WO2019062853A1 - 一种cd4辅助性t细胞表位融合肽及其疫苗 - Google Patents

一种cd4辅助性t细胞表位融合肽及其疫苗 Download PDF

Info

Publication number
WO2019062853A1
WO2019062853A1 PCT/CN2018/108331 CN2018108331W WO2019062853A1 WO 2019062853 A1 WO2019062853 A1 WO 2019062853A1 CN 2018108331 W CN2018108331 W CN 2018108331W WO 2019062853 A1 WO2019062853 A1 WO 2019062853A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
epitope
seq
fusion peptide
group
Prior art date
Application number
PCT/CN2018/108331
Other languages
English (en)
French (fr)
Inventor
徐建青
黄杨
张晓燕
Original Assignee
苏州工业园区唯可达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州工业园区唯可达生物科技有限公司 filed Critical 苏州工业园区唯可达生物科技有限公司
Priority to JP2020539127A priority Critical patent/JP7146926B2/ja
Priority to EP18861153.7A priority patent/EP3715359A4/en
Priority to US16/650,318 priority patent/US11352411B2/en
Publication of WO2019062853A1 publication Critical patent/WO2019062853A1/zh
Priority to CN201910505212.8A priority patent/CN110205340A/zh
Priority to PCT/CN2019/090877 priority patent/WO2020062930A1/zh
Priority to CN202410006185.0A priority patent/CN117987471A/zh

