectrum vaccines against coronaviruses, particularly SARS-CoV-2.

[0143]

[0174] Of course, many changes and modifications can be made to the above-described embodiments of the invention without departing from the scope thereof. Therefore, in order to promote the advancement of science and useful arts, the present invention has been disclosed and is intended to be limited only by the appended claims.

Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:

26.

2. A nucleic acid encoding the peptide of claim 1.

3. An antibody or T cell receptor capable of specifically recognizing and / or binding to the peptide of claim 1.

4. The antibody or T cell receptor of claim 3 , wherein the peptide is bound to an MHC molecule.

5. A recombinant host cell comprising one component selected from the group consisting of the peptide of claim 1, the nucleic acid of claim 2, and the antibody or T cell receptor of claim 3 or 4.

6. Activated T lymphocytes that selectively recognize cells presenting the peptide of claim 1.

7. A pharmaceutical composition for use in the prevention or treatment of pathogenic infection in a subject, comprising at least one active ingredient selected from the group consisting of a peptide described in claim 1, a nucleic acid described in claim 2, an antibody or T cell receptor described in claim 3, and an activated T lymphocyte described in claim 6.

8. A pharmaceutical composition for use in the prevention or treatment of a pathogenic infection in a subject, the pharmaceutical composition comprising a recombinant host cell according to claim 5.

9. The pharmaceutical composition of claim 7 , further comprising a pharmaceutically acceptable carrier, an adjuvant, a pharmaceutically acceptable excipient and / or a stabilizer.

10. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, an adjuvant, a pharmaceutically acceptable excipient and / or a stabilizer.

11. 8. The pharmaceutical composition of claim 7, wherein the pathogenic infection is induced by a coronavirus.

12. 9. The pharmaceutical composition of claim 8, wherein the pathogenic infection is induced by a coronavirus.

13. 12. The pharmaceutical composition of claim 11, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

14. 13. The pharmaceutical composition of claim 12, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

15. A complex comprising the peptide of claim 1 bound to an MHC molecule.

16. 16. The complex of claim 15, comprising two or more peptides of claim 1 and two or more MHC molecules.

17. 17. The complex of claim 16, wherein the MHC molecule is associated with a dextran backbone.

18. 17. A method for collecting data to determine the presence or absence of a current or previous coronavirus infection in an individual, comprising the steps of contacting a peptide according to claim 1 or a conjugate according to claim 15 or 16 with a sample obtained from the individual, and determining the presence or absence of binding between the peptide or conjugate and a molecule contained in the sample, wherein the molecule is an antibody or an antibody fragment containing an antigen-binding site, or a T-cell receptor.

19. 17. A method for identifying coronavirus-specific T cells, the method comprising the steps of contacting a peptide according to claim 1 or a complex according to claim 15 or 16 with a T cell receptor or with a sample obtained from an individual, and determining the presence or absence of binding between the peptide or complex and the T cell receptor or a T cell receptor contained in the sample, wherein the T cell receptor or the T cell receptor contained in the sample is on the surface of or contained in a T cell.

20. 20. The method of claim 19, wherein the individual is currently infected with the coronavirus or was previously infected with the coronavirus but is not currently infected.

Images