WO2022083585A1 - 一种蛋白多糖结合物及其应用 - Google Patents

一种蛋白多糖结合物及其应用 Download PDF

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WO2022083585A1
WO2022083585A1 PCT/CN2021/124705 CN2021124705W WO2022083585A1 WO 2022083585 A1 WO2022083585 A1 WO 2022083585A1 CN 2021124705 W CN2021124705 W CN 2021124705W WO 2022083585 A1 WO2022083585 A1 WO 2022083585A1
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polysaccharide
protein
conjugate
tetanus toxin
proteoglycan
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PCT/CN2021/124705
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English (en)
French (fr)
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祝先潮
陈华根
熊细双
李颖
王娟娟
刘畅
夏清风
毛意芝
王著
谌恩华
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上海瑞宙生物科技有限公司
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Priority to CA3196222A priority Critical patent/CA3196222A1/en
Priority to KR1020237017056A priority patent/KR20230123464A/ko
Priority to AU2021367018A priority patent/AU2021367018A1/en
Priority to EP21882001.7A priority patent/EP4234572A1/en
Priority to US18/032,784 priority patent/US20230405104A1/en
Priority to CN202180071656.0A priority patent/CN116507359A/zh
Priority to JP2023548992A priority patent/JP2023546740A/ja
Publication of WO2022083585A1 publication Critical patent/WO2022083585A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present application relates to the field of biomedicine, in particular to a proteoglycan conjugate and its application.
  • the proteoglycan conjugates comprise polysaccharides from Streptococcus pneumoniae and tetanus toxin protein truncations.
  • the proteoglycan conjugate can improve the immunogenicity of Streptococcus pneumoniae capsular polysaccharide.
  • Streptococcus pneumoniae (Streptococcus pneumoniae, pneumococcus), formerly known as "pneumococcus", is a gram-positive diplococcus with a capsule. Polysaccharides are important pathogenic factors. Pneumococcal disease is one of the serious public health problems worldwide. The World Health Organization estimates that in 2005, 1.6 million people died of pneumococcal disease every year, including 700,000 to 1 million children under the age of 5, most of whom live in developing countries. It can be seen that pneumococcus has been seriously endangering the health of children. In developed countries, pneumococcal disease mainly comes from children under 2 years of age and the elderly, as well as immunocompromised people of all age groups.
  • Pneumococcal polysaccharide conjugate vaccine is formed by coupling pneumococcal polysaccharide and carrier protein, and the carrier protein plays a very important role in improving the immunogenicity of pneumococcal polysaccharide.
  • the protection effect is not achieved and terminated (Marilla G Lucero et al, The Pediatric Infectious Disease Journal. 28(6):455 462, JUN 2009) (Jan Poolman et al, Vaccine. 2009 May 21;27(24):3213-22). Therefore, there is an urgent need to develop protocols that can induce more immunogenic S. pneumoniae vaccines.
  • the present application provides a tetanus tetanus protein variant, the tetanus tetanus protein variant comprising the C fragment of the tetanus tetanus toxin protein and a partial fragment of the translocation region of the tetanus tetanus toxin protein, the protein
  • the variant can be used as a carrier protein to bind to S. pneumoniae polysaccharide and has the effect of improving the immunogenicity of S. pneumoniae polysaccharide.
  • the present application also provides proteoglycan conjugates comprising polysaccharides from Streptococcus pneumoniae and tetanus toxin protein truncations, said polysaccharide conjugates having significantly better immunogenicity.
  • the present application also provides a method for improving immunogenicity, the method comprising the steps of: providing a proteoglycan conjugate comprising a polysaccharide from Streptococcus pneumoniae and a truncate toxin protein truncate, and the method can Improve Streptococcus pneumoniae immunogenicity.
  • the application provides a tetanus toxin protein variant comprising the C fragment of the tetanus toxin protein and a partial fragment of the translocation region of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises the T cell epitope P2 of the translocation region of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises amino acids 829-864 of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-8.
  • the C fragment of the tetanus toxin protein comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the tetanus toxin protein variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-2.
  • the present application provides proteoglycan conjugates comprising a polysaccharide from Streptococcus pneumoniae and a tetanus toxin truncate (TTD).