Links

Images

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001186—MAGE
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the fields of molecular biology and immunology.
  • the present invention relates to a CD4 helper T cell epitope fusion peptide, and more particularly to a vaccine comprising the epitope fusion peptide and use thereof.
  • T helper cells are T cells that play an important role in the immune system, especially in the adaptive immune system. They help the activity of other immune cells by releasing T cell cytokines. These cells help to suppress or regulate the immune response. They are required for the conversion of B cell antibody classes, activation and growth of cytotoxic T cells, and maximal bactericidal activity of phagocytic cells such as macrophages.
  • CD4 + T cells are typically pre-defined as helper T cells in the immune system.
  • CD4 + T cells will aid these cells through a combination of cell-to-cell interactions (eg, CD40 (protein) and CD40L) and cytokines.
  • helper T cells can be seen in HIV, a virus that primarily infects CD4 + T cells.
  • HIV acquired immunodeficiency syndrome
  • loss of functional CD4 + T cells leads to a stage of infection symptoms known as acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • continuous treatment may delay the time of this. If AIDS occurs, treatment can also better manage the AIDS process.
  • lymphopenia that cause loss or dysfunction of CD4 + T cells. These diseases produce similar symptoms, many of which are fatal.
  • an antigenic epitope refers to a chemical group on the surface of an antigen that determines antigen specificity. Epitopes can be recognized by the immune system (especially antibodies, B cells or T cells). The region of an antibody that recognizes an epitope is called a "paratope” or an “antibody determinant.” Although the antigenic epitope usually refers to a part of a substance such as a foreign protein, an epitope recognized by the autoimmune system is classified as an antigenic epitope.
  • the epitopes of protein antigens are classified into two types, conformational epitopes and linear epitopes, depending on their structure and interaction with the paratope.
  • conformational epitope consists of a discrete portion of the antigenic amino acid sequence, such that the interaction between the paratope and the epitope is based on the three dimensional features and shape of the surface, or the tertiary structure of the antigen.
  • Most antigenic epitopes belong to conformational epitopes.
  • a linear epitope is composed of a contiguous sequence of antigenic amino acids, and the interaction with the antigen is based on its primary structure.
  • the T cell epitope consists mainly of a short peptide consisting of 8-17 amino acids and appears on antigen-presenting cells (APCs). This epitope will be associated with major histocompatibility.
  • the complex combines to form a complex and bind to the corresponding T cell epitope receptor, thereby activating T cells and producing a corresponding cellular immune response (Shimonkevitz et al., 1984; Babbitt et al., 1985; Buus et Al., 1986; Townsend and Bodmer, 1989).
  • T-cell epitope represented by the class I major histocompatibility complex is usually composed of a polypeptide of 8 to 11 amino acids in length, and the major tissue phase II is composed.
  • the capacitive complex exhibits a relatively longer T cell epitope, consisting of 13-17 amino acids.
  • a helper T cell epitope refers to a type of T cell epitope in a T cell epitope that is formed by binding to an MHC molecule and which is recognized by a CD4 helper T cell receptor.
  • the Th epitope binds primarily to molecules present on the surface of antigen-presenting cells (APCs) encoded by class II genes of the major histocompatibility complex (MHC).
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • the source of the helper T cell epitope of the peptide immunogen is the carrier protein covalently coupled to the peptide, but the coupling process can introduce other problems, such as modification of the antigenic determinant during the coupling process and targeting of the peptide.
  • Induction of antibodies against vectors at the expense of antibodies Schoutze, MP, Leclerc, C. Jolivet, M. Audibert, F. Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J Immunol. 1985, 135, 2319-2322; DiJohn, D., Torrese, J. Murillo, J. Herrington, DA et al. Effect of priming with carrier on response to conjugate vaccine. The Lancet.
  • Carrier-priming leads to hapten-specific suppression. Nature 285:664; Schutze, MP, Leclerc, C., Jolivet, M., Audibert, F., and Chedid, L. 1985. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines.
  • the immunogen must contain a helper T cell epitope, in addition to the epitope to be recognized by the surface Ig or the receptor present on the cytotoxic T cell. It should be appreciated that these types of epitopes can be very different. For B cell epitopes, conformation is important because B cell receptors bind directly to the native immunogen. In contrast, epitopes recognized by T cells are independent of the conformational integrity of the epitope and consist of a short sequence of about 9 amino acids for CTL and a slightly longer sequence for helper T cells (less limited length) composition.
  • these epitopes can be contained in the binding cleavage of a class I or class II molecule, respectively, and then the complex can bind to the T cell receptor.
  • the binding sites for class II molecules are open at both ends, allowing for as little as 8 amino acid residues reported (Fahrer, AM, Geysen, HM, White, DO, Jackson, DC and Brown, LEAnalysis of the requirements for Class II-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to HEd J. Immunol. 1995.
  • the Th epitope stimulates and activates helper T cells, and accordingly, promotes activation of CD8 T cells and B cells, ultimately enhancing the immune response.
  • the Th epitope in addition to activating an immune response against itself, the Th epitope can also effectively aid in the immune response of other antigens or epitopes associated with it. Therefore, a heterologous strong Th epitope can be fused to the immunogen of interest, whereby the immunogenicity of the immunogen of interest can be improved.
  • PADRE pan HLA DR-binding Epitope
  • Th epitope P2 derived from tetanus toxin is also commonly used to conjugate with the immunogen of interest to enhance immunogenicity (Panina-Bordignon P et al., Eur J Immunol, 1989, 19: 2237). -42; La Rosa, Corinna et al., The Journal of infectious diseases, 2012, 205: 1928-304).
  • the Th epitope used to enhance immunogenicity is generally heterologous, in other words, the vaccine subject does not have a high level of immune response against the Th epitope itself. Therefore, when vaccinating a subject with a strong Th epitope as described above, it is likely that the immune system of the vaccine subject is initially exposed to such a Th epitope, and the activation of the receptor immune system is directed against such Th epitope and purpose.
  • the epitopes of the immunogen are substantially synchronized, and the generation time and number of T cells for such Th epitopes are similar to those of the target immunogen, and thus the effect on the immunogen for the purpose of assistance is thus limited.
  • Th epitopes Especially for weakly immunogenic tumor antigens, the auxiliary role of such Th epitopes is more difficult to play.
  • the direct use of strong Th epitopes can activate tumor antigens, but the level of cellular immune response stimulated is still low and cannot meet the needs of tumor vaccines (Ghaffari-Nazari H et al., PLoS ONE, 2015, 10 (11) ):e0142563).
  • Th epitope strategies are needed to increase the immunogenicity of the immunogen of interest, particularly some weak immunogens, such as tumor antigens.
  • the present invention utilizes a strong Th epitope derived from Cytomegalovirus (CMV) and Influvirus (Flu) to obtain an epitope fusion peptide to increase the immunogenicity of the immunogen of interest.
  • CMV Cytomegalovirus
  • Flu Influvirus
  • epitope fusion peptide refers to a peptide formed by joining together several epitopes.
  • the "important immunogen” refers to an immunogen, such as an antigen, which is an immunologically active substance, preferably a protein, for achieving an immune response.
  • Another object of the present invention is to provide a fusion protein of the epitope fusion peptide and the immunogen of interest.
  • the present invention provides a CD4 helper T cell epitope fusion peptide comprising a cytomegalovirus epitope and/or an influenza virus epitope.
  • the epitope fusion peptide comprises one or more selected from the group consisting of cytomegalovirus epitopes set forth in SEQ ID NO: 1-10, and/or is selected from the group consisting of SEQ ID NO: One or more of the influenza virus epitopes shown in 11-23.
  • the epitope fusion peptide is one or more selected from the group consisting of a cytomegalovirus epitope represented by SEQ ID NO: 1-10, and/or is selected from the group consisting of SEQ ID NO: One or more of the influenza virus epitopes shown in Figures 11-23.
  • the epitope fusion peptide consists of 5 or 10 cytomegalovirus epitopes and/or consists of 8 or 13 influenza virus epitopes, such as the epitope fusion peptides set forth in SEQ ID NO: 34 or 44.
  • the epitope fusion peptide consists of 13 influenza virus epitopes, such as the epitope fusion peptide set forth in SEQ ID NO:48.
  • the epitope fusion peptide induces a humoral or cellular immune response.
  • the present invention also provides a fusion protein of the epitope fusion peptide and the immunogen of interest.
  • the invention also provides a polynucleotide encoding the epitope fusion peptide and/or the fusion protein.
  • the immunogen of interest is any one or more immunogens.
  • the immunogen of interest is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a part of a virus or virus, a bacterium, a parasite or other intact microorganism.
  • the antigen is a tumor antigen or an infection associated antigen.
  • the tumor antigen is selected from one or more of a lung cancer antigen, a testicular cancer antigen, a melanoma antigen, a liver cancer antigen, a breast cancer antigen, or a prostate cancer antigen.
  • the tumor antigen is selected from one or more of a LAGE antigen, a MAGE antigen or a NY-ESO-1 antigen.
  • the LAGE antigen is LAGE-1 and the MAGE antigen is MAGE-A3.
  • the tumor antigen comprises LAGE-1, MAGE-A3 and NY-ESO-1.
  • the amino acid sequence of LAGE-1 is set forth in SEQ ID NO: 24, the amino acid sequence of MAGE-A3 is set forth in SEQ ID NO: 25, and the amino acid sequence of NY-ESO-1 is SEQ ID NO: NO:26.
  • the infection-associated antigen is selected from one or more of an HIV antigen, an influenza virus antigen or an HBV antigen.
  • the fusion protein is as set forth in one of SEQ ID NOs: 55-58.
  • Another object of the present invention is to provide an immunological composition
  • an immunological composition comprising a therapeutically effective amount of an epitope fusion peptide, a fusion protein and/or a polynucleotide according to the present invention, and a pharmaceutically acceptable carrier.
  • the immunological composition is a vaccine.
  • Another object of the present invention is to provide a kit comprising an epitope fusion peptide, a fusion protein, a polynucleotide and/or an immunological composition according to the present invention, and instructions for its use.
  • the invention also provides the use of an epitope fusion peptide, fusion protein, polynucleotide and/or immunological composition according to the invention in the manufacture of a medicament or vaccine for increasing the immunogenicity of an immunogen of interest.
  • the present invention also provides a method for increasing the immunogenicity of an immunogen of interest using an epitope fusion peptide according to the present invention, comprising a CD4 helper T cell epitope having a strong immune response in a vaccine subject or population.
  • a fusion protein formed by fusion with an immunogen of interest. The method is specifically as follows:
  • the above epitope is fused to form an epitope fusion peptide
  • the epitope fusion peptide is fused with the immunogen of interest to form a fusion protein
  • the fusion protein is expressed and made into a vaccine
  • the expression vector can be in the form of a DNA vaccine vector.
  • the above vaccine is inoculated to a vaccine subject, and an appropriate adjuvant such as incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant or the like may be selected at the time of inoculation.
  • an appropriate adjuvant such as incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant or the like may be selected at the time of inoculation.
  • step (1) of the method of use further comprises the step of examining the MHC phenotype of the vaccine subject.
  • examining the MHC phenotype of the vaccine subject comprises examining the MHC class II gene subtype of the vaccine subject.
  • the invention also provides a method of treating or preventing a condition in a subject in need thereof, comprising administering a therapeutically effective amount of an epitope fusion peptide, fusion protein, immunological composition and/or polynucleotide of the invention.
  • the condition is selected from one or more of a malignant tumor, a bacterial and a viral chronic infection.
  • the malignant tumor is breast cancer or colon cancer.
  • the DNA vaccine vector is primed, the protein vaccine vector is boosted, and more preferably, the pVKD1.0-CI-LMNB DNA vaccine is primed and the LMNB-I13 protein is boosted.
  • the epitope fusion peptide provided by the invention can greatly improve the cellular immune response level of the target immunogen, especially the weak immunogen, and overcome the immune tolerance of the immune system to the antigen, especially to the tumor antigen or the infection-associated antigen.
  • Figure 1 and Figure 2 are plasmid maps and double restriction enzyme identification maps of the DNA vaccine vector pVKD1.0-hLMN carrying the LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences, respectively.
  • Figure 3 and Figure 4 are plasmid maps and double restriction enzyme digestion of DNA vaccine vector pVKD1.0-hLMN-CTB with LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin B subunit coding sequence, respectively.
  • Figure 5 and Figure 6 are plasmid maps and double restriction enzyme maps of the DNA vaccine vector pVKD1.0-CI with CMV and influenza virus CD4 epitope coding sequences, respectively.
  • Figure 7 and Figure 8 are DNA vaccine vectors pVKD1.0- with LAGE-1, MAGE-A3 and NY-ESO-1 antigens and cholera toxin B subunits, and coding sequences derived from CMV and influenza virus CD4 epitopes, respectively. Plasmid map and double enzyme digestion map of CI-LMNB.
  • Figure 9 and Figure 10 are plasmid maps and double restriction enzyme maps of the prokaryotic vector pET-30a(+)-LMN carrying the LAGE-1, MAGE-A3 and NY-ESO-1 antigen coding sequences, respectively.
  • Figure 11 and Figure 12 are plasmid maps and double enzymes of the prokaryotic vector pET-30a(+)-LMN-CTB with LAGE-1, MAGE-A3 and NY-ESO-1 antigens and the coding sequence of cholera toxin B subunit, respectively. Cut the identification map.
