WO2021256795A1 - Épitope du nouveau coronavirus et son utilisation - Google Patents

Épitope du nouveau coronavirus et son utilisation Download PDF

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WO2021256795A1
WO2021256795A1 PCT/KR2021/007379 KR2021007379W WO2021256795A1 WO 2021256795 A1 WO2021256795 A1 WO 2021256795A1 KR 2021007379 W KR2021007379 W KR 2021007379W WO 2021256795 A1 WO2021256795 A1 WO 2021256795A1
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cells
epitope
present
coronavirus
seq
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신의철
최승진
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한국과학기술원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to novel coronavirus epitopes and various uses of compositions comprising them.
  • Corona virus is a representative virus that causes lethal infectious disease in modern civilization.
  • Severe Acute Respiratory Syndrome also known as SARS
  • MERS Middle East Respiratory Syndrome
  • MERS spread from the Middle East to the world, resulting in a death rate of about 36%.
  • COVID-19 novel coronavirus disease 2019 (COVID-19)
  • COVID-19 novel coronavirus disease 2019 (COVID-19)
  • Beta-coronavirus is the source of a major new virus that enters centuries, and it is expected that it will appear periodically in the future. Rather than developing a vaccine every time a novel coronavirus appears, it is very important to develop a vaccine that can cover all beta coronaviruses, so the inventors of the present invention developed a method containing the beta corona virus shared CR (conserved region) gene. We have come to discover the optimal epitope for developing a DNA vaccine. If humans have such a vaccine, they will be able to cope with the novel coronavirus much more leisurely, and it is expected that they will be able to be free from the novel coronavirus that threatens the survival of civilization as well as the improvement of human health and health care. .
  • One object of the present invention is to provide a coronavirus-specific epitope.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the epitope and an expression vector comprising the molecule.
  • Another object of the present invention is to provide an antigen-presenting cell loaded with the epitope and capable of activating T cells for the treatment of coronavirus infection.
  • Another object of the present invention is to provide a T cell activated by the antigen-presenting cell loaded with the epitope.
  • Another object of the present invention is to provide a method for activating T cells for the treatment of coronavirus infection.
  • Another object of the present invention is to provide a composition for T cell activation comprising the epitope.
  • Another object of the present invention is to provide an immunogenic composition and a vaccine composition comprising the epitope.
  • Another object of the present invention is to provide a method for preventing or treating a coronavirus infection comprising administering an effective amount of a composition for activating T cells comprising the epitope.
  • corona virus (Coronavirus) specific epitope.
  • epitope is also referred to as an epitope, and refers to a specific portion of an antigen that allows the immune system, such as antibodies, B cells, T cells, etc. to identify the antigen.
  • the epitope of a protein antigen is largely divided into a three-dimensional epitope and a linear epitope according to its shape and the mode of action with the antigen-binding site.
  • a conformational epitope is composed of a discontinuous amino acid sequence of an antigen, and reacts with the three-dimensional structure of the antibody-antigen binding site. Most of them correspond to conformational epitopes.
  • coronavirus is four in (alpha, beta, gamma, delta) corona virus subfamily (Coronavirinae) of corona bayireoseugwa (Coronaviridae), people with RNA virus gene size 27 to 32kb in the present invention It is known to cause respiratory and digestive system infections in animals. It is easily transmitted mainly through mucosal transmission and droplet transmission. Humans usually cause mild respiratory infections, but rarely fatal infections. Diarrhea in cattle and pigs, and respiratory diseases in chickens. It is divided into the classifications in Table 1 below (Centers for Disease Control, 2020). Among the four genera, alpha and beta infect humans and animals, and gamma and delta are reported to infect only animals.
  • Virus name Genus host Symptom HCoV-229E Alpha Human mild respiratory symptoms HCoV-NL63 Alpha Human mild respiratory symptoms SARS-CoV Beta Human severe respiratory symptoms MERS-CoV Beta Human severe respiratory symptoms HCoV-OC43 Beta Human mild respiratory symptoms HCoV-HKU1 Beta Human Pneumonia symptoms SARS-CoV-2 Beta Human Mild respiratory symptoms Severe cases can cause shortness of breath
  • the "beta coronavirus” corresponds to a zoonotic infection as one of four genera of coronaviruses in the subfamily Coronavirus.
  • beta coronaviruses include Severe Acute Respiratory Syndrome (SARS; SARS-CoV), Middle East Respiratory Syndrome (MERS; MERS-CoV), and novel coronavirus infection (COVID-19; SARS-CoV).