  • TTD tetanus toxin truncate
  • the polysaccharide is derived from Streptococcus pneumoniae capsular polysaccharide.
  • the polysaccharide possesses more than one S. pneumoniae serotype.
  • the polysaccharide is selected from any of the following S. pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A , 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein, wherein the polysaccharide is selected from any of the following S. pneumoniae serotypes: 1, 3 , 5, 6A, 6B, 7F, 10A, 12F, 15B, 19A, 19F and 33F.
  • the C fragment of the tetanus toxin protein comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein and a partial fragment of the translocation region of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises the broad-spectrum T cell epitope P2 of the translocation region of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises amino acids 829-864 of the tetanus toxin protein.
  • the partial fragment of the translocation region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-8.
  • the tetanus toxin protein truncate comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.
  • the mass ratio of the polysaccharide to the tetanus toxin protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 1 to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 3 to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 5 polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6A polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6B polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 7F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 10A polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 12F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 15B polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19A to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 33F to the protein truncate is 0.4-2.5.
  • the proteoglycan conjugate can be coupled to the polysaccharide and the protein variant by a coupling method.
  • the method of coupling comprises any of the following methods: hydrogen bromide method, CDAP method, reduced amine method.
  • the present application provides a method for improving immunogenicity, comprising the steps of: providing a proteoglycan conjugate comprising a polysaccharide from Streptococcus pneumoniae and a tetanus toxin protein truncate.
  • the polysaccharide is derived from Streptococcus pneumoniae capsular polysaccharide.
  • the polysaccharide possesses more than one S. pneumoniae serotype.
  • the polysaccharide is selected from any of the following S. pneumoniae serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A , 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein, wherein the polysaccharide is selected from any of the following S. pneumoniae serotypes: 1, 3 , 5, 6A, 6B, 7F, 10A, 12F, 15B, 19A, 19F and 33F.
  • the C fragment of the tetanus protein comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the tetanus toxin protein truncate comprises the C fragment of the tetanus toxin protein and a partial fragment of the tetanus toxin protein translocation region.
  • the partial fragment of the translocation region comprises the broad-spectrum T cell epitope P2 of the translocation region of the tetanus toxin protein.
  • the partial fragment of the translocation region of the tetanus toxin protein comprises amino acids 829-864 of the tetanus toxin protein.
  • the tetanus toxin protein truncate comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.
  • the mass ratio of the Streptococcus pneumoniae polysaccharide to the tetanus toxin protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 1 to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 3 to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 5 polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6A polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6B polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 7F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 10A polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 12F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae type 15B polysaccharide to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19A to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19F to the protein truncate is 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 33F to the protein truncate is 0.4-2.5.
  • the method comprises conjugating the polysaccharide and the protein variant using a conjugation method.
  • the method of coupling comprises any of the following methods: hydrogen bromide method, CDAP method, reduced amine method.
  • the increasing the immunogenicity of bacterial polysaccharide comprises that the proteoglycan conjugate is more immunogenic than the polysaccharide CRM197 conjugate.
  • the higher immunogenicity is detected in animal immunization experiments.
  • the animal immunization test comprises the following steps: formulating the proteoglycan conjugate and adjuvant into an immunizing antigen.
  • the injection method of the immunizing antigen comprises intraperitoneal injection, subcutaneous injection, intramuscular injection and/or intravenous injection.
  • the animal immunization test comprises the following steps: ELISA detection of antibodies in the serum of the immunized animals obtained.
  • the animal immunization test comprises the following steps: performing an opsonophagocytic bactericidal test on the serum obtained from the immunized animal.
  • the animals comprise mice, rats and/or rabbits.
  • the adjuvant comprises aluminum hydroxide, aluminum phosphate, and/or Freund's adjuvant, among others.
  • the application provides a nucleic acid molecule comprising encoding the tetanus toxin protein variant.
  • the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NOs: 4-6.
  • the application provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the nucleic acid molecule or the vector.
  • the present application also provides a carrier protein comprising the tetanus toxin protein variant.