  • Figure 13 and Figure 14 are plasmid maps and double restriction enzyme maps of the prokaryotic vector pET-30a(+)-CMV Th derived from the CMV epitope coding sequence, respectively.
  • Figure 15 and Figure 16 are prokaryotic vectors pET-30a(+)-CMV10-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • the plasmid map and the double restriction enzyme identification map are prokaryotic vectors pET-30a(+)-CMV10-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • the plasmid map and the double restriction enzyme identification map are prokaryotic vectors pET-30a(+)-CMV10-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • Figure 17 and Figure 18 are plasmid maps and double restriction enzyme maps of the prokaryotic vector pET-30a(+)-CMV Th derived from the influenza virus epitope coding sequence, respectively.
  • Figure 19 and Figure 20 are prokaryotic vectors pET-30a(+)-Influ8-, respectively, derived from the influenza virus epitope and the LAGE-1, MAGE-A3 and NY-ESO-1 antigens and the cholera toxin B subunit coding sequence. Plasmid map and double restriction map of LMNB.
  • Figure 21 and Figure 22 are prokaryotic vectors pET-30a(+)-Influ13-, respectively, derived from the influenza virus epitope and the LAGE-1, MAGE-A3 and NY-ESO-1 antigens and the cholera toxin B subunit coding sequence. Plasmid map and double restriction map of LMNB.
  • Figure 23 shows the results of cellular immune response assays in animal immunization experiments.
  • Figure 24 and Figure 25 are prokaryotic vectors pET-30a(+)-CMV5-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • the plasmid map and the double restriction enzyme identification map are prokaryotic vectors pET-30a(+)-CMV5-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • the plasmid map and the double restriction enzyme identification map are prokaryotic vectors pET-30a(+)-CMV5-LMNB with coding sequences derived from CMV epitopes and LAGE-1, MAGE-A3 and NY-ESO-1 antigens, and cholera toxin B subunit, respectively.
  • Figure 26 is a graph showing the results of cellular immune response test in the animal immunization experiment in Example 12.
  • Figure 27 is a graph showing tumor growth in mice of Example 13.
  • Figure 28 and Figure 29 are the tumor-free survival status and overall survival status of the mice in Example 13, respectively.
  • Figure 30 is a graph showing the tumor growth of each treatment group in a 4T1-hNY-ESO-1 mouse tumor model.
  • Figure 31 is a graph showing the tumor growth of each treatment group in a CT26-hLAGE-1 mouse tumor model.
  • the LAGE-1, MAGE-A3 and NY-ESO-1 amino acid sequences are shown in SEQ ID NOS: 24-26, respectively.
  • the above amino acid sequence of the antigen was optimized by the online codon optimization software (http://www.jcat.de/) into a nucleotide sequence preferred by the mammalian codon, as shown in SEQ ID NOs: 27-29, respectively.
  • the DNA vaccine vector pVKD1.0-hLMN (plasmid map shown in Figure 1), which can express the fusion protein antigen, was constructed and identified by sequencing and then stored in the library.
  • the vector pVKD1.0-hLMN was identified using the restriction enzymes Sal I and BamH I (the enzyme digestion system is shown in Table 1), and the restriction enzyme verification map is shown in Fig. 2 .
  • Enzyme digestion system volume Plasmid pVKD1.0-hLMN 3 ⁇ L, about 1 ⁇ g Sal I ( ⁇ , part number 1080A) 1 ⁇ L BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the mammalian codon-optimized sequence (SEQ ID NO: 31) of the amino acid sequence (SEQ ID NO: 30) of Cholera toxin subunit B (CTB) and its eukaryotic expression vector pVKD1.0-CTB Suzhou Industrial Park is only available from Biotech Co., Ltd.
  • CTB Cholera toxin subunit B
  • pVKD1.0-CTB eukaryotic expression vector
  • primers were designed (see Table 2).
  • the CTB gene fragment was amplified by PCR, and then the corresponding fragment was recovered by gel.
  • the CTB fragment was inserted into the linearized vector pVKD1.0-hLMN by homologous recombination.
  • the DNA vaccine vector pVKD1.0-hLMN-CTB (plasmid map shown in Figure 3) was constructed and identified by sequencing and then stored in the library.
  • the vector pVKD1.0-hLMN-CTB was identified using the restriction enzymes Sal I and BamH I (the enzyme digestion system is shown in Table 3), and the restriction enzyme verification map is shown in Fig. 4 .
  • Enzyme digestion system volume Plasmid pVKD1.0-hLMN-CTB 3 ⁇ L, about 1 ⁇ g Sal I ( ⁇ , part number 1080A) 1 ⁇ L BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the strong Th epitope of CMV includes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104; influenza virus Strong Th epitopes include HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127, and M1-210.
  • the epitopes selected in Table 4 cover most subtypes of MHC class II molecules in the population and also cover mouse MHC II subtype molecules. Then, the selected epitopes pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221 are connected in series.
  • An epitope fusion peptide of CMV virus and influenza virus is formed, the amino acid sequence of which is shown in SEQ ID NO: 34, and the epitope fusion peptide is optimized by mammalian codon, and the nucleic acid sequence is shown as SEQ ID NO:35.
  • the nucleic acid sequence was synthesized by Suzhou Yuxun Biotechnology Co., Ltd., and then inserted into the DNA vaccine vector pVKD1.0 (Suzhou Industrial Park Weida Biotechnology Co., Ltd.) by molecular biological methods well known in the art.
  • the vector pVKD1.0-CI (plasmid map shown in Figure 5) was correctly identified by sequencing and then stored in the library.
  • the vector pVKD1.0-CI was identified using restriction endonucleases Pst I and Bgl II (the enzyme digestion system is shown in Table 5), and the enzyme digestion verification spectrum is shown in Fig. 6.
  • Epitope name source Amino acid sequence Pp65-11 CMV LLQTGIHVRVSQPSL (SEQ ID NO: 1) Pp65-71 CMV IIKPGKISHIMLDVA (SEQ ID NO: 2) Pp65-92 CMV EHPTFTSQYRIQGKL (SEQ ID NO: 3) Pp65-123 CMV AGILARNLVPMVATV (SEQ ID NO: 4) Pp65-128 CMV KYQEFFWDANDIYRI (SEQ ID NO: 5) Pp65-57 CMV KVYLESFCEDVPSGK (SEQ ID NO: 6) Pp65-62 CMV TLGSDVEEDLTMTRN (SEQ ID NO: 7) Pp65-30 CMV PLKMLNIPSINVHHY (SEQ ID NO: 8) Pp65-112 CMV ACTSGVMTRGRLKAE (SEQ ID NO: 9) Pp65-104 CMV TERKTPRVTGGGAMA (SEQ ID NO: 10) HA203 Influ NQRALYH
  • HA375 Influ SGYAADQKSTQNAINGITNKVN (SEQ ID NO: 15)
  • NP24 Influ EIRASVGKMIDGIGRFYI (SEQ ID NO: 16)
  • NP95 Influ PIYRRVDGKWMRELVLY (SEQ ID NO: 17)
  • NP221 Influ RMCNILKGKFQTAAQRAM (SEQ ID NO: 18)
  • HA434 Influ IWTYNAELLVLLENERT SEQ ID NO: 19
  • HA440 Influ ELLVLLENERTLDFHDS (SEQ ID NO: 20)
  • Enzyme digestion system volume Plasmid pVKD1.0-CI 3 ⁇ L, about 1 ⁇ g Pst I ( ⁇ , Item No. 1073A) 1 ⁇ L Bgl II ( ⁇ , Item No. 1021A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • Enzyme digestion system volume Plasmid pVKD1.0-CI-LMNB 3 ⁇ L, about 1 ⁇ g Bam HI (Bao Bio, Item No. 1010A) 1 ⁇ L EcoR V ( ⁇ , Item No. 1042A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the LAGE-1, MAGE-A3 and NY-ESO-1 amino acid sequences are shown in SEQ ID NOS: 24-26, respectively.
  • the amino acid sequence of the antigen is optimized by the online codon optimization software (http://www.jcat.de/) into a nucleotide sequence of codon usage preference of E. coli, and the nucleotide sequence thereof is SEQ ID NO: 38, respectively. -40 is shown. After synthesis by Suzhou Yuxun Biotechnology Co., Ltd., it was inserted into the prokaryotic expression vector pET-30a(+) (Novagen, Cat. No. 69909) by the molecular biology method well known in the art.
  • the prokaryotic expression construct pET-30a(+)-LMN (plasmid map shown in Figure 9) was constructed and identified by sequencing and then stored in the library.
  • the vector pET-30a(+)-LMN was identified by restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 8), and the restriction enzyme verification map is shown in Fig. 10.
  • Enzyme digestion system volume Plasmid pET-30a(+)-LMN 3 ⁇ L, about 1 ⁇ g Nco I ( ⁇ , Item No. 1160A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the cholera toxin B subunit CTB amino acid sequence (SEQ ID NO: 30) and its prokaryotic codon optimized nucleic acid sequence (SEQ ID NO: 41) were all provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd. Primers were designed (see Table 9), and pET-30a(+)-CTB (Suzhou Industrial Park Weida Biotechnology Co., Ltd.) was used as a template to amplify nucleic acid fragments containing CTB coding sequences by PCR. For details, see Ex Taq. Enzyme (Bao Bio, Item No. RR001B) reagent instructions.
  • nucleic acid fragment was inserted into pET-30a(+)-LMN vector by homologous recombination to construct pET-30a(+)-LMN-CTB vector (plasmid map as shown in Figure 11).
  • Library. The vector pET-30a(+)-LMN-CTB was identified by restriction endonucleases Nco I and Xho I (the enzyme digestion system is shown in Table 10), and the restriction enzyme verification map is shown in Fig. 12.
  • Enzyme digestion system volume Plasmid pET-30a(+)-LMN-CTB 3 ⁇ L, about 1 ⁇ g Nco I ( ⁇ , Item No. 1160A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • Th epitopes derived from CMV, pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112, and Pp65-104, which are ligated together, and the amino acid sequence consisting of SEQ ID NO: 44, wherein the sequence "EFELRRQ" in SEQ ID NO: 44 is caused by the introduction of a cleavage site, It is a commonly used technology for fusion construction.
  • the Th epitope amino acid sequence was optimized by the online codon optimization software (http://www.jcat.de/) into the nucleotide sequence of the E.
  • coli codon usage preference SEQ ID NO: 45
  • the Biotechnology Co., Ltd. was inserted into the prokaryotic expression vector pET-30a (+) (Novagen, Cat. No. 69909) by the molecular biology method well-known in the art and inserted into the multiple cloning site Nco I and Xho I.
  • the prokaryotic expression construct pET-30a(+)-CMV Th (plasmid map shown in Figure 13) expressing the fusion protein antigen was identified and sequenced.
  • the vector pET-30a(+)-CMV Th was identified by restriction endonucleases Mlu I and Xho I (the enzyme digestion system is shown in Table 11), and the restriction enzyme verification map is shown in Fig. 14.
  • CMV Th1 contains 5 CMV Th epitopes, which are formed by pp65-11, pp65-71, pp65-92, pp65-123, and pp65-128 in series
  • CMV Th2 contains 5 CMVs.
  • the Th epitope consists of pp65-57, pp65-62, pp65-30, pp65-112 and pp65-104.
  • Three restriction enzymes such as EcoR I, Sac I and Sal I were introduced between CMV Th1 and CMV Th2. Site.
  • Enzyme digestion system volume Plasmid pET-30a(+)-CMV Th 3 ⁇ L, about 1 ⁇ g Mlu I ( ⁇ , Item No. 1071A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the primers were designed (see Table 12), and the pET-30a(+)-LMN-CTB in Example 5 was used as a template to amplify the nucleic acid fragment containing the LMN-CTB coding sequence by PCR. For details, see Ex Taq Enzyme (Po Biological, item number RR001B) Reagent instructions. This nucleic acid fragment was then inserted into Notp and Xho I on the pET-30a(+)-CMV Th vector of Example 6 by molecular biology methods well known in the art to construct pET-30a(+)-CMV10. - LMNB vector (plasmid map is shown in Figure 15, and sequenced and identified correctly.
  • the vector pET-30a(+)-CMV10-LMNB was identified by restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 13).
  • the restriction enzyme digestion map is shown in Figure 16.
  • pET-30a(+)-CMV10-LMNB contains the CMV Th1 and CMV Th2 fragments, i.e., the vector contains all 10 CMV Th epitopes in Table 4.
  • the epitopes are pp65-11, pp65-71, pp65-92, pp65-123, pp65-128, pp65-57, pp65-62, pp65-30, pp65-112, and pp65-104.
  • Primer sequence 6F (SEQ ID NO: 46) GCGCGGCCGCGACGACAAGGCCATGGCT 6R (SEQ ID NO: 47) GCCTCGAGGTTAGCCATAGAGATAGC
  • Enzyme digestion system volume Plasmid pET-30a(+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • Th epitopes derived from influenza virus HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, NP221, HA434, HA440, NP324, M1-127 and M1-210, were selected. They are ligated together and the amino acid sequence consisting is shown in SEQ ID NO:48. The amino acid sequence containing the influenza virus Th epitope was optimized to the nucleotide sequence of the E.
  • the multiple cloning sites Nco I and Xho I on the prokaryotic expression vector pET-30a(+) were inserted by molecular biology methods well known in the art.
  • the prokaryotic expression construct pET-30a(+)-Influ Th (plasmid map shown in Figure 17), which can express the fusion protein antigen, was constructed and identified by sequencing and then stored in the library.
  • the vector pET-30a(+)-Influ Th was identified with the restriction enzymes Nco I and Xho I (the enzyme digestion system is shown in Table 14), and the restriction enzyme verification map is shown in Fig. 18.
  • Influ Th1 contains Th epitopes of eight influenza viruses, which are composed of HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95 and NP221 in series, and Influ Th2 contains 5 influenzaes.
  • the Th epitope of the virus is composed of HA434, HA440, NP324, M1-127 and M1-210, and three restriction enzyme cleavage sites such as EcoR I, Sac I and Sal I are introduced between Influ Th1 and Influ Th2.
  • Enzyme digestion system volume Plasmid pET-30a(+)-Influ Th 3 ⁇ L, about 1 ⁇ g Nco I ( ⁇ , Item No. 1160A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the primers were designed (see Table 15), and the pET-30a(+)-LMN-CTB in Example 5 was used as a template to amplify the nucleic acid fragment containing the LMN-CTB coding sequence by PCR. For details, see Ex Taq Enzyme (Po Biological, item number RR001B) Reagent instructions. The nucleic acid fragment was then inserted between Not I and Sal I on the pET-30a(+)-Influ Th vector of Example 7 by molecular biology methods well known in the art to construct pET-30a(+)-Influ8. -LMNB vector (containing 8 influenza virus Th epitopes, plasmid map as shown in Figure 19), identified by sequencing and then stored in the library.
  • -LMNB vector containing 8 influenza virus Th epitopes, plasmid map as shown in Figure 19
  • the vector pET-30a(+)-Influ8-LMNB was identified by restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 16), and the restriction enzyme verification map is shown in Fig. 20.
  • the pET-30a(+)-Influ8-LMNB vector contains the Influ Th1 fragment, which contains HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95 and NP221 including Table 4. 8 influenza virus Th epitopes.
  • Primer sequence 7F1 (SEQ ID NO: 50) GCGCGGCCGCGTTAGCCATAGAGATAGC 7R1 (SEQ ID NO: 51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
  • Enzyme digestion system volume Plasmid pET-30a(+)-Influ8-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the primers were designed (see Table 17), and the pET-30a(+)-LMN-CTB in Example 5 was used as a template to amplify the nucleic acid fragment containing the LMN-CTB coding sequence by PCR. For details, see Ex Taq Enzyme (Po Biological, item number RR001B) Reagent instructions.
  • the nucleic acid fragment was then inserted into Not-I and Xho I on the pET-30a(+)-Influ Th vector of Example 6 by molecular biology methods well known in the art to construct pET-30a(+)- Influ13-LMNB vector (containing 13 influenza virus Th epitopes, plasmid map as shown in Figure 21), was identified by sequencing and then stored in the library.