  • CoV-2), human coronavirus OC43 (HCoV-OC43), or human coronavirus HKU1 (HCoV-HKU1) are known to exist.
  • the coronavirus genome has a 5' cap structure and a 3' poly A tail.
  • RNA Single-stranded positive-sense RNA within 30 kb with The genomic RNA is used as a template to directly translate the polyprotein 1a/1ab (pp1a/pp1ab), encodes a non-structural protein (nsp) and replicates in double-membrane vesicles (DMV) -forms the transcription-transcription complex (RTC) (J Virol. 2006; 80(12): 5927-5940.).
  • sgRNAs subgenomic RNAs
  • mRNAs subgenomic messenger RNAs
  • Transcription termination and subsequent acquisition of leader RNA occurs in transcriptional regulatory sequences located between open reading frames (ORFs). This negative-stranded sgRNA is used as a template for subgenomic mRNA production.
  • a typical coronavirus genome and subgenome in the present invention contains at least 6 ORFs and encodes 16 nsp (nsp1 to nsp16), except for gamma coronaviruses, which lack nsp1 by about 2/3 of the total genome length.
  • the terminus encodes four major structural proteins: the spike (S), membrane (M), envelope (E) and nucleocapsid (N) proteins.
  • S spike
  • M membrane
  • E envelope
  • N nucleocapsid
  • other coronaviruses also encode specialized structural and helper proteins such as HE protein, 3a/b protein and 4a/b protein.
  • the Orf1ab protein is a non-structural protein (nsp) related to RNA synthesis of a virus and is known not to be exposed to the outside of a virion.
  • nsp non-structural protein
  • Orf1ab of five beta coronaviruses consists of nsp1 to nsp16, and most of nsp have been reported to play a role in coronavirus replication, but the entire role has not yet been revealed.
  • the "nonstructural protein” refers to a protein that is not a structural protein as a kind of protein. Amino acids are biopolymers linked by peptide bonds, and non-structural proteins are mostly spherical and often have specific functions.
  • the viral nonstructural protein refers to a protein encoded by a virus or a protein that does not constitute a part of a virus particle. They control the host and various enzymes and transcription factors the virus uses to replicate itself, such as viral proteases (such as 3CL/nsp5), RNA replicase or other template-directed polymerases. means for doing so.
  • the "coronavirus-specific epitope” is derived from the protein antigen of the coronavirus, and preferably corresponds to the protein antigen present in the alpha corona virus or beta corona virus, more preferably the beta corona virus.
  • the corona virus-specific epitope includes at least one epitope recognized by a T-cell receptor, and preferably, as an epitope present in beta corona virus, the epitope is in Orf1ab protein of 5 types of beta coronavirus. It may be a non-structural protein included.
  • the epitope provided in the present invention may be present in a region conserved between a plurality of corona viruses shown in Table 1 or Table 2 above.
  • the conserved region is a region in which the sequences of SARS-CoV-2 and other five proteins of beta corona virus (Orf1ab, Spike, Envelope, Membrane, Nucleocapsid) match, and the beta corona A conserved region of a virus.
  • the epitope may preferably be a sequence located in the Orf1ab protein, more preferably nsp 12 or a fragment thereof in the Orf1ab protein; nsp 13 or a fragment thereof; nsp 14 or a fragment thereof; and nsp 16 or a fragment thereof; may be any one or more conserved sequences selected from the group consisting of.
  • the epitope may be a conserved sequence of nsp 12 or a fragment thereof, which is a conserved sequence having an RNA-dependent RNA polymerase function, preferably consisting of SEQ ID NOs: 1 to 3 Any one or more sequences selected from the group may be included.
  • the epitope may be a conserved sequence of nsp 13 or a fragment thereof, which is a conserved sequence having a helicase function, preferably any one or more sequences selected from the group consisting of SEQ ID NOs: 4 to 8 may be included.
  • the epitope may be a conserved sequence of nsp 14 or a fragment thereof, which is a conserved sequence having a 3'-5' exonuclease function, preferably SEQ ID NO: 9 and SEQ ID NO: At least one sequence out of 10 may be included.
  • the epitope may be an nsp 16 conserved sequence or a fragment thereof, which is a conserved sequence having 2'-O-ribose methyltransferases (2'-O-ribose methyltransferases) functions, preferably a fragment thereof.
  • the sequence of SEQ ID NO: 11 may be included.