  • the present application also provides a pharmaceutical composition comprising the proteoglycan conjugate and optionally a pharmaceutically acceptable adjuvant.
  • the present application also provides the use of the tetanus toxin protein variant for preparing a medicine.
  • the present application also provides the use of the polysaccharide conjugate for preparing a medicine.
  • the medicament is used to prevent and/or treat Streptococcus pneumoniae disease.
  • the Streptococcus pneumoniae disease comprises pneumonia, sepsis, meningitis and/or otitis media.
  • the present application also provides a vaccine comprising the proteoglycan conjugate, the pharmaceutical composition and/or optionally a pharmaceutically acceptable adjuvant.
  • the present application also provides a method for preventing Streptococcus pneumoniae disease, comprising administering the proteoglycan conjugate, the nucleic acid molecule, the vector, the cell, the The carrier protein, the pharmaceutical composition and/or the vaccine.
  • the Streptococcus pneumoniae disease comprises pneumonia, sepsis, meningitis and/or otitis media.
  • the present application also provides the use of the polysaccharide conjugate as an antigen to prepare an antibody.
  • the antibodies are used for diagnostic typing of isolated strains.
  • the present application also provides a kit comprising the polysaccharide conjugate.
  • the kit is used for diagnostic typing of isolated strains.
  • Figure 1 shows the electropherogram of the tetanus toxin protein truncated (TTD) expression plasmid described in the present application.
  • Figure 2 shows the electropherogram of the TTD described in the present application after being expressed in E. coli.
  • FIG. 3 shows the electropherogram of the purified TTD described in the present application.
  • FIG. 4 shows the SEC-HPLC profile of the purified TTD described in this application.
  • Figure 5 shows the antibody titers in mouse serum after immunization with the type 3 polysaccharide-TTD-1, 2, 3 and type 3 polysaccharide-CRM197 conjugates described in the present application.
  • Figure 6 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the type 3 polysaccharide-TTD-1 and type 3 polysaccharide-CRM197 conjugates described in the present application.
  • Figure 7 shows the antibody titers in mouse serum after immunization with the type 6B polysaccharide-TTD-1 and type 6B polysaccharide-CRM197 conjugates described in the present application.
  • Figure 8 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the type 6B polysaccharide-TTD-1 and type 6B polysaccharide-CRM197 conjugates described in the present application.
  • Figure 9 shows the antibody titers in mouse serum after immunization with the 15B polysaccharide-TTD-3 and 15B polysaccharide-CRM197 conjugates described in the present application.
  • Figure 10 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the 15B-type polysaccharide-TTD-3 and 15B-type polysaccharide-CRM197 conjugates described in the present application.
  • Figure 11 shows the antibody titers in mouse serum after immunization with the type 6A polysaccharide-TTD-3 and type 6A polysaccharide-CRM197 conjugates described in the present application.
  • Figure 12 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the type 6A polysaccharide-TTD-3 and type 6A polysaccharide-CRM197 conjugates described in the present application.
  • Figure 13 shows the antibody titers in rabbit serum after immunization with the type 7F polysaccharide-TTD-1 conjugate and the type 7F polysaccharide-CRM197 conjugate described in the present application.
  • Figure 14 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the 7F polysaccharide-TTD-3 and 7F polysaccharide-CRM197 conjugates described in the present application.
  • Figure 15 shows the antibody titers in rabbit serum after immunization with the type 10A polysaccharide-TTD-2 conjugate and the type 10A polysaccharide-CRM197 conjugate described in the present application.
  • Figure 16 shows the opsonophagocytic function of specific antibodies in mouse serum after immunization with the type 10A polysaccharide-TTD-2 and type 10A polysaccharide-CRM197 conjugates described in the present application.
  • Figure 17 shows the antibody titers in rabbit serum after immunization with the type 19A polysaccharide-TTD-1 conjugate and the type 19A polysaccharide-CRM197 conjugate described in the present application.
  • Figure 18 shows the opsonophagocytic function of specific antibodies in rabbit serum after immunization with the type 19A polysaccharide-TTD-1 conjugate and the type 19A polysaccharide-CRM197 conjugate described in the present application.