  • the vector pET-30a(+)-CMV10-LMNB was identified by restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 18), and the restriction enzyme verification map is shown in Fig.
  • the pET-30a(+)-Influ13-LMNB vector contains Influ Th1 and Influ Th2 fragments, including HA203, NP438, NS1-84, M1-181, HA375, NP24, NP95, and Eight influenza virus Th epitopes including NP221 and five influenza virus Th epitopes including HA434, HA440, NP324, M1-127 and M1-210 in Table 4.
  • the vector included a total of all 13 influenza virus Th epitopes in Table 4.
  • Primer sequence 7F2 (SEQ ID NO: 52) GCCTCGAGGTTAGCCATAGAGATAGCA 7R2 (SEQ ID NO: 53) GCGCGGCCGCGACGACAAGGCCATGGCTATG
  • Enzyme digestion system volume Plasmid pET-30a(+)-Influ13-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the prokaryotic expression vector pET-30a(+)-LMN constructed in Example 4 the prokaryotic expression vector pET-30a(+)-LMN-CTB constructed in Example 5, and the prokaryotic expression vector pET constructed in Example 6 were respectively prepared.
  • LMNB prokaryotic expression vectors pET-30a(+)-Influ8-LMNB and pET-30a(+)-Influ13- constructed in Example 7 LMNB was transformed into BL21(DE3) competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., item number CB105, see transformational method for competent cell instructions), prepared according to the "PET System Manual" (TB055 8th Edition02/99, Novagen) Recombinant protein LMN (the amino acid sequence of which is shown in SEQ ID NO: 59), LMNB (the amino acid sequence of which is shown by SEQ ID NO: 54), LMNB-C10 (the amino acid sequence of which is shown by SEQ ID NO: 58), LMNB -I8 (the amino acid sequence of which is shown in SEQ ID NO: 55) and LMNB-13 (the amino acid sequence of which is shown in SEQ ID NO:
  • the concentration of the recombinant protein prepared by the BCA method is 1 mg/mL.
  • the recombinant protein endotoxin content ⁇ 1EU/mg was determined by gel method (Xiamen ⁇ reagent experimental plant Co., Ltd., item number G011000), which meets the requirements of animal experiments.
  • the vaccine information prepared in Examples 2, 3 and Example 8 is shown in Table 19.
  • the DNA vaccine vector pVKD1.0 was provided by Suzhou Industrial Park Weiyue Co., Ltd., DNA vaccine pVKD1.0-NP (expressed from strain A/Shanghai/02/2013 (H7N9)) influenza antigen NP (NCBI reference sequence: YP_009118476 .1) Provided by Suichang Company of Suzhou Industrial Park, protein vaccine VP1 (Enterovirus 71 type VP1 protein, see Chinese patent application 201310088364.5) was provided by Suzhou Industrial Park only.
  • mice Sixteen female BAL B/c mice, 6-8 weeks old, were purchased from the Animal Experimental Center of Suzhou University and were housed in the SPF animal room of the Animal Experimental Center of Suzhou University. Experimental animal grouping and vaccination planning are shown in Table 20. All DNA vaccines were injected into the anterior muscle of the calf, 100 ⁇ g/head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously 10 ⁇ g/head. Two weeks after the last immunization, the mice were sacrificed and serum and spleen cells were collected for enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA).
  • ELISPOT enzyme-linked immunospot assay
  • ELISA enzyme-linked immunosorbent assay
  • the mouse IFN- ⁇ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the method is described in the BD IFN- ⁇ ELISPOT kit.
  • the stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP), which was synthesized by Shanghai Kep peptide Biotechnology Co., Ltd., and the final concentration was 10 ⁇ g/mL.
  • the positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin (inomysin) were purchased from Sigma, USA.
  • the method of ELISA is well known to those skilled in the art and is briefly described below.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory. Reorganized LMN and NY-ESO-1 are provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd.
  • the protein was coated with NaHCO 3 buffer (pH 9.6) at 4 ° C overnight at a coating concentration of 10 ⁇ g/mL. After blocking with phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) for 30 minutes at 37 ° C, and washing with phosphate buffer (PBST) containing 0.5% Tween 20 for 5 times, The mouse serum was incubated for 1 hour at room temperature with an initial dilution of 1:100.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Tween 20 phosphate buffer
  • the primers were designed (see Table 21), and the pET-30a(+)-LMN-CTB in Example 5 was used as a template to amplify the nucleic acid fragment containing the LMN-CTB coding sequence by PCR. For details, see Ex Taq Enzyme (Po Biological, item number RR001B) Reagent instructions. The nucleic acid fragment was then inserted into Notp and Sal I on the pET-30a(+)-CMV Th vector of Example 6 by molecular biology methods well known in the art to construct pET-30a(+)-CMV5. - LMNB vector (plasmid map as shown in Figure 24), identified by sequencing and then stored in the library.
  • the vector pET-30a(+)-CMV5-LMNB was identified by restriction endonucleases BamH I and Xho I (the enzyme digestion system is shown in Table 22), and the restriction enzyme verification map is shown in Fig. 25.
  • pET-30a(+)-CMV5-LMNB contained a CMV Th1 fragment, i.e., the vector contained the first five CMV Th epitopes in Table 4. These epitopes are pp65-11, pp65-71, pp65-92, pp65-123, and pp65-128.
  • Primer sequence 7F1 (SEQ ID NO: 50) GCGCGGCCGCGTTAGCCATAGAGATAGC 7R1 (SEQ ID NO: 51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
  • Enzyme digestion system volume Plasmid pET-30a(+)-CMV10-LMNB 3 ⁇ L, about 1 ⁇ g BamH I (Bao Bio, Item No. 1010A) 1 ⁇ L Xho I ( ⁇ , Item No. 1094A) 1 ⁇ L Digestion buffer 1 ⁇ L ddH 2 O Supplement to 10 ⁇ L
  • the prokaryotic expression vector pET-30a(+)-CMV5-LMNB constructed in Example 10 was transformed into BL21(DE3) competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. CB105, as described in Example 8).
  • BL21(DE3) competent cells Teiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. CB105, as described in Example 8.
  • the transformation method refer to the Competent Cell Instructions
  • prepare the recombinant protein LMNB-C5 (the amino acid sequence is shown in SEQ ID NO: 57) according to the "PET System Manual" (TB055 8th Edition 02/99, Novagen). , -80 ° C to save.
  • the concentration of the recombinant protein prepared by the BCA method is 1 mg/mL.
  • the recombinant protein endotoxin content ⁇ 1EU/mg was determined by gel method (Xiamen ⁇ reagent experimental plant Co., Ltd., item number G011000), which meets the requirements of animal experiments.
  • the vaccine information is shown in Table 19.
  • the DNA vaccine pVKD1.0-CI (Example 3) was provided by Suzhou Industrial Park Weida.
  • mice Twenty female BAL B/c mice, 6-8 weeks old, were purchased from the Animal Experimental Center of Suzhou University and housed in the SPF animal room of the Animal Experimental Center of Suzhou University. Experimental animal grouping and vaccination planning are shown in Table 23. All DNA vaccines were injected into the anterior muscle of the calf, 100 ⁇ g/head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously 10 ⁇ g/head. Two weeks after the last immunization, the mice were sacrificed and serum and spleen cells were collected for enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA).
  • ELISPOT enzyme-linked immunospot assay
  • ELISA enzyme-linked immunosorbent assay
  • the mouse IFN- ⁇ ELISPOT kit was purchased from BD, USA (Cat. No. 551083), and the method is described in the BD IFN- ⁇ ELISPOT kit.
  • the stimulating peptide was NY-ESO-1 41# peptide (WITQCFLPVFLAQPP), which was synthesized by Shanghai Kep Biotech Co., Ltd., and the final concentration was 10 ⁇ g/mL.
  • the positive stimuli phorbol-12-myristate-13-acetate (PMA) and ionomycin (inomysin) were purchased from Sigma, USA.
  • the method of ELISA is well known to those skilled in the art and is briefly described below.
  • the 96-well microtiter plate was purchased from Jianghai Glass Instrument Factory. Reorganized LMN and NY-ESO-1 are provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd.
  • the protein was coated with NaHCO 3 buffer (pH 9.6) at 4 ° C overnight at a coating concentration of 10 ⁇ g/mL. After blocking with phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) for 30 minutes at 37 ° C, and washing with phosphate buffer (PBST) containing 0.5% Tween 20 for 5 times, The mouse serum was incubated for 1 hour at room temperature with an initial dilution of 1:100.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Tween 20 phosphate buffer
  • Fig. 26 The results of the cellular immune response test are shown in Fig. 26.
  • the priming pVKD1.0-CI-LMNB DNA vaccine, LMNB-C5 protein boost (ie, group C in Example 11) and LMNB-C10 protein boost (ie, group D in Example 11) had the best immune effect.
  • group C the inclusion of 5 Th epitopes (group C) and 10 Th epitopes (group D) of CMV virus can significantly increase the level of cellular immune response of weak immunogens.
  • the vaccine information prepared in Examples 2, 3 and Example 8 is shown in Table 19.
  • the DNA vaccine vector pVKD1.0 was provided by Suzhou Industrial Park Weiyue Co., Ltd., DNA vaccine pVKD1.0-NP (expressed from strain A/Shanghai/02/2013 (H7N9)) influenza antigen NP (NCBI reference sequence: YP_009118476 .1) Provided by Suzhou Industrial Park Wei Da Biotechnology Co., Ltd., protein vaccine VP1 (Enterovirus 71 VP1 protein, see Chinese patent application 201310088364.5) was provided by Suzhou Industrial Park Wei Da Biotechnology Co., Ltd.
  • mice Sixty female BAL B/c mice, 6-8 weeks old, were purchased from the Animal Experimental Center of Suzhou University and kept in the SPF animal room of the Animal Experimental Center of Suzhou University. Experimental animal grouping and vaccination planning are shown in Table 24. All DNA vaccines were injected into the anterior muscle of the calf, 100 ⁇ g/head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously 10 ⁇ g/head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice The tumor growth of each group of immunized mice is shown in Fig. 27.
  • the control group (group A) mice all showed tumors on the 14th day after the tumor attack (ie, after tumor inoculation), and rapidly grew.
  • the tumor growth of each immunized group lags behind the control group.
  • the LMNB-I13 booster group (group D) and the LMNB-I13 and LMNB-C10 mixed booster group (group E) have the slowest tumor growth, so These two groups of vaccines work best.
  • mice tumor-free survival analysis was also performed, and the results are shown in Fig. 28.
  • the median number of tumor-free survival (TFS) of the control group A mice was 14 days.
  • the tumor-free survival of the mice in each vaccine immunization group was significantly higher than that in the control group. This indicates that all vaccines can improve the tumor-free survival of mice after immunization.
  • the D group with the I13 epitope fusion peptide, the E group and the F group with the I13 and C10 epitope fusion peptides had the best effect, and the highest doubling of the tumor-free survival of the mice.
  • the vaccine group with the I13 epitope fusion peptide significantly improved tumor-free survival and increased tumor-free survival by approximately 40% compared to the conventional vaccine group (Group B). It is indicated that the inclusion of 13 Th epitopes or 10 CMV Th epitopes of influenza virus can greatly improve the tumor protection effect of tumor vaccine.
  • mice overall survival analysis was also performed, and the results are shown in FIG. Among them, the median overall survival (OS) of the control group A mice was 35 days. The overall survival of the mice in each vaccine immunization group was significantly higher than that in the control group, indicating that all vaccines can improve the survival of mice after immunization. Among them, the D group with the I13 epitope fusion peptide, the E group and the F group with the I13 and C10 epitope fusion peptides had the best effect, and the overall survival increased by 83%.
  • OS median overall survival
  • the vaccine group with the I13 epitope fusion peptide significantly increased the tumor-free survival with a maximum increase of 28%, indicating that the influenza virus contained 13 Th Epitopes or 10 CMV Th epitopes can greatly enhance the tumor protection effect of tumor vaccines.
  • Example 9 See Example 9 for the vaccine involved.
  • Experimental animal grouping and vaccination planning are shown in Table 25. All DNA vaccines were injected into the anterior muscle of the calf, 100 ⁇ g/head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously 10 ⁇ g/head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice The tumor growth of each group of immunized mice is shown in FIG. Among them, the control group (group A) mice all showed tumors on the 14th day after the tumor attack (ie, after tumor inoculation), and rapidly grew. Compared with the untreated control group (group A), the LMNB-I13 protein vaccine treatment group (group C) had the slowest tumor growth. Moreover, on day 22 after tumor challenge in mice, the tumor size of the mice treated with LMNB-I13 protein vaccine was significantly smaller than that of the control group (group A), and there was a significant difference in tumor size between the two groups until day 30. By day 35, tumor growth began to accelerate in group C mice, possibly related to the cessation of LMNB-I13 protein vaccine. This result indicates that the LMNB-I13 protein vaccine can inhibit tumor growth in mice.
  • Example 9 for the vaccine involved.
  • Experimental animal grouping and vaccination planning are shown in Table 26. All DNA vaccines were injected into the anterior muscle of the calf, 100 ⁇ g/head. All protein vaccines were fully emulsified with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and injected subcutaneously 10 ⁇ g/head.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice Two weeks after the last immunization, the mice were subcutaneously inoculated with tumor cell CT26-hLAGE-1 stably transfected cell line (provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd.), and the inoculation dose was 1 ⁇ 10 5 cells/only.
  • mice The tumor growth of each group of immunized mice is shown in Figure 31. Since the untreated control group (group A) mice failed to successfully inoculate the tumor after tumor attack (ie, after tumor inoculation), they were not included in the analysis and compared. Parallel vaccine control group (group B) and LMNB-I13 treatment group (group C) mice. Compared with B, the tumor growth of mice in group C was slower, and on the 22nd day after tumor attack in mice, the tumor size of mice treated with LMNB-I13 protein vaccine was significantly smaller than that of the parallel vaccine control group (group B) until the first At 30 days, there was also a significant difference in tumor size between the two groups.
  • mice in the C group began to accelerate in the late stage of tumor growth, which may be related to the cessation of vaccination with the LMNB-I13 protein vaccine. This result indicates that the LMNB-I13 protein vaccine can inhibit tumor growth in mice.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Pulmonology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种CD4辅助性T细胞表位融合肽、其编码核酸以及包含其的免疫组合物。所述表位融合肽包含巨细胞病毒表位和/或流感病毒表位。所述表位融合肽能大幅度提高目的免疫原，尤其是弱免疫原的细胞免疫应答水平，是克服免疫系统对抗原免疫耐受性，尤其是肿瘤抗原或感染相关抗原的有效手段，适用于高效增强疫苗的效力。