  • the epitope may include any one or more sequences selected from the group consisting of SEQ ID Nos: 12 to 20 shown in Table 3 below.
  • sequence list sequence information SEQ ID NO: 12 IFVDGVPFV SEQ ID NO: 13 FVDGVPFVV SEQ ID NO: 14 RLANECAQV SEQ ID NO: 15 SLAIDAYPL SEQ ID NO: 16 LYYQNNVFM SEQ ID NO: 17 YYQNNVFMS SEQ ID NO: 18 NRARTVAGV SEQ ID NO: 19 ARTVAGVSI SEQ ID NO: 20 NRFNVAITR
  • the epitope of the present invention may include substitution, insertion, or addition of 1 or 2 amino acids in any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 12 to 20, but is not limited thereto.
  • the amino acid to be substituted, inserted or added may be a neutral amino acid, an acidic amino acid, a basic amino acid, or an aromatic amino acid.
  • the neutral amino acid is glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), cysteine (cysteine; C), methionine (M), proline (P) or threonine (T), wherein the acidic amino acid is aspartic acid (D), glutamic acid (E) ), may be asparagine (N) or glutamine (Q), and the basic amino acid may be lysine (K), arginine (R) or histidine (H), and the aromatic amino acid may be phenylalanine (F), tryptophan (W) or tyrosine (Y).
  • amino acid substitution refers to the replacement of one amino acid residue with another amino acid residue.
  • Suitable amino acid substitutions can be made conservatively by amino acid substitutions having similar hydrophobicity, polarity, and R-chain length to one another. Examples of conservative amino acid substitutions are shown in Table 4 below.
  • the epitope provided in the present invention may activate T cells, preferably cytotoxic T cells, to induce T cell immunity.
  • the "T-cell immunity” refers to cellular immunity or cell-mediated immunity, and is a concept corresponding to humoral immunity involving antibodies. It refers to an immune process that destroys non-self cells. It is an immune response including activation of phagocytosis, antigen-specific cytotoxic T cells, and secretion of various cytokines in response to antigen.
  • an antigen binds to the antigen recognition epitope of mature cytotoxic T cells
  • an antigen-specific immune response occurs.
  • cytotoxic T cells and target cells bind, vesicles containing proteolytic enzymes move from the cytoplasm of cytotoxic T cells to target cells, and the vesicles are secreted through exocytosis.
  • the secreted enzymes when the perforin proteins that pierce the cell membrane bind to the cell membrane of the target cell, apoptosis is induced.
  • activation of the T cells induces a cell-mediated response.
  • the reaction induced by exposure to an antigen for the first time is called the primary immune response, and the response induced by re-exposure to the same antigen is called the secondary immune response induced by immune memory.
  • Immune memory is formed by the generation of antigen-specific memory cells after the primary immune response to an antigen, and when exposed to the same antigen after several months or years, a faster and stronger immune response is induced by the generated memory cells.
  • This T cell immune response recognizes antigens as well as non-structural proteins with relatively few mutations as well as envelope proteins with many mutations. Therefore, the present invention finds a well-conserved T cell antigen without mutation in the beta coronavirus from a non-structural protein and utilizes it as a vaccine antigen. This exists
  • the epitope is HLA-A, HLA-B, HLA-C, HLA-E so that T cells, preferably memory T cells, extracted from human blood can have efficacy.
  • the epitope in the present invention which has a high binding affinity for HLA-A*02:01, may be an epitope, which is a peptide represented by any one of SEQ ID NOs: 12 to 15; It has a high binding affinity to HLA-A*24:02, and may be an epitope, which is a peptide represented by any one of SEQ ID NO: 16 and SEQ ID NO: 17; Alternatively, it may be an epitope that has a high binding affinity for HLA-B*27:05 and is a peptide represented by any one of SEQ ID NOs: 18 to 20.
  • the NetMHCpan 4.0 EL algorithm may be used to predict whether an epitope binds to a specific HLA allele, but is not limited thereto.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA-I human MHC class I genes including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G and ⁇ 2-microglobulin loci.
  • HLA-II HLA Class II
  • HLA Class II means HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA- Refers to human MHC class II genes including DM, HLA-DOA and HLA-DOB loci.
  • nucleic acid molecule encoding the epitope provided in the present invention.
  • Nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of a polypeptide provided in the present invention is translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), and all of these are also included in the nucleic acid molecule of the present invention.
  • ORF open reading frame
  • an expression vector comprising the isolated nucleic acid molecule provided by the present invention.