  • Figure 19 shows the antibody titers in rabbit serum after immunization with the type 1 polysaccharide-TTD-3 conjugate and the type 1 polysaccharide-CRM197 conjugate described in the present application.
  • Figure 20 shows the opsonophagocytic function of specific antibodies in rabbit serum after immunization with the type 1 polysaccharide-TTD-3 conjugate and the type 1 polysaccharide-CRM197 conjugate described in the present application.
  • Figure 21 shows the antibody titers in mouse serum after immunization with the 5, 12F, 33F, 19F polysaccharide-TTD and the corresponding polysaccharide-CRM197 conjugates described in the present application.
  • tetanus bacillus also known as “clostridium tetani” generally refers to bacteria capable of causing infectious, toxic diseases. It can affect the nervous system of an infected person by synthesizing a toxin protein.
  • the toxin protein produced by the tetanus bacillus may be called “tetanus bacillus toxin protein", “tetanus bacillus toxoid” or "tetanus spasm toxin”.
  • the tetanus toxin protein may comprise three fragments, A, B and C, and the molecular weight of each fragment may be about 50 kDa.
  • the A fragment can be a catalytic region with endopolypeptidase activity.
  • the B fragment can be a translocation domain.
  • a C fragment can be a domain that has the ability to bind to a receptor (Ana C. Calvo, Int. J. Mol. Sci. 2012, 13, 6883-6901; doi: 10.3390/ijms13066883).
  • the B fragment can comprise the amino acid sequence set forth in SEQ ID NO:7.
  • the B fragment can comprise the amino acid sequence set forth in SEQ ID NO:8.
  • the C fragment can comprise the amino acid sequence set forth in SEQ ID NO:3.
  • tetanus toxin protein translocation region may refer to the B fragment of said tetanus toxin protein.
  • tetanus toxin protein C fragment may refer to the C fragment of said tetanus toxin protein.
  • protein variant generally refers to a compound having sequence homology to a native biologically active protein or polypeptide.
  • Protein variants described herein may include proteins with altered amino acid sequences by addition (including insertions), deletions, modifications and/or substitutions of one or more amino acid residues, while retaining at least one organism of the parent sequence active.
  • a variant can be at least about 0%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity.
  • Variants may be naturally occurring or non-naturally occurring.
  • Non-naturally occurring variants can be generated using techniques known in the art. Protein variants may contain conservative or non-conservative amino acid substitutions, deletions or additions. In the present application, the "tetanus toxin protein variant" may refer to a protein with a partial amino acid sequence of the tetanus toxin protein deleted.
  • the term "truncation” generally refers to anything less than the whole.
  • the "tetanus toxin protein truncation (TTD)” may refer to a compound whose amino acid sequence is less than the entire sequence of the tetanus toxin protein.
  • the truncation is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95% less than the parent protein About 96%, at least about 97%, at least about 98%, or at least about 99% of the amino acid sequence.
  • Truncations may be naturally occurring or non-naturally occurring. Non-naturally occurring truncations can be generated using techniques known in the art.
  • T cell epitope generally refers to a polypeptide or protein fragment that T lymphocytes can recognize.
  • the "T cell epitope P2" generally refers to a polypeptide of the tetanus toxin protein that can be recognized by T cells, which may comprise p2, p4 and/or p30.
  • p2 may have a DR molecule that recognizes all MHCs (molecules of the histocompatibility complex).
  • the T cell epitope p2 may comprise the amino acid sequence of positions 830-844 of the tetanus toxin protein.
  • p30 can bind to a large number of different MHC II, showing that it can be recognized by T cells and has immunogenic properties.
  • the T cell epitope p30 may comprise the amino acid sequence of positions 947-967 of the tetanus toxin protein.
  • the T cell epitope p4 may comprise the amino acid sequence of tetanus toxin protein 1273-1284.
  • a protein variant may comprise the "T cell epitope p2" region.
  • Streptococcus pneumoniae capsular polysaccharide mainly induces the body's T cell-independent immune response.
  • the immune response may include the process of immune cells recognizing, activating, proliferating, and differentiating antigen molecules to produce immune substances to produce specific immune effects after the body is stimulated by antigens.