Description

一种CD4辅助性T细胞表位融合肽及其疫苗 技术领域
本发明属于分子生物学和免疫学领域。具体地，本发明涉及一种CD4辅助性T细胞表位融合肽，尤其涉及包含该表位融合肽的疫苗及其应用。
背景技术
T辅助细胞(Th细胞)是一种在免疫系统中起重要作用的T细胞，特别是在适应性免疫系统中。它们通过释放T细胞细胞因子来帮助其他免疫细胞的活性。这些细胞有助于抑制或调节免疫反应。它们在B细胞抗体类别的转换、细胞毒性T细胞的活化和生长以及吞噬细胞如巨噬细胞的杀菌活性最大化中是必需的。
成熟Th细胞表达蛋白CD4，称为CD4 +T细胞。通常将这样的CD4 +T细胞作为免疫系统内的辅助T细胞进行预定义的处理。例如，当抗原呈递细胞在II类MHC上表达抗原时，CD4 +细胞将通过细胞与细胞相互作用的组合(例如CD40(蛋白质)和CD40L)和细胞因子来帮助这些细胞。
可以从HIV(一种主要感染CD4 +T细胞的病毒)中看到辅助T细胞的重要性。在艾滋病毒感染的晚期阶段，功能性CD4 +T细胞的丧失导致被称为获得性免疫缺陷综合征(AIDS)的感染症状阶段。当血液或其他体液早期发现HIV病毒时，连续治疗可能会延迟发生这种情况的时间。如果发生艾滋病，治疗也可以更好地管理艾滋病的过程。还有其他罕见的疾病，如淋巴细胞减少症，导致CD4 +T细胞的缺失或功能障碍。这些疾病产生类似的症状，其中许多是致命的。
抗原表位(antigenic epitope)，简称“表位”(epitope)，也称为“抗原决定簇”(antigenic determinant)，是指抗原表面上决定抗原特异性的化学基团。抗原表位可被免疫系统(尤其是抗体、B细胞或者T细胞)所识别。抗体中能识别抗原表位的区域叫做“互补位”或“抗体决定簇”。尽管通常抗原表位是指外来蛋白质等物质的其中一部分，但只要能被自身免疫系统所识别的表位，也被归为抗原表位。
蛋白质抗原的表位根据它们的结构以及与互补位的交互作用，被分为构象表位和线性表位这两种类型。其中构象表位由抗原氨基酸序列中的不连续部分组成，因此互补位和抗原表位的交互作用是基于表面的三维特征和形状，或者是抗原的三级结构。大部分的抗原表位都属于构象表位。与此相反，线性表位是由一段连续的抗原氨基酸序列构成，与抗原的交互作用的基础是其一级结构。
T细胞表位(T cell epitope)主要是由8-17氨基酸组成的短肽构成，出现在抗原呈递细胞(antigen-presenting cell,APC)上，这种抗原表位将会和主要组织相容性复合体(MHC)相结合形成复合体，并与相应的T细胞表位受体结合，从而活化T细胞，产生相应细胞免疫应答(Shimonkevitz et al.,1984；Babbitt et al.,1985；Buus et al.,1986；Townsend and Bodmer,1989)。与表位结合的MHC主要有两类分子，其中，I类主要组织相容性复合体所呈现的T细胞抗原表位通常是由8至11个氨基酸长度的多肽组成，而II类主要组织相容性复合体所呈现的T细胞抗原表位相对更长，由13-17个氨基酸组成。
辅助T细胞表位(Th epitope，即Th表位)是指T细胞表位中的，与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别的一类T细胞表位。Th表位主要与存在于由主要组织相容性复合体(MHC)的II类基因编码的抗原呈递细胞(antigen-presenting cell,APC)表面上的分子结合。然后，II类分子和肽表位的复合物被T辅助淋巴细胞表面上的特异性T细胞受体(TCR)识别。以这种方式，在MHC分子的环境中呈现抗原表位的T细胞可以被激活，并为B淋巴细胞分化提供必要的信号。传统上，肽免疫原的辅助T细胞表位的来源是肽共价偶联的载体蛋白，但是该偶联过程可引入其他问题，例如在偶联过程中抗原决定簇的修饰和以针对肽的抗体为代价的针对载体的抗体的诱导(Schutze,M.P.,Leclerc,C.Jolivet,M.Audibert,F.Chedid,L.Carrier-induced epitopic suppression,a major issue for future synthetic vaccines.J Immunol.1985,135,2319-2322；DiJohn,D.,Torrese,J.R.Murillo,J.Herrington,D.A.et al.Effect of priming with carrier on response to conjugate vaccine.The Lancet.1989,2,1415-1416)。此外，在制备中使用无关蛋白引入质量控制问题。合适的载体蛋白的选择在设计肽疫苗时非常重要，其选择受到其大规模生产的毒性和可行性等因素的限制。这种方法还有其他限制，包括可偶联的肽负载的大小和可以安全施用的载体的剂量(Audibert,F.a.C.,L.1984.Modern approaches to vaccines.Molecular and chemical basis of virus virulence and immunogenicity.,Cold Spring Harbor Laboratory,New York.)。虽然载体分子允许诱导强的免疫应答，但它们也与不利影响相关，例如抑制抗肽抗体应答(Herzenberg,L.A.and Tokuhisa,T.1980.Carrier-priming leads to hapten-specific suppression.Nature 285:664；Schutze,M.P.,Leclerc,C.,Jolivet,M.,Audibert,F.,and Chedid,L.1985.Carrier-induced epitopic suppression,a major issue for future synthetic vaccines.J Immunol 135:2319；Etlinger,H.M.,Felix,A.M.,Gillessen,D.,Heimer,E.P.,Just,M.,Pink,J.R., Sinigaglia,F.,Sturchler,D.,Takacs,B.,Trzeciak,A.,and et al.,1988.Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite,Plasmodium falciparum.J Immunol 140:626)。
一般来说，免疫原必须包含辅助T细胞表位，除了将被表面Ig识别的表位或存在于细胞毒性T细胞上的受体之外。应该意识到，这些类型的表位可能是非常不同的。对于B细胞表位，构象是重要的，因为B细胞受体直接与天然免疫原结合。相比之下，由T细胞识别的表位不依赖于表位的构象完整性，并且由针对CTL的约9个氨基酸的短序列和针对辅助T细胞的稍长的序列(长度限制较小)组成。这些表位的唯一要求是它们可以分别容纳在I类或II类分子的结合裂口中，并且然后复合物能够与T细胞受体结合。II类分子的结合位点在两端是开放的，允许与报道的短至8个氨基酸残基(Fahrer,A.M.,Geysen,H.M.,White,D.O.,Jackson,D.C.and Brown,L.E.Analysis of the requirements for class II-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to HEd J.Immunol.1995.155:2849-2857)的表位结合的肽的长度变化更大(Brown,J.H.,T.S.Jardetzky,J.C.Gorga,L.J.Stern,R.G.Urban,J.L.Strominger and D.C.Wiley.1993.Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.Nature 364:33)。
Th表位能刺激并活化辅助T细胞，相应地，促进CD8T细胞和B细胞活化，最终提高免疫应答。实质上，Th表位除了能活化针对自身的免疫应答外，还能有效帮助与之关联的其他抗原或表位的免疫应答。因此，可以将异源性的强Th表位与目的免疫原融合，据此可以提高目的免疫原的免疫原性。一种称之为“PADRE(pan HLA DR-binding Epitope))”的人造强Th表位已用于多个疫苗的融合构造，以提高相关免疫原的免疫应答水平(del Guercio et al.,Vaccine,1997,15:441.；Franke,E.D.et al.,Vaccine,1999,17:1201；Jeff Alexander et al.,J Immunol,2000,164(3)1625-1633；Jeff Alexander et al.,Vaccine,2004,22:2362.；La Rosa,Corinna et al.,The Journal of infectious diseases,2012,205:1294-304)。此外，源于破伤风毒素(Tetanus toxin)的强Th表位P2也常用于与目的免疫原偶联，以提高免疫原性(Panina-Bordignon P et al.,Eur J Immunol,1989,19:2237-42；La Rosa,Corinna et al.,The Journal of infectious diseases,2012,205:1294-304)。
但一般而言，用于提高免疫原性的Th表位通常为异源性的，换言之，疫苗受试者体内不会存在针对该Th表位自身的高水平的免疫应答。因此， 使用上述的强Th表位接种疫苗受试者时，很可能的情况是疫苗受试者的免疫系统是初次接触此类Th表位，受体免疫系统活化针对此类Th表位和目的免疫原的表位基本上是同步的，针对此类Th表位的T细胞的生成时间和数量与目的免疫原相似，这样的话，对于帮助目的免疫原的效果会因此受限。特别是对于弱免疫原性的肿瘤抗原，此类Th表位的辅助作用更难发挥。事实上，直接采用强Th表位虽然能活化肿瘤抗原，但其激发的细胞免疫应答水平仍较低，不能满足肿瘤疫苗的需要(Ghaffari-Nazari H et al.,PLoS ONE,2015,10(11):e0142563)。
因此，需要新的Th表位策略来提高目的免疫原免疫原性，特别是一些弱的免疫原，例如肿瘤抗原。
发明内容
本发明的目的是提供一种CD4辅助性T细胞表位融合肽，通过该表位融合肽，目的免疫原的免疫原性得以提高。
进一步地，本发明利用来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位获得表位融合肽来提高目的免疫原的免疫原性。
为了本发明的目的以下定义下列术语。
“表位融合肽”是指将若干表位连接在一起所形成的一个肽。
“目的免疫原”是指为实现某种免疫应答，所采用的免疫原，包括抗原等具有免疫学活性的物质，优选为蛋白。
本发明的另一目的是提供一种所述表位融合肽和所述目的免疫原的融合蛋白。
为实现上述目的，本发明提供一种CD4辅助性T细胞表位融合肽，其包含巨细胞病毒表位和/或流感病毒表位。
在本发明的一个实施方案中，所述表位融合肽包含选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种。
在本发明的一个实施方案中，所述表位融合肽由选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种组成。优选地，所述表位融合肽由5个或10个巨细胞病毒表位和/或由8个或13个流感病毒表位组成，例如SEQ ID NO:34或44所示的表位融合肽。最优选地，所述表位融合肽由13个流感病毒表位组成，例如SEQ ID NO:48所示的表位融合肽。
优选地，所述表位融合肽诱导体液或细胞免疫应答。
本发明还提供了所述表位融合肽和目的免疫原的融合蛋白。
本发明还提供了编码所述表位融合肽和/或所述融合蛋白的多核苷酸。
在本发明的一个实施方案中，所述目的免疫原为任意一种或多种免疫原。优选的，所述目的免疫原为肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物。在本发明的一个实施方案中，所述抗原为肿瘤抗原或感染相关抗原。
在本发明的一个实施方案中，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种。
在本发明的一个实施方案中，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。优选地，LAGE抗原为LAGE-1，MAGE抗原为MAGE-A3。进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1。优选地，所述LAGE-1的氨基酸序列如SEQ ID NO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示。
在本发明的一个实施方案中，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种。
优选地，所述融合蛋白如SEQ ID NO：55-58中之一所示。
本发明的另一目的是提供一种免疫组合物，所述免疫组合物包含治疗有效量的根据本发明的表位融合肽、融合蛋白和/或多核苷酸，以及药学上可接受的载体。优选地，该免疫组合物为疫苗。
本发明的另一目的是提供一种药盒，所述药盒包含根据本发明的表位融合肽、融合蛋白、多核苷酸和/或免疫组合物，以及其使用说明。
本发明还提供了根据本发明的表位融合肽、融合蛋白、多核苷酸和/或免疫组合物在制备提高目的免疫原的免疫原性的药物或疫苗中用途。
本发明还提供了一种使用根据本发明的表位融合肽提高目的免疫原的免疫原性的方法，包括将疫苗受试者或人群中已有较强免疫应答的CD4辅助性T细胞表位与目的免疫原融合形成的融合蛋白。其方法具体为：
(1)选择一个或多个CD4辅助性T细胞的表位，所述表位与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别，并且在疫苗接种前，疫苗受试者体内已经存在针对所述表位中至少一个表位产生了T细胞免疫应答；
(2)将上述表位融合制成表位融合肽，将所述表位融合肽与目的免疫原融合制成融合蛋白，表达融合蛋白并制成疫苗，其表达载体形式可以是DNA疫苗载体形式，可以是蛋白疫苗载体形式，还可以是病毒疫苗载 体形式；
(3)将上述疫苗接种给疫苗受试者，接种时可以选择适当的佐剂，例如不完全弗氏佐剂，完全弗氏佐剂，氢氧化铝佐剂等。
进一步地，所述使用方法的步骤(1)还包括检查疫苗受试者MHC表型的步骤。优选地，检查疫苗受试者MHC表型包括检查疫苗受试者的MHC II类基因亚型型别。
本发明还提供了一种治疗或预防有需要的受试者的病症的方法，其包括给予治疗有效量的本发明的表位融合肽、融合蛋白、免疫组合物和/或多核苷酸。优选地，所述病症选自恶性肿瘤、细菌和病毒性慢性感染中的一种或多种。优选地，所述恶性肿瘤为乳腺癌或结肠癌。优选地，在该方法中，初免使用DNA疫苗载体，加强免疫使用蛋白疫苗载体，更优选地，初免使用pVKD1.0-CI-LMNB DNA疫苗，加强免疫使用LMNB-I13蛋白。本发明提供的表位融合肽能大幅度提高目的免疫原，尤其是弱免疫原的细胞免疫应答水平，是克服免疫系统对抗原免疫耐受性，尤其是对肿瘤抗原或感染相关抗原免疫耐受性的有效手段，适用于高效增强疫苗的效力。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1和图2分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的DNA疫苗载体pVKD1.0-hLMN的质粒图谱和双酶切鉴定图。
图3和图4分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的DNA疫苗载体pVKD1.0-hLMN-CTB的质粒图谱和双酶切鉴定图。
图5和图6分别是带有源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI的质粒图谱和双酶切鉴定图。
图7和图8分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位，和源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI-LMNB的质粒图谱和双酶切鉴定图。
图9和图10分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的原核载体pET-30a(+)-LMN的质粒图谱和双酶切鉴定图。
图11和图12分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-LMN-CTB的质粒图谱和双酶切鉴定图。
图13和图14分别是带有源于CMV表位编码序列的原核载体pET-30a(+)-CMV Th的质粒图谱和双酶切鉴定图。
图15和图16分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV10-LMNB的质粒图谱和双酶切鉴定图。
图17和图18分别是带有源于流感病毒表位编码序列的原核载体pET-30a(+)-CMV Th的质粒图谱和双酶切鉴定图。
图19和图20分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ8-LMNB的质粒图谱和双酶切鉴定图。
图21和图22分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ13-LMNB的质粒图谱和双酶切鉴定图。
图23为动物免疫实验中的细胞免疫应答检测结果。
图24和图25分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV5-LMNB的质粒图谱和双酶切鉴定图。
图26为实施例12中的动物免疫实验中的细胞免疫应答检测结果。
图27为实施例13中的小鼠肿瘤生长情况。
图28和图29分别是实施例13中的小鼠无瘤存活状况和总体存活状况。
图30为4T1-hNY-ESO-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长情况。
图31为CT26-hLAGE-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长情况。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
实施例1 DNA疫苗pVKD1.0-hLMN构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将上述抗原氨基酸序列优化成哺乳动物密码子使用偏好的核苷酸序列，分别如SEQ ID NO:27-29所示。经上海捷瑞生物技术有限公司合成后，通过本领域熟知的方法克隆至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司提供)上的多克隆位点Sal I与BamH I之间，构建成可表达融合蛋白抗原的DNA疫苗载体 pVKD1.0-hLMN(质粒图谱如图1)，经测序鉴定正确后入库。用限制内切酶Sal I与BamH I鉴定载体pVKD1.0-hLMN(酶切体系如表1)，其酶切验证图谱如图2所示。
表1：质粒pVKD1.0-hLMN的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例2 DNA疫苗pVKD1.0-hLMN-CTB构建
霍乱毒素B亚单位(Cholera toxin subunit B，简称CTB)的氨基酸序列(SEQ ID NO:30)的哺乳动物密码子优化序列(SEQ ID NO:31)及其真核表达载体pVKD1.0-CTB由苏州工业园区唯可达生物科技有限公司提供。以pVKD1.0-CTB为模板，设计引物(见表2)，通过PCR扩增CTB基因片段，然后凝胶回收相应片段，通过同源重组法将CTB片段插入线性化载体pVKD1.0-hLMN相应位置上，构建成DNA疫苗载体pVKD1.0-hLMN-CTB(质粒图谱如图3)，经测序鉴定正确后入库。用限制内切酶Sal I与BamH I鉴定载体pVKD1.0-hLMN-CTB(酶切体系如表3)，其酶切验证图谱如图4所示。
表2：实施例2中的引物
Figure PCTCN2018108331-appb-000001
表3：质粒pVKD1.0-hLMN-CTB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN-CTB 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例3 DNA疫苗pVKD1.0-CI-LMNB构建
在免疫表位数据库(IEDB，http://www.iedb.org)上获取来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位(见表4)，其中，CMV的强Th表位包括pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104；流感病毒的强Th表位包括HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127和M1-210。表4中选择的表位覆盖人群中MHC II类分子多数亚型，而且也覆盖小鼠MHC II亚型分子。然后将所选的表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221串联在一起，形成一段CMV病毒和流感病毒的表位融合肽，其氨基酸序列如SEQ ID NO:34所示，该表位融合肽经哺乳动物密码子优化后，核酸序列如SEQ ID NO:35所示。送苏州泓迅生物科技有限公司合成该核酸序列，然后通过本领域熟知的分子生物学方法将该核酸序列插入至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司)上，形成载体pVKD1.0-CI(质粒图谱如图5)，经测序鉴定正确后入库。用限制内切酶Pst I与Bgl II鉴定载体pVKD1.0-CI(酶切体系如表5)，其酶切验证谱如图6所示。
表4：实施例3中的Th表位
表位名称 来源 氨基酸序列
pp65-11 CMV LLQTGIHVRVSQPSL(SEQ ID NO:1)
pp65-71 CMV IIKPGKISHIMLDVA(SEQ ID NO:2)
pp65-92 CMV EHPTFTSQYRIQGKL(SEQ ID NO:3)
pp65-123 CMV AGILARNLVPMVATV(SEQ ID NO:4)
pp65-128 CMV KYQEFFWDANDIYRI(SEQ ID NO:5)
pp65-57 CMV KVYLESFCEDVPSGK(SEQ ID NO:6)
pp65-62 CMV TLGSDVEEDLTMTRN(SEQ ID NO:7)
pp65-30 CMV PLKMLNIPSINVHHY(SEQ ID NO:8)
pp65-112 CMV ACTSGVMTRGRLKAE(SEQ ID NO:9)
pp65-104 CMV TERKTPRVTGGGAMA(SEQ ID NO:10)
HA203 Influ NQRALYHTENAYVSVVS(SEQ ID NO:11)
NP438 Influ SDMRAEIIKMMESARPE(SEQ ID NO:12)
NS1-84 Influ ALASRYLTDMTIEEMSR(SEQ ID NO:13)
M1-181 Influ LASTTAKAMEQMAGSSE(SEQ ID NO:14)
HA375 Influ SGYAADQKSTQNAINGITNKVN(SEQ ID NO:15)
NP24 Influ EIRASVGKMIDGIGRFYI(SEQ ID NO:16)
NP95 Influ PIYRRVDGKWMRELVLY(SEQ ID NO:17)
NP221 Influ RMCNILKGKFQTAAQRAM(SEQ ID NO:18)
HA434 Influ IWTYNAELLVLLENERT(SEQ ID NO:19)
HA440 Influ ELLVLLENERTLDFHDS(SEQ ID NO:20)
NP324 Influ HKSQLVWMACNSAAFED(SEQ ID NO:21)
M1-127 Influ CMGLIYNRMGAVTTESA(SEQ ID NO:22)
M1-210 Influ RQMVQAMRAIGTHPSSSTGLKND(SEQ ID NO:23)
表5：质粒pVKD1.0-CI的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI 3μL，约1μg
Pst I(宝生物，货号1073A) 1μL
Bgl II(宝生物，货号1021A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
最后以实施例2中的载体pVKD1.0-hLMN-CTB为模板，设计引物(见表6)，通过PCR扩增hLMN-CTB目的基因片段，然后通过本领域熟知的分子生物学方法将该基因片段插入该pVKD1.0-CI载体上Not I和Bam HI酶切位点之间，构建成DNA疫苗载体pVKD1.0-CI-LMNB(质粒图谱如图7)，经测序鉴定正确后入库。用限制内切酶Bam HI与EcoR V鉴定载体pVKD1.0-CI-LMNB(酶切体系如表7)，其酶切验证图谱如图8所示。
表6：实施例3中的引物
Figure PCTCN2018108331-appb-000002
表7：质粒pVKD1.0-CI-LMNB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI-LMNB 3μL，约1μg
Bam HI(宝生物，货号1010A) 1μL
EcoR V(宝生物，货号1042A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例4 LMN原核表达载体构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将所述抗原氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列，其核苷酸序列分别如SEQ ID NO:38-40所示。经苏州泓迅生物科技有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成原核表达构建体pET-30a(+)-LMN(质粒图谱如图9)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN(酶切体系如表8)，其酶切验证图谱如图10所示。
表8：质粒pET-30a(+)-LMN的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例5 LMN-CTB原核表达载体构建
霍乱毒素B亚单位CTB氨基酸序列(SEQ ID NO:30)及其原核密码子优化核酸序列(SEQ ID NO:41)均由苏州工业园区唯可达生物科技有限公司提供。设计引物(见表9)，以pET-30a(+)-CTB(苏州工业园区唯可达生物科技有限公司)为模板，用PCR方法扩增含有CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过同源重组的方式将该核酸片段插入pET-30a(+)-LMN载体上，构建成pET-30a(+)-LMN-CTB载体(质粒图谱如图11)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN-CTB(酶切体系如表10)，其酶切验证图谱如图12所示。
表9：实施例5中的引物
Figure PCTCN2018108331-appb-000003
表10：实施例5中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN-CTB 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例6含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
从表4中选取10个来源于CMV的Th表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:44所示，其中，该SEQ ID NO:44中的一段序列“EFELRRQ”是因为引入了酶切位点导致的，属于融合构建常用的技术。通过在线密码子优化软件(http://www.jcat.de/)将所述Th表位氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:45)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-CMV Th(质粒图谱如图13)，经测序鉴定正确后入库。用限制内切酶Mlu I和Xho I鉴定载体pET-30a(+)-CMV Th(酶切体系如表11)，其酶切验证图谱如图14所示。
其中，如图13所示，CMV Th1含有5个CMV的Th表位，由pp65-11、pp65-71、pp65-92、pp65-123和pp65-128串联而成，CMV Th2含有5个CMV的Th表位，由pp65-57、pp65-62、pp65-30、pp65-112和pp65-104组成，CMV Th1和CMV Th2之间引入了EcoR I、Sac I和Sal I等3个限制酶酶切位点。
表11：质粒pET-30a(+)-CMV Th的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV Th 3μL，约1μg
Mlu I(宝生物，货号1071A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(见表12)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Xho I之间，构建成pET-30a(+)-CMV10-LMNB载体(质粒图谱如图15，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表13)，其酶切验证图谱如图16所示。如图15所示，pET-30a(+)-CMV10-LMNB含有CMV Th1和CMV Th2片段，即该载体含有表4中所有10个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104。
表12：实施例6中的引物设计
引物 序列
6F(SEQ ID NO:46) GCGCGGCCGCGACGACAAGGCCATGGCT
6R(SEQ ID NO:47) GCCTCGAGGTTAGCCATAGAGATAGC
表13：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例7含有LMN-CTB与Influ Th表位融合蛋白的原核表达载体构建
从表4中选取13个来源于流感病毒的Th表位HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127 和M1-210，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:48所示。通过在线密码子优化软件(http://www.jcat.de/)将所述含有流感病毒Th表位的氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:49)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-Influ Th(质粒图谱如图17)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-Influ Th(酶切体系如表14)，其酶切验证图谱如图18所示。
其中，如图17所示，Influ Th1含有8个流感病毒的Th表位，由HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221串联而成，Influ Th2含有5个流感病毒的Th表位，由HA434、HA440、NP324、M1-127和M1-210组成，Influ Th1和Influ Th2之间引入了EcoR I、Sac I和Sal I等3个限制酶酶切位点。
表14：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ Th 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(见表15)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例7中的pET-30a(+)-Influ Th载体上的Not I和Sal I之间，构建成pET-30a(+)-Influ8-LMNB载体(含8个流感病毒Th表位，质粒图谱如图19)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-Influ8-LMNB(酶切体系如表16)，其酶切验证图谱如图20所示。如图19所示，pET-30a(+)-Influ8-LMNB载体含有Influ Th1片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位。
表15：实施例7中的引物
引物 序列
7F1(SEQ ID NO:50) GCGCGGCCGCGTTAGCCATAGAGATAGC
7R1(SEQ ID NO:51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
表16：质粒pET-30a(+)-Influ8-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ8-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
设计引物(见表17)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段分别插入实施例6中的pET-30a(+)-Influ Th载体上的Not I和Xho I之间，构建成pET-30a(+)-Influ13-LMNB载体(含13个流感病毒Th表位，质粒图谱如图21)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表18)，其酶切验证图谱如图22所示。
如图21所示，pET-30a(+)-Influ13-LMNB载体含有Influ Th1和Influ Th2片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位和包括表4中的HA434、HA440、NP324、M1-127和M1-210在内的5个流感病毒Th表位。该载体总共包括表4中的所有13个流感病毒Th表位。
表17：实施例7中的引物设计
引物 序列
7F2(SEQ ID NO:52) GCCTCGAGGTTAGCCATAGAGATAGCA
7R2(SEQ ID NO:53) GCGCGGCCGCGACGACAAGGCCATGGCTATG
表18：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ13-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例8融合蛋白的表达纯化
分别将实施例4中构建的原核表达载体pET-30a(+)-LMN、实施例5中构建的原核表达载体pET-30a(+)-LMN-CTB、实施例6中构建的原核表达载体pET-30a(+)-CMV5-LMNB和pET-30a(+)-CMV10-LMNB、实施例7中构建的原核表达载体pET-30a(+)-Influ8-LMNB和pET-30a(+)-Influ13-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)分别制备重组蛋白LMN(其氨基酸序列如SEQ ID NO:59所示)、LMNB(其氨基酸序列如SEQ ID NO:54所示)、LMNB-C10(其氨基酸序列如SEQ ID NO:58所示)、LMNB-I8(其氨基酸序列如SEQ ID NO:55所示)和LMNB-13(其氨基酸序列如SEQ ID NO:56所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量<1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例9动物免疫实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买16只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表20。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均 在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽(WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO 3缓冲液(pH 9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serum albumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5四甲基联苯胺(3，3，5，5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度为50。
表19：疫苗信息
Figure PCTCN2018108331-appb-000004
Figure PCTCN2018108331-appb-000005
细胞免疫应答检测结果如图23所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-I13蛋白加强(即实施例8中的D组)免疫效果最好，显著性高于其平行对照(B组)以及LMNB-I8加强组(C组))。而且LMNB-I13蛋白加强组的细胞免疫应答水平与平行对照组(B组)相比，提高了将近3倍。表明装有流感病毒13个Th表位(D组)能大幅度提高弱免疫原的细胞免疫应答水平。
实施例10含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
设计引物(见表21)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Sal I之间，构建成pET-30a(+)-CMV5-LMNB载体(质粒图谱如图24)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV5-LMNB(酶切体系如表22)，其酶切验证图谱如图25所示。如图24所示，pET-30a(+)-CMV5-LMNB含有CMV Th1片段，即该载体含有表4中前5个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123和pp65-128。
表21：实施例10中的引物设计
引物 序列
7F1(SEQ ID NO:50) GCGCGGCCGCGTTAGCCATAGAGATAGC
7R1(SEQ ID NO:51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
表22：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH 2O 补至10μL
实施例11融合蛋白的表达纯化
如实施例8所述，将实施例10中构建的原核表达载体pET-30a(+)-CMV5-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)制备重组蛋白LMNB-C5(其氨基酸序列如SEQ ID NO:57所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量<1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例12动物免疫实验
疫苗信息如表19。DNA疫苗pVKD1.0-CI(实施例3)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表23。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽 (WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO 3缓冲液(pH 9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serum albumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5四甲基联苯胺(3，3，5，5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度为50。
表23：分组与免疫规划
Figure PCTCN2018108331-appb-000006
细胞免疫应答检测结果如图26所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-C5蛋白加强(即实施例11中的C组)和LMNB-C10蛋白加强(即实施例11中的D组)免疫效果最好，显著性高于其平行对照(B组)。表明装有CMV病毒5个Th表位(C组)和10个Th表位(D组)均能大幅度提高弱免疫原的细胞免疫应答水平。
实施例13肿瘤预防动物实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体 pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达生物科技有限公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达生物科技有限公司提供。
从苏州大学动物实验中心购买60只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表24。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表24：分组与免疫规划
Figure PCTCN2018108331-appb-000007
各组免疫小鼠肿瘤生长情况如图27所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。各免疫组小鼠 肿瘤生长滞后于对照组，其中，LMNB-I13加强免疫组(D组)小鼠和LMNB-I13与LMNB-C10混合加强免疫组(E组)小鼠肿瘤生长最慢，因此，此两组疫苗效果最好。
此外，还进行了小鼠无瘤存活分析，结果如图28所示。其中，对照组A组小鼠的无瘤存活(TFS)中位数为14天。各疫苗免疫组小鼠无瘤存活均显著高于对照组。表明所有疫苗均能提高免疫后小鼠的无瘤存活期。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，最高增加了一倍的小鼠无瘤存活期。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组)显著提高了无瘤存活期，提高了约40％的无瘤存活。表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
最后，还进行了小鼠总体存活分析，结果如图29所示。其中，对照组A组小鼠的总体存活(OS)中位数为35天。各疫苗免疫组小鼠总体存活均显著高于对照组，表明所有疫苗均能提高免疫后小鼠的存活。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，总体存活最高增加了83％。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组和F组)显著增加了无瘤存活期，最高增加了28％，表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
实施例14肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表25。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，分别在肿瘤细胞接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表25：分组与免疫规划
Figure PCTCN2018108331-appb-000008
各组免疫小鼠肿瘤生长情况如图30所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。与未治疗的对照组(A组)相比，LMNB-I13蛋白疫苗治疗组(C组)小鼠肿瘤生长最慢。而且，在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于对照组(A组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。到第35天，C组小鼠肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。
实施例15肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表26。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞CT26-hLAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×10 5细胞/只，分别在肿瘤细胞接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm 3)＝长×宽 2/2。当小鼠肿瘤体积超过2000mm 3时，对小鼠处死。
表26：分组与免疫规划
Figure PCTCN2018108331-appb-000009
各组免疫小鼠肿瘤生长情况如图31所示，由于未治疗对照组(A组)小鼠在攻瘤后(即肿瘤接种后)有小鼠未能成功接种肿瘤，故未纳入分析，比较了平行疫苗对照组(B组)和LMNB-I13治疗组(C组)小鼠。与B相比，C组小鼠肿瘤生长较慢，而且在在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于平行疫苗对照组(B组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。同样地，在CT26小鼠模型也观察到C组小鼠在后期肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。

Claims (15)

  1. 一种CD4辅助性T细胞表位融合肽，其包含巨细胞病毒表位和/或流感病毒表位。
  2. 根据权利要求1所述的表位融合肽，所述表位融合肽包含选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种。
  3. 根据权利要求1或2所述的表位融合肽，所述表位融合肽由选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种组成；
    优选地，所述表位融合肽由5个或10个巨细胞病毒表位和/或由8个或13个流感病毒表位组成，例如SEQ ID NO:34或44所示的表位融合肽；
    优选地，所述表位融合肽由13个流感病毒表位组成，例如SEQ ID NO:48所示的表位融合肽。
  4. 根据权利要求1-3任一项所述的表位融合肽，所述表位融合肽诱导体液或细胞免疫应答。
  5. 一种融合蛋白，其包含权利要求1-4任一项所述的表位融合肽和目的免疫原。
  6. 根据权利要求5所述的融合蛋白，其中，所述目的免疫原选自肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物；优选地，所述抗原为肿瘤抗原或感染相关抗原；进一步优选地，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种；优选地，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种；进一步优选地，所述LAGE抗原为LAGE-1，所述MAGE抗原为MAGE-A3；优选地，所述LAGE-1的氨基酸序列如SEQ ID NO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示；还进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1；优选地，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种；优选地，所述融合蛋白如SEQ ID NO：55-58中之一所示。
  7. 一种多核苷酸，其编码权利要求1-4任一项的所述表位融合肽或权利要求5或6所述的融合蛋白。
  8. 一种免疫组合物，其包含预防或治疗有效量的根据权利要求1-4任 一项的所述的表位融合肽、权利要求或5或6所述的融合蛋白和/或权利要求7所述的多核苷酸，以及药学上可接受的载体。
  9. 根据权利要求8的免疫组合物，其为疫苗。
  10. 一种药盒，其包含权利要求1-4任一项的所述的表位融合肽、权利要求或5或6所述的融合蛋白、权利要求7所述的多核苷酸和/或权利要求8或9所述的免疫组合物，以及其使用说明。
  11. 权利要求1-4任一项的所述的表位融合肽、权利要求或5或6所述的融合蛋白、权利要求7所述的多核苷酸和/或权利要求8或9所述的免疫组合物在制备提高目的免疫原的免疫原性的药物中的用途。
  12. 权利要求1-4任一项的所述的表位融合肽、权利要求或5或6所述的融合蛋白、权利要求7所述的多核苷酸和/或权利要求8或9所述的免疫组合物在制备提高目的免疫原的免疫原性的疫苗中的用途。
  13. 权利要求1-4任一项的所述的表位融合肽、权利要求或5或6所述的融合蛋白、权利要求7所述的多核苷酸和/或权利要求8或9所述的免疫组合物在制备治疗或预防有需要的受试者的病症的药物中的用途。
  14. 根据权利要求13所述的用途，所述的病症选自恶性肿瘤、细菌和病毒性慢性感染中的一种或多种。
  15. 根据权利要求14所述的用途，所述恶性肿瘤为乳腺癌或结肠癌。
PCT/CN2018/108331 2017-09-29 2018-09-28 一种cd4辅助性t细胞表位融合肽及其疫苗 WO2019062853A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2020539127A JP7146926B2 (ja) 2017-09-29 2018-09-28 Ｃｄ４ヘルパーｔ細胞エピトープの融合ペプチドおよびそのワクチン
EP18861153.7A EP3715359A4 (en) 2017-09-29 2018-09-28 CD4-HELPER-T-CELL PITOP-FUSION PEPTIDE AND VACCINE WITH IT
US16/650,318 US11352411B2 (en) 2017-09-29 2018-09-28 Fusion peptides of CD4 helper T cell epitopes and vaccines thereof
CN201910505212.8A CN110205340A (zh) 2017-09-29 2019-06-12 一种重组病毒载体、包含其的免疫组合物以及用途
PCT/CN2019/090877 WO2020062930A1 (zh) 2017-09-29 2019-06-12 一种重组病毒载体、包含其的免疫组合物以及用途
CN202410006185.0A CN117987471A (zh) 2017-09-29 2019-06-12 一种重组病毒载体、包含其的免疫组合物以及用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/CN2017/104401 WO2019061297A1 (zh) 2017-09-29 2017-09-29 一种cd4辅助性t细胞表位融合肽及其疫苗
CNPCT/CN2017/104401 2017-09-29

Publications (1)

Publication Number Publication Date
WO2019062853A1 true WO2019062853A1 (zh) 2019-04-04

Family

ID=65900409

Family Applications (3)

Application Number Title Priority Date Filing Date
PCT/CN2017/104401 WO2019061297A1 (zh) 2017-09-29 2017-09-29 一种cd4辅助性t细胞表位融合肽及其疫苗
PCT/CN2018/108331 WO2019062853A1 (zh) 2017-09-29 2018-09-28 一种cd4辅助性t细胞表位融合肽及其疫苗
PCT/CN2019/090877 WO2020062930A1 (zh) 2017-09-29 2019-06-12 一种重组病毒载体、包含其的免疫组合物以及用途

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/104401 WO2019061297A1 (zh) 2017-09-29 2017-09-29 一种cd4辅助性t细胞表位融合肽及其疫苗

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/090877 WO2020062930A1 (zh) 2017-09-29 2019-06-12 一种重组病毒载体、包含其的免疫组合物以及用途

Country Status (5)

Country Link
US (2) US11352411B2 (zh)
EP (1) EP3715359A4 (zh)
JP (1) JP7146926B2 (zh)
CN (4) CN115850506A (zh)
WO (3) WO2019061297A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019061297A1 (zh) * 2017-09-29 2019-04-04 苏州工业园区唯可达生物科技有限公司 一种cd4辅助性t细胞表位融合肽及其疫苗
GB201804468D0 (en) * 2018-03-21 2018-05-02 Valo Therapeutics Oy PeptiCRAd Cancer Therapy
CN110923266A (zh) * 2019-11-07 2020-03-27 苏州工业园区唯可达生物科技有限公司 一种重组病毒载体、包含其的免疫组合物以及用途
WO2021087851A1 (zh) * 2019-11-07 2021-05-14 苏州工业园区唯可达生物科技有限公司 一种重组病毒载体、包含其的免疫组合物以及用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1659436A (zh) * 2002-02-28 2005-08-24 英特塞尔股份公司 分离配体的方法
WO2008157419A2 (en) * 2007-06-13 2008-12-24 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunogenic peptides of influenza virus
CN101652385A (zh) * 2006-05-30 2010-02-17 塞缪尔·博戈奇 Replikin肽及其用途
WO2011046996A2 (en) * 2009-10-13 2011-04-21 The Johns Hopkins University Consensus sequence for influenza a virus
WO2013093514A2 (en) * 2011-12-23 2013-06-27 Retroscreen Virology Ltd Vaccines - peptides
WO2013093512A2 (en) * 2011-12-23 2013-06-27 Retroscreen Virology Ltd Vaccine - screening method

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9808932D0 (en) 1998-04-27 1998-06-24 Chiron Spa Polyepitope carrier protein
TWI229679B (en) * 1998-06-20 2005-03-21 United Biomedical Inc Artificial T helper cell epitopes as immune stimulators for synthetic peptide immunogens
RS49960B (sr) * 1998-09-15 2008-09-29 Pharmexa A/S., Postupak za regulisanje smanjenja aktivnosti osteoprotegerinskog liganda
HUP0103976A3 (en) * 1998-10-05 2008-04-28 Pharmexa As Novel methods for therapeutic vaccination
EP1467751A2 (en) * 2002-01-17 2004-10-20 Pharmexa A/S Immunogenic cea
WO2003075951A2 (en) 2002-03-11 2003-09-18 Pharmexa A/S Novel application of vaccination against tnf-alpha
CA2490449A1 (en) * 2002-06-25 2003-12-31 City Of Hope Adjuvant-free peptide vaccine
TW200407162A (en) 2002-08-30 2004-05-16 Glaxo Group Ltd Vaccine
CN1517437B (zh) * 2003-01-15 2010-06-09 张聪慧 特异性治疗肿瘤或胞内感染的疫苗及应用
ITMI20031201A1 (it) 2003-06-13 2004-12-14 Istituto Giannina Gaslini Epitopi della proteina pp65 di citomegalovirus
GB0409940D0 (en) 2004-05-04 2004-06-09 Glaxosmithkline Biolog Sa Vaccine
CA2603052A1 (en) * 2005-03-31 2006-10-05 Pharmexa A/S Immunogenic egfr peptides comprising foreign t cell stimulating epitope
AU2006312847A1 (en) * 2005-11-09 2007-05-18 Pharmexa A/S Therapeutic vaccines targeting HMGB1
HUE030139T2 (en) * 2006-12-27 2017-04-28 Univ Emory Preparations and procedures for treating inflammations
AU2008207025B2 (en) 2007-01-15 2012-08-23 Glaxosmithkline Biologicals Sa Vaccine
HUE025149T2 (hu) 2007-08-02 2016-01-28 Biondvax Pharmaceuticals Ltd Multimer multiepitóp influenza vakcinák
US8580276B2 (en) * 2009-06-05 2013-11-12 City Of Hope Genetically stable recombinant modified vaccinia ankara (rMVA) vaccines and methods of preparation thereof
CN101579527B (zh) * 2009-06-24 2010-10-27 山东大学齐鲁医院 中国人群hla特异人巨细胞病毒多表位腺病毒核酸疫苗
EP2470554B1 (en) 2009-08-26 2017-06-28 Selecta Biosciences, Inc. Compositions that induce t cell help
CN102600466B (zh) * 2012-03-23 2014-06-11 河南农业大学 抗甲型流感病毒新型通用表位疫苗及其制备方法
AU2016355926A1 (en) 2015-11-18 2018-05-31 Immunovaccine Technologies Inc. Adjuvanting systems and water-free vaccine compositions comprising a polyi:C polynucleotide adjuvant and a lipid-based adjuvant
WO2019061297A1 (zh) * 2017-09-29 2019-04-04 苏州工业园区唯可达生物科技有限公司 一种cd4辅助性t细胞表位融合肽及其疫苗
CN110923266A (zh) * 2019-11-07 2020-03-27 苏州工业园区唯可达生物科技有限公司 一种重组病毒载体、包含其的免疫组合物以及用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1659436A (zh) * 2002-02-28 2005-08-24 英特塞尔股份公司 分离配体的方法
CN101652385A (zh) * 2006-05-30 2010-02-17 塞缪尔·博戈奇 Replikin肽及其用途
WO2008157419A2 (en) * 2007-06-13 2008-12-24 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunogenic peptides of influenza virus
WO2011046996A2 (en) * 2009-10-13 2011-04-21 The Johns Hopkins University Consensus sequence for influenza a virus
WO2013093514A2 (en) * 2011-12-23 2013-06-27 Retroscreen Virology Ltd Vaccines - peptides
WO2013093512A2 (en) * 2011-12-23 2013-06-27 Retroscreen Virology Ltd Vaccine - screening method

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
AUDIBERT, F. A. C., L.: "Molecular and chemical basis of virus virulence and immunogenicity", 1984, COLD SPRING HARBOR LABORATORY, article "Modern approaches to vaccines"
BROWN, J. H.T. S. JARDETZKYJ. C. GORGAL. J. STEMR. G. URBANJ. L. STROMINGERD. C. WILEY: "Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1", NATURE, vol. 364, 1993, pages 33, XP002132608, DOI: 10.1038/364033a0
DEL GUERCIO ET AL., VACCINE, vol. 15, 1997, pages 441
DIJOHN, D.TORRESE, J. R.MURILLO, J.HERRINGTON, D. A. ET AL.: "Effect of priming with carrier on response to conjugate vaccine", THE LANCET, vol. 2, 1989, pages 1415 - 1416
ETLINGER, H. M.FELIX, A. M.GILLESSEN, D.HEIMER, E. P.JUST, M.PINK, J.R.SINIGAGLIA, F.STURCHLER, D.TAKACS, B.TRZECIAK, A. ET AL.: "Assessment in humans of a synthetic peptide-based vaccine against the sporozoite stage of the human malaria parasite, Plasmodium falciparum", J IMMUNOL, vol. 140, 1988, pages 626
FAHRER, A. M.GEYSEN, H. M.WHITE, D. O.JACKSON, D. C.BROWN, L. E.: "Analysis of the requirements for class II-restricted T-cell recognition of a single determinant reveals considerable diversity in the T-cell response and degeneracy of peptide binding to HEd", J. IMMUNOL., vol. 155, 1995, pages 2849 - 2857
FRANKE, E. D. ET AL., VACCINE, vol. 17, 1999, pages 1201
GHAFFARI-NAZARI H ET AL., PLOS ONE, vol. 10, no. 11, 2015, pages e0142563
HERZENBERG, L. A.TOKUHISA, T.: "Carrier-priming leads to hapten-specific suppression", NATURE, vol. 285, 1980, pages 664
JEFF ALEXANDER ET AL., J IMMUNOL, vol. 164, no. 3, 2000, pages 1625 - 1633
JEFF ALEXANDER ET AL., VACCINE, vol. 22, 2004, pages 2362
LA ROSA, CORINNA ET AL., THE JOURNAL OF INFECTIOUS DISEASES, vol. 205, 2012, pages 1294 - 304
NAYAK, J. L. ET AL.: "Analyses of the Specificity of CD 4 T Cells During the Primary Immune Response to Influenza Virus Reveals Dramatic MHC-Linked Asymmetries in Reactivity to Individual Viral Proteins", VIRAL IMMUNOLOGY, vol. 23, no. 2, 31 December 2010 (2010-12-31), pages 169 - 180, XP055587934 *
PANINA-BORDIGNON P ET AL., EUR J IMMUNOL, vol. 19, 1989, pages 2237 - 42
RICHARDS, K. A. ET AL.: "The Memory Phase of the CD 4 T- Cell Response to Influenza Virus Infection Maintains its Diverse Antigen Specificity", IMMUNOLOGY, vol. 133, 31 December 2011 (2011-12-31), pages 246 - 256, XP055587940 *
SCHUTZE, M. P.LECLERC, C. JOLIVETM. AUDIBERTF. CHEDID: "L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines", J IMMUNOL., vol. 135, 1985, pages 2319 - 2322, XP002067525
SCHUTZE, M. P.LECLERC, C.JOLIVET, M.AUDIBERT, F.CHEDID, L.: "Carrier-induced epitopic suppression, a major issue for future synthetic vaccines", J IMMUNOL, vol. 135, 1985, pages 2319, XP002067525
See also references of EP3715359A4

Also Published As

Publication number Publication date
CN115850506A (zh) 2023-03-28
JP7146926B2 (ja) 2022-10-04
JP2020534874A (ja) 2020-12-03
WO2019061297A1 (zh) 2019-04-04
EP3715359A1 (en) 2020-09-30
CN109575142B (zh) 2022-07-12
CN110205340A (zh) 2019-09-06
US11352411B2 (en) 2022-06-07
US20220002750A1 (en) 2022-01-06
WO2020062930A1 (zh) 2020-04-02
US20200216516A1 (en) 2020-07-09
EP3715359A4 (en) 2021-06-23
CN117987471A (zh) 2024-05-07
CN109575142A (zh) 2019-04-05

Similar Documents

Publication Publication Date Title
WO2019062853A1 (zh) 一种cd4辅助性t细胞表位融合肽及其疫苗
JP2023524054A (ja) ベータコロナウイルスの予防と治療
JP5200201B2 (ja) 外来抗原に対する宿主の寛容を壊すためのキメラ抗原
EP2247306B1 (en) Immunogenic control of tumours and tumour cells
KR102228843B1 (ko) Cd4+ t-세포 반응의 증강을 위한 변형된 에피토프
US11352416B2 (en) Mosaic chimeric viral vaccine particle
CN113015541A (zh) 具有新氧化还原酶基序的免疫原性肽
JP5792630B2 (ja) Ｐａｎ−ｄｒ結合ポリペプチドおよびその使用
WO2022127825A1 (zh) 针对新型冠状病毒感染的疫苗组合物
JP2010535504A5 (zh)
CN101402674A (zh) 附睾蛋白酶抑制剂的功能肽段及应用
CN109575141A (zh) 一种cd4辅助性t细胞表位融合肽及其疫苗
CN103626877A (zh) 含ny-eso-1的融合蛋白、制备方法及应用
CN106543287A (zh) 构象表位疫苗和应用
He et al. Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from β-hCG subunit
CN106581667B (zh) 一种多房棘球蚴亚单位疫苗LTB-Emy162的设计、制备方法和应用
WO2023010176A1 (en) Vaccine construct and uses thereof
WO2017008638A1 (zh) 一种嵌合诺如病毒p颗粒及其制备和应用
CN113318225B (zh) 肿瘤免疫增强剂及其制法和应用
CN105198982B (zh) 基于il-6的抗原表位及其应用
CN101161287A (zh) 一种携人分子佐剂的融合蛋白避孕疫苗及其制备方法
WO2023187366A1 (en) Immunogenic compositions for the prevention of influenza a
WO2024220728A1 (en) Compositions and methods for raising mucosal and systemic immune responses against complement anaphylatoxins for inflammatory disease
CN118717960A (zh) 细胞因子纳米颗粒抗原
AU3183701A (en) Method for preparing a polypeptide soluble in an aqueous solvent in the absence of detergent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18861153

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020539127

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018861153

Country of ref document: EP

Effective date: 20200429