  • the "vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked.
  • a vector which refers to circular double-stranded DNA into which additional DNA segments can be ligated.
  • a phage vector Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors are episomal mammalian vectors having a bacterial origin of replication).
  • vectors may integrate into the genome of the host cell upon entry into the host cell, thereby being replicated along with the host genome.
  • some vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”.
  • expression vectors useful in recombinant DNA techniques often exist in the form of plasmids.
  • plasmid and vector may be used interchangeably, since a plasmid is the most commonly used form among vectors.
  • the expression vector in the present invention include pCDNA vectors widely used commercially, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmid; phage such as lambda, lambdoid, M13, Mu, p1 P22, Q ⁇ , T-even, T2, T3, T7; It may be selected from the group consisting of plant viruses, but is not limited thereto, and any expression vector known to those skilled in the art as an expression vector can be used in the present invention, and the selection of an expression vector depends on the properties of a target host cell.
  • the vector When introducing a vector into a host cell, it can be carried out by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can use
  • An introduction method suitable for the expression vector and host cell can be selected and used.
  • the vector contains one or more selection markers, but is not limited thereto, and selection is possible depending on whether a product is produced using a vector that does not contain a selection marker.
  • the selection of the selection marker is selected by the desired host cell, and the present invention is not limited thereto since it uses a method already known to those skilled in the art.
  • the nucleic acid molecule of the present invention may be fused by inserting a tag sequence onto an expression vector.
  • the tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag facilitating purification known to those skilled in the art can be used in the present invention.
  • a host cell transfected with the expression vector provided in the present invention.
  • the term "host cell” includes individual cells or cell cultures that may or have been recipients of the vector(s) for the incorporation of a polypeptide insert.
  • a host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation.
  • Host cells include cells transfected in vivo with the polypeptide(s) herein.
  • the host cell may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; yeast cells and fungal cells such as P.
  • insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells) or PERC.6 (Human Retinal Cells) animal cells; Or, it may be a plant cell, but is not limited thereto, and any cell that can be used as a host cell known to those skilled in the art is available.
  • a T cell comprising a coronavirus-specific epitope provided in the present invention, a nucleic acid molecule encoding the same, an expression vector containing the nucleic acid molecule, or a host cell transformed with the expression vector It relates to a composition for activation of
  • activation of T cells means monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., having a T cell receptor that recognizes at least one coronavirus antigen peptide). For example, it refers to a population of T cells (with clones encoding different TCRs).
  • Activated T cells include, but are not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells, regulatory T cells, and memory T cells. It may contain the above subtypes, preferably memory T cells (memory T cells).
  • the activated T cells prevent the progression to severe infection and enable early recovery by directly removing virus-infected cells.
  • the coronavirus-specific epitope provided in the present invention may be loaded antigen presenting cells (antigen presenting cells, APC).
  • the epitope may include any one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, but preferably include any one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 can
  • the antigen-presenting cells may include one or more of dendritic cells (DC), B cells, and macrophages, but preferably dendritic cells.
  • DC dendritic cells
  • B cells B cells
  • macrophages but preferably dendritic cells.
  • the "dendritic cells” are members of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their unique conformation and high expression levels of surface class I and class II molecules, proteins that present antigenic peptides to T cells.
  • DCs, other APCs, and T cells can be conveniently isolated or derived (eg differentiated) from peripheral blood and from multiple tissue sources, such as peripheral blood mononuclear cells (PBMC) derived from peripheral blood.
  • PBMC peripheral blood mononuclear cells
  • the antigen-presenting cells can induce the differentiation and proliferation of coronavirus antigen-specific T cells, preferably memory T cells, thereby removing infected cells, preventing progression to severe infection, and early recovery.
  • the present invention relates to T cells activated by the antigen presenting cells provided in the present invention.
  • the T cells are monoclonal (eg, encoding the same TCR) or polyclonal (eg, having clones encoding different TCRs) having a T cell receptor that recognizes a coronavirus antigen peptide.
  • a T cell population may include at least one selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells, regulatory T cells, and memory T cells, but is not limited thereto. It may contain one or more subtypes of non-T cells, but preferably memory T cells.
  • the "memory T cells” are T cells differentiated from T cells or activated T cells that have previously encountered and reacted with their specific antigen.
  • coronavirus-specific memory T cells make up a small fraction of the total T-cell quantity, they perform an important function in the surveillance of coronavirus-infected cells during an individual's entire lifespan.
  • the specific memory T cells encounter an infected cell expressing their specific coronavirus antigen, the memory T cells are immediately activated and clonally expanded.
  • the activated and proliferated T cells differentiate into effector T cells, killing infected cells with high efficiency.
  • Memory T cells are important for establishing and maintaining antigen-specific responses of T cells.
  • the activated T cell preferably the activated memory T cell, can specifically recognize the antigen of the coronavirus-infected cell, thereby preventing the progression to a severe infection and treating it at an early stage.
  • an epitope provided in the present invention, a nucleic acid molecule encoding the same, an expression vector containing the nucleic acid molecule, a host cell transformed with the expression vector, and an antigen-presenting cell loaded with the epitope , or provides an immunogenic composition against a coronavirus comprising T cells activated by the antigen-presenting cells.
  • the term "immunogenicity” refers to the property of inducing an immune response when a high molecular substance (eg, a protein drug) enters the body. It is also taken to refer to the ability of a vaccine to induce mediated immunity or immunological memory. Immunogenicity can be predicted from assessing the reactivity of antibodies or T cells. In general, immunogenicity is performed before a clinical trial to evaluate the effectiveness of a large-scale vaccine, and is used as an endpoint in an initial clinical trial to predict the effectiveness of a vaccine.
  • a high molecular substance eg, a protein drug
  • the corona virus may be an alpha corona virus, a beta corona virus, a gamma corona virus or a delta corona virus, but preferably an alpha corona virus or a beta corona virus, more preferably a beta corona virus .
  • an epitope provided in the present invention, a nucleic acid molecule encoding the same, an expression vector containing the nucleic acid molecule, a host cell transformed with the expression vector, and an antigen-presenting cell loaded with the epitope Or, it provides a vaccine composition for preventing or treating coronavirus infection comprising T cells activated by the antigen-presenting cells.
  • the vaccine is a vaccine comprising some sequences in the conserved region of the coronavirus, and if it corresponds to a function of activating T cells to induce memory T cells, it is not limited thereto.
  • the epitope included as an active ingredient in the vaccine may be used in combination with a carrier in order to maintain its three-dimensional structure or increase efficiency as a vaccine composition.
  • the carrier can be used without limitation as long as it is compatible with the body and can achieve the desired effect in the present invention.
  • it may be selected from serum albumin, peptide, immunoglobulin, hemocyanin, polysaccharide, and the like, but is not limited thereto.
  • a vaccine adjuvant may be further added as an active ingredient in the vaccine, and the adjuvant may be used without limitation as long as it is a pharmacological or immunological substance known in the art.
  • the adjuvant may be used without limitation as long as it is a pharmacological or immunological substance known in the art.
  • sugars or amino acids as stabilizers mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, cymosin, interleukin, etc. can be used as adjuvants.
  • the corona virus may be an alpha corona virus, a beta corona virus, a gamma corona virus or a delta corona virus, but preferably an alpha corona virus or a beta corona virus, more preferably a beta corona virus .
  • the present invention relates to a method of activating T cells by the antigen presenting cells (APC) provided in the present invention.
  • the T cells for activation of the T cells, may be co-cultured with the antigen-presenting cells loaded with the coronavirus-specific epitope of the present invention.
  • the T cells can be obtained from autologous sources, that is, from various sources including those derived from the subject of interest, preferably from peripheral blood mononuclear cells (PBMC) derived from peripheral blood. More preferably, it can be obtained from the non-adherent portion of the peripheral blood mononuclear cells.
  • PBMC peripheral blood mononuclear cells
  • the non-adherent portion of the PBMC can be obtained by density gradient centrifugation of a peripheral blood sample, which is cultured with or without an anti-CD3 antibody (such as OKT3) with at least one cytokine can be obtained by
  • the T cells are monoclonal (eg, encoding the same TCR) or polyclonal (eg, having clones encoding different TCRs) having a T cell receptor that recognizes a coronavirus antigen peptide.
  • a T cell population may include at least one selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, ⁇ T cells, regulatory T cells, and memory T cells, but is not limited thereto. It may contain one or more subtypes of non-T cells, but preferably memory T cells.
  • the T cell and the antigen-presenting cell may be derived from the same individual, such as an individual infected with a corona virus, preferably a beta coronavirus, but is not limited thereto.
  • the T cells are combined with the antigen-presenting cells of the present invention and 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, may be co-cultured for any one or more of 26, 28 or 30 days, preferably 1 to 21 days, 1 to 14 days, 2 to 10 days, 2 to 5 days, 2 to 5 days, It may be co-cultured for 3 days, 5 days, 7 days, 10 days, 14 days, 16 days, 18 days or 21 days, but is not limited thereto.
  • T cells when co-culturing the T cells and the antigen-presenting cells of the present invention, one or more cytokines are added to promote the activation, maturation and/or proliferation of T cells, and for subsequent differentiation into memory T cells, T cells can be primed.
  • a method for preventing or treating a coronavirus infection comprising administering to a subject in need of administration a composition for activating T cells comprising a coronavirus-specific epitope in an effective amount is about
  • the epitope may include any one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 20, but preferably include any one or more sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 can
  • the contents of the epitope, the composition for activating T cells, and the corona virus are the same as described above, and thus are omitted to avoid excessive complexity of the present specification.
  • the "administration" means providing a composition for activating T cells comprising a predetermined epitope of the present invention to a subject by any suitable method.
  • the "subject" in need of the administration may include both mammals and non-mammals.
  • mammals include humans, non-human primates such as chimpanzees, other apes or monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs or cats; laboratory animals such as rodents such as rats, mice or guinea pigs, but are not limited thereto.
  • non-mammal in the present invention may include, but are not limited to, birds or fish.
  • the epitope included as an active ingredient in the present invention may be used in a form combined with a carrier in order to maintain its three-dimensional structure or increase efficiency as a vaccine composition.
  • the carrier can be used without limitation as long as it is compatible with the body and can achieve the desired effect in the present invention.
  • it may be selected from serum albumin, peptide, immunoglobulin, hemocyanin, polysaccharide, and the like, but is not limited thereto.
  • the formulation of the composition administered as described above in the present invention is not particularly limited, and may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and a liquid formulation for oral or parenteral administration immediately before use. It may be administered in a solid form preparation intended to be converted into However, the present invention is not limited thereto.
  • a vaccine adjuvant may be further added together with the composition of the present invention during the administration, and the adjuvant may be used without limitation as long as it is a pharmacological or immunological substance known in the art.
  • the adjuvant may be used without limitation as long as it is a pharmacological or immunological substance known in the art.
  • sugars or amino acids as stabilizers mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, cymosin, interleukin, etc. can be used as adjuvants.
  • a pharmaceutically acceptable carrier may be additionally administered together with the composition for T cell activation containing the epitope of the present invention.
  • the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a pigment, a flavoring agent, etc., in the case of oral administration, and in the case of an injection, a buffer, Preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used.
  • the dosage form of the composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, in the case of oral administration, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. can be prepared in the form of, and in the case of injection, it can be prepared in the form of unit dose ampoules or multiple doses. have.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.
  • the administration route of the composition for activating T cells containing the epitope according to the present invention is not limited thereto, but is not limited thereto, but may include oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • compositions of the present invention may also be administered in the form of suppositories for rectal administration.
  • a "pharmaceutically effective amount” refers to an amount sufficient of an agent to provide a desired biological result. The result may be reduction and/or alleviation of the signs, symptoms or causes of a disease, or any other desirable change in the biological system.
  • an “effective amount” for prophylactic or therapeutic uses is the amount of the composition for activating T cells disclosed herein required to provide a clinically significant reduction in disease.
  • An appropriate “effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Accordingly, the expression “effective amount” generally refers to the amount in which the active substance has a therapeutic effect.
  • the active substance is a memory T cell activator and a preventive, ameliorating or therapeutic agent for coronavirus.
  • the composition of the present invention may vary depending on several factors including the activity of the specific epitope used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 100 mg/kg or 0.001 per day to 100 mg/kg may be administered. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
  • the composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
  • composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy, and biological response modifiers.
  • the present invention is able to cover even novel coronaviruses that cannot be covered by antibodies by finding the well-conserved T-cell antigen from the non-structural protein in the beta-coronavirus and using it as a vaccine antigen.
  • FIG 1 shows the 11 conserved sequence structures of the Orf1ab protein of 5 beta coronaviruses according to an embodiment of the present invention.
  • Figure 2 is a diagram showing the structure of the upper 2% of the position of the RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, nsp 12) as a conserved sequence of the beta corona virus according to an embodiment of the present invention.
  • FIG. 3 is a diagram showing the structure of the top 2% sequence of the helicase (Helicase, nsp 13) position as a conserved sequence of the beta corona virus according to an embodiment of the present invention.
  • FIG. 4 is a diagram showing the structure of the upper 2% sequence of the 3'-5' nuclease (exonuclease, nsp 14) position as a conserved sequence of the beta corona virus according to an embodiment of the present invention.
  • FIG. 5 is a diagram showing the top 2% sequence structure of the 2'-O-ribose methyltransferase (2'-O-ribose methyltransferases, nsp 16) position as a conserved sequence of beta corona virus according to an embodiment of the present invention. to be.
  • FIG. 6 is a diagram confirming the results of stimulating T cells and measuring the secretion of TNF cytokines using PBMCs of non-SARS-CoV-2 in vitro according to an embodiment of the present invention.
  • T cell functional avidity T cell responses of non-SARS-CoV-2 non-exposed subjects according to an embodiment of the present invention by measuring EC50 values.
  • a corona virus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • nucleic acid molecule encoding a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • nucleic acid molecule encoding a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • an expression vector comprising a nucleic acid molecule encoding a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • a host cell transformed with an expression vector comprising a nucleic acid molecule encoding a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 is about
  • composition for activating T cells comprising a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • an antigen-presenting cell loaded with a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • a method for activating T cells by an antigen-presenting cell loaded with a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20. will be.
  • an immunogenic composition for coronavirus comprising a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20.
  • a vaccine composition for preventing or treating a coronavirus infection comprising a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20. will be.
  • a composition for activating T cells comprising a coronavirus-specific epitope comprising any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 20 to a subject in need of administration is effective It relates to a method for preventing or treating coronavirus infection, comprising administering in an amount.
  • peripheral blood mononuclear cells obtained from 200 healthy Koreans before the 2015 MERS outbreak were mononuclear cells (PBMC) were provided from Kyungpook National University, and the immune response of IFN- ⁇ secreting T cells to CR peptide in normal Korean PBMCs was analyzed.
  • PBMCs from 200 healthy Koreans were collected through a cohort study conducted by the Department of Preventive Medicine, Yonsei University College of Medicine, and this study was approved as a prospective study after review by the Clinical Research Ethics Review Committee. After hearing, only those who agreed to this were included in the study.
  • the present inventors analyzed the sequence of SARS-CoV-2 and five other beta coronavirus proteins (Orf1ab, Spike, Envelope, Membrane, Nucleocapsid) using BLAST (Basic Local Alignment Search Tool).
  • Orf1ab protein was subjected to multiple sequence alignment using the NCBI Multiple Sequence Alignment Viewer (see Table 6).
  • the normalized entropy score was calculated using Shanon's information theoretic entropy of 'Bio3D' (R package).
  • a conserved sequence was defined as a case where the normalized entropy score calculated from 30 consecutive amino acids was 26 or higher.
  • Orf1ab is a non-structural protein (nsp) related to the RNA synthesis of the virus and cannot be a target of the humoral immunity because it is not exposed to the outside of the virion, but cellular immunity The reaction may also specifically respond to these non-structural proteins. Therefore, this research team tried to identify the conserved sequence of SARS-CoV-2, which can be a CD8 T lymphocyte target. 11 conserved sequences were found in the Orf1ab protein of 5 types of beta coronaviruses (see Fig. 1). ).
  • SEQ ID NOs #1-3 are RNA-dependent RNA polymerase (nsp 12), SEQ ID NOs #4-8 are helicase (nsp 13), SEQ ID NOs #9-10 is 3'-5' nuclease (exonuclease, nsp 14), SEQ ID NO: 11 was confirmed to be located in 2'-O-ribose methyltransferase (2'-O-ribose methyltransferases, nsp 16) (Fig. see 1).
  • Example 3 Epitope discovery in the conserved sequence of beta corona virus
  • HLA class I types include HLA-A*02:01, HLA-A*24:02, HLA-B*07:02 and HLA- T cell epitopes presented by B*27:05 were analyzed.
  • NetMHCpan EL 4.0 algorithm http://www.cbs.dtu.dk/services/NetMHC/ was used to predict the epitope sequence for CD8 T lymphocytes from the conserved sequence of the beta coronavirus.
  • the epitope was predicted for a total of four HLA class I types (HLA-A*02:01, HLA-A*24:02, HLA-B*07:02, HLA-B*27:05). In each HLA class I type, the top 2% epitope was judged to be significant.
  • NetMHCpan 4.0 EL algorithm was used for each of the four HLA class I types (HLA-A*02:01, HLA-A*24:02, HLA-B*07:02, HLA-B*27:05) in each conserved sequence.
  • HLA-A*02:01, HLA-A*24:02, HLA-B*07:02, HLA-B*27:05 the sequence of the top 2% is shown in FIGS. 2 to 5 .
  • HLA location epitope sequence 1-log50k (aff) Affinity (nM) Combination level SEQ ID NO: HLA-A*02:01 #One IFVDGVPFV 0.532 157.98 WB SEQ ID NO: 12 HLA-A*02:01 #One FVDGVPFVV 0.785 10.20 SB SEQ ID NO: 13 HLA-A*02:01 #2-3 RLANECAQV 0.633 52.91 WB SEQ ID NO: 14 HLA-A*02:01 #3 SLAIDAYPL 0.719 20.82 SB SEQ ID NO: 15 HLA-A*24:02 #3 LYYQNNVFM 0.409 601.76 WB SEQ ID NO: 16 HLA-A*24:02 #3 YYQNNVFMS 0.382 797.90 WB SEQ ID NO: 17 HLA-B*27:05 #2-2 NRARTVAGV 0.432 467.68 WB SEQ ID NO: 18 HLA-B*27:05 #2-2 ARTVAGVSI
  • Example 1 It was attempted to verify whether normal Korean PBMCs collected in Example 1 exhibit IFN- ⁇ -secreting T-cell immune responses to CR peptides.
  • the CR peptide part as an antigen for stimulating T cells was prepared and used in the form of an overlapping peptide (OLP), and each OLP consists of 15 amino acids, adjacent peptides and 10 Amino acids were designed to overlap and used.
  • OLP overlapping peptide
  • the immune response of CR peptide antigen-specific T cells was confirmed through T cell stimulation using OLP.
  • IFN- ⁇ secretion ability of T cells was measured through ELISPOT assay.
  • ELISPOT assay In order to increase the sensitivity during ELISPOT assay, 500,000 to 700,000 PBMCs per well of a 96-well plate were added and tested.
  • PBMCs showing a strong T-cell immune response to the CR peptide were selected, stimulated with two different epitope peptides with one amino acid, and analyzed by intracellular cytokine staining.
  • the functional affinity of T cell responses to SARS-CoV-2 CR peptides (conserved region peptides) or S peptides (spike peptides) using PBMCs from people who had never been exposed to SARS-CoV-2 ( functional avidity) was measured to confirm suitability as a T cell vaccine.
  • the CR peptide sequence used in the experiment was SEQ ID NOs: 1 to 3, and 15 mer overlapping peptides of three sequences were prepared, respectively, and the experiment was conducted after mixing.
  • PBMCs were obtained by recruiting 9 non-SARS-CoV-2 non-exposed individuals, and the following additional experiments were performed in vitro.
  • the functional affinity of the T cell response was measured by diluting the concentration of the antigenic peptide (serial dilution), stimulating T cells in vitro, measuring the secretion level of TNF cytokines, and then calculating the EC50 (effective concentration 50). (See FIG. 6 ), and the measurement results are compared and shown in FIG. 7 . Referring to the above results, it was confirmed that the functional affinity of T cells for SARS-CoV-2 CR peptide in non-SARS-CoV-2 non-exposed subjects was significantly higher than that of T cell functional affinity for SARS-CoV-2 S peptide.
  • T-cell epitope of the beta-coronavirus conserved region (CR) is used, not only the highly mutated envelope protein but also the non-structural protein with relatively little mutation is recognized as an antigen and used as a vaccine antigen by inducing a T-cell immune response It is possible.

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Abstract

La présente invention concerne un épitope de lymphocyte T pour des peptides de région conservée (CR) qui ont bien été conservés dans le coronavirus bêta, qui est connu pour provoquer une infection chez l'être humain. Lorsqu'il est utilisé, la présente invention peut être utilisée en tant que vaccin à lymphocytes T capable de faire face à un nouveau coronavirus bêta contre lequel des anticorps classiques ne peuvent pas entreprendre une action due à des mutations dans la protéine d'enveloppe du virus.
PCT/KR2021/007379 2020-06-15 2021-06-14 Épitope du nouveau coronavirus et son utilisation WO2021256795A1 (fr)

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KR20190070629A (ko) * 2017-12-13 2019-06-21 전북대학교산학협력단 HBD2(human beta-defensin 2) 또는 상기 유전자와 융합된 메르스 코르나 바이러스의 에피토프 단백질을 포함하는 메르스 코로나바이러스의 예방 또는 치료용 조성물

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