  • the immune response may include a series of physiological responses such as antigen presentation, activation of lymphocytes, formation of immune molecules, and generation of immune effects.
  • Streptococcus pneumoniae can be divided into different serotypes according to the antigenicity of capsular polysaccharides. In certain instances, Streptococcus pneumoniae may contain 91 serotypes depending on the capsular polysaccharide antigen.
  • the main pathogenic serotypes of Streptococcus pneumoniae can include the following 24 species, namely: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the capsular polysaccharide of Streptococcus pneumoniae can comprise repeating oligosaccharide units, which can comprise up to 8 saccharide residues. In certain instances, the capsular polysaccharide can be a full-length polysaccharide. In certain instances, the capsular polysaccharide may be one oligosaccharide unit or a sugar chain of repeating oligosaccharide units that is shorter than the native length.
  • Streptococcus pneumoniae disease generally refers to diseases caused by Streptococcus pneumoniae infecting the body, including but not limited to childhood pneumonia, meningitis, bacteremia, acute otitis media, and sinusitis.
  • carrier protein generally refers to a protein, protein homolog or polypeptide that can bind to a saccharide or polysaccharide from a microorganism, carry the microbial saccharide or polysaccharide into a subject and elicit an immune response. Conjugation of the saccharide with the carrier protein enhances the immunogenicity of the saccharide, as this converts the saccharide from a thymus independent antigen to a thymus dependent antigen, which can elicit immune memory. Thymus-dependent antigens can also be called T-cell-dependent antigens; thymus-independent antigens can also be called T-cell-independent antigens.
  • proteoglycan conjugate refers to a single structural substance produced by combining the carrier protein with a saccharide or polysaccharide from bacteria.
  • conjugation refers to the covalent or non-covalent association of two moieties of substances to form a single structure, wherein the first moiety is an antigen, especially a polysaccharide, and the second moiety is an immunogen Sexual carriers, such as carrier proteins.
  • the binding can be achieved through covalent chemical bonds between molecules or through the use of linking groups such as adipic acid dihydrazide.
  • the term "immunogenicity” generally refers to properties capable of eliciting an immune response, including but not limited to properties capable of stimulating cell activation, proliferation, differentiation, production of immune effector antibodies, and sensitized lymphocytes.
  • the higher immunogenicity includes, but is not limited to, higher antibody titers, more serotype-specific antibodies, and higher bactericidal efficiency in the subject's serum after immunizing the subject.
  • polysaccharide to protein mass ratio can usually be used to measure the degree of glycosylation modification of the protein in the conjugate. Different mass ratios affect the immunogenicity of the conjugate. Both free polysaccharide and free protein belong to the binding reaction. The free capsular polysaccharide in the conjugate will reduce the immune response of the conjugate, and the excess free protein will also have an inhibitory effect on the immune response. In some cases, the molecular size distribution of the conjugate is directly related to the immunogenicity of the product, and is also an important indicator for measuring the binding process and the stability of the conjugate.
  • the term "vector” generally refers to a vector that contains the regulatory sequences necessary for the transcription and translation of a cloned nucleic acid molecule or molecules, and thereby allows transcription and cloning of the nucleic acid molecule.
  • the vector may contain one or more regulatory sequences operably linked to the nucleic acid molecule, which may be selected according to the type of host cell used. Regulatory sequences include promoters, enhancers, and other expression control elements, such as polyadenylation (poly(A)+) sequences.
  • Additional vector components may include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selection genes, and a transcription termination sequence.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a subject in need thereof.
  • the pharmaceutical composition described in the present application can comprise the proteoglycan conjugate described in the present application and a pharmaceutically acceptable carrier.
  • the term "vaccine” refers to an agent or composition containing an active ingredient effective to induce a therapeutic level of immunity in a subject against a particular pathogen or disease.
  • the vaccine may comprise bacterial polysaccharides and carrier proteins.
  • the vaccine may also contain other immunogenic active components.
  • the combination of bacterial polysaccharide and protein can also be used as an active component in a multicomponent vaccine.
  • the "vaccine” may also comprise a pharmaceutical composition, and thus generally includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not contain other active ingredients.
  • it may be a combination vaccine that additionally includes other components that induce an immune response (eg, other proteins against bacterial polysaccharides and/or against other infectious agents).
  • ELISA generally refers to an enzyme-linked immunosorbent assay. It may contain various protocols for immunodetection.
  • ELISA methods can include sandwich ELISA, bridging ELISA, ELISA direct method, ELISA indirect method, and the like.
  • ELISA immunoassays may be manual assays or may be performed by automated means.
  • S. pneumoniae polysaccharide immunogenicity can be assessed by ELISA.
  • opsonophagocytic bactericidal assay is also referred to as "OPA", which usually refers to an assay in which antibodies and complements promote phagocytic phagocytosis of particulate antigens such as bacteria by phagocytes.
  • the antibodies comprise antibodies produced from a subject immunized with an antigen.
  • the antibodies are contained in serum.
  • the phagocytic cells include phagocytic cells or phagocytic cells differentiated from cells with differentiation capacity, such as HL (Hela cells)-60.
  • the bacteria comprise Streptococcus pneumoniae.
  • the terms "subject” or “individual” or “animal” or “patient” are used interchangeably to refer to a subject, eg, a mammalian subject, in need of administration of the pharmaceutical compositions of the present application.
  • Animal subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, and the like, eg, mice, eg, rats, eg, rabbits.
  • the rabbit may be a large rabbit, for example, a New Zealand white rabbit.
  • homologous sequence can include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence.
  • a homologue will contain the same active site and the like as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a tetanus toxin variant (Tetanus Toxin Domain, TTD), which is compared with the amino acid sequence of the wild-type tetanus toxin protein (NBCI accession number WP011100836).
  • TTD Tetanus Toxin Domain
  • the tetanus toxin protein variant comprises the C fragment of the tetanus toxin protein and a partial fragment of the translocation region of the tetanus toxin protein.
  • the tetanus tetanus protein variant is obtained by removing the N-terminal amino acid sequence of the tetanus tetanus toxin protein through the technology of genetic engineering recombinant protein to obtain a new protein fragment that does not contain the N-terminal partial amino acid sequence.
  • the A fragment at the N-terminus of the tetanus toxin protein can exert a toxic effect by inhibiting the release of neurotransmitters, so in the present application, the toxic N-terminal fragment is removed by techniques commonly used in the art.
  • the C fragment of the tetanus toxin protein comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the C fragment of the tetanus toxin protein can comprise at least about 70% (eg, at least about 75%, at least about 80%, at least about 85%) of the amino acid sequence set forth in SEQ ID NO: 3 %, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% %, or at least about 100%) amino acid sequences of sequence identity.
  • the partial fragment of the translocation region of the tetanus toxin protein comprises the T cell epitope P2 of the translocation region of the tetanus toxin protein.
  • the partial fragment of the tetanus toxin protein translocation region comprises amino acids 829-864 of the tetanus toxin protein.
  • the partial fragment of the tetanus toxin protein translocation region may comprise 1-36 amino acids of the tetanus toxin protein translocation region, for example: 1 amino acid, 2 amino acids, 3 amino acids , 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids , 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, or 36 amino acids.
  • amino acids 829-864 are amino acid residues 1-36 of the amino acid sequence set forth in SEQ ID NO:1.
  • the partial fragment of the tetanus toxin protein translocation region comprises: amino acids 830-864, amino acids 831-864, amino acids 832-864 of the tetanus toxin protein , amino acids 833-864, amino acids 834-864, amino acids 835-864, amino acids 836-864, amino acids 837-864, amino acids 838-864, amino acids 839-864, Amino acids 840-864, amino acids 841-864, amino acids 842-864, amino acids 843-864, amino acids 844-864, amino acids 845-864, amino acids 846-864, Amino acid 847-864, amino acid 848-864, amino acid 849-864, amino acid 850-864, amino acid 851-864, amino acid 852-864, amino acid 853-864, amino acid 854 -Amino 864, Amino 855-864, Amino 856-864, Amino 857-864, Amino 858-864, Amin
  • the partial fragment of the translocation region of the tetanus toxin protein may comprise the amino acid sequence set forth in SEQ ID NO:7.
  • the partial fragment of the translocation region of the tetanus toxin protein may comprise the amino acid sequence set forth in SEQ ID NO:8.
  • the tetanus toxin protein can comprise at least about 70% (eg, at least about 75%, at least about 80%) of the amino acid sequence shown in any one of SEQ ID NOs: 1-3 , at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% , at least about 99%, or at least about 100%) amino acid sequences of sequence identity.
  • the protein variant may comprise at least about 70% (eg, about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, such as at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) and all at least about 70% (such as about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%) of said translocated region fragments , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%).
  • at least about 70% eg, about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 9
  • the protein variant comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-2. In certain instances, the protein variant comprises at least about 70% (eg, at least about 75%, at least about 80%, at least about 80%) of the amino acid sequence as set forth in any of SEQ ID NOs: 1-2 about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about amino acid sequences of about 99%, or at least about 100%) sequence identity.
  • the protein variant comprises at least about 70% (eg, at least about 75%, at least about 80%, at least about 80%) of the amino acid sequence as set forth in any of SEQ ID NOs: 1-2 about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 9
  • various additions, deletions and substitutions can be made to the protein variant amino acid sequence to generate the variant without adversely affecting the carrier capacity of the molecule.
  • conservative and semi-conservative amino acid substitutions can be made.
  • Exemplary conservative amino acid substitutions include, but are not limited to, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; cysteine to serine; Amino amide to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine Acid is valine or isoleucine; lysine is arginine, glutamine or glutamic acid; methionine is converted to leucine or isoleucine; phenylalanine is converted to tyrosine acid, leucine or methionine; serine to threonine; threon
  • suitable regions for mutation of various purposes include C-terminal and N-terminal regions.
  • the application provides nucleic acid molecules comprising encoding the tetanus toxin protein variant.
  • the nucleic acid sequence encoding a TTD comprises a wild-type TTD nucleic acid sequence, sequence fragment, complementary sequence, and/or a homologue thereof.
  • the nucleic acid sequence encoding a TTD comprises a mutation, truncation, substitution mutation or translocation of a wild-type TTD nucleic acid sequence.
  • the nucleic acid encoding sequence expressed by the TTD protein is codon-optimized.
  • the nucleic acid coding sequence of the TTD protein is shown in any one of SEQ ID NOs: 4-6.
  • the codon-optimized TTD nucleotide sequence may comprise at least about 70% (eg, at least about 75%, at least about 80%) of the nucleotide sequence set forth in any of SEQ ID NOs: 4-6 %, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% %, at least about 99%, or at least about 100%) nucleotide sequences of sequence identity.
  • the C-segment nucleic acid encoding sequence of the tetanus toxin protein is codon-optimized.
  • the nucleic acid coding sequence of the C fragment of the tetanus toxin protein is shown in SEQ ID NO:6.
  • the C fragment of the codon-optimized tetanus toxin protein can comprise at least about 70% (eg, at least about 75%, at least about 80%, at least about 80%) of the nucleotide sequence set forth in SEQ ID NO:6 about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about nucleotide sequences of about 99%, or at least about 100%) sequence identity.
  • the codon optimization can increase the protein expression level of the TTD.
  • the protein expression level is improved, the sequence integrity is improved, the sequence correctness is improved and/or the amplification condition is improved by at least about 10% (for example, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or higher).
  • the TTD protein may comprise the T cell epitope p2.
  • the T cell epitope p2 may comprise amino acids 830 to 844 of the tetanus toxin protein.
  • the TTD protein can comprise at least about 70% (eg, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%) of the amino acid sequence of the T cell epitope p2 about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) Nucleotide sequences of sequence identity.
  • the TTD protein can be expressed in high yield by cloning its expression sequence into a protein expression vector.
  • the host of the protein expression vector can be an expression system commonly used in the art, for example, E. coli expression system, Pichia expression system, or Bacillus subtilis expression system, insect cell expression system, plant cell expression system system and/or mammalian expression system.
  • the protein expression system can include an E. coli expression system.
  • the application provides a vector comprising the nucleic acid molecule.
  • the vector may comprise the nucleic acid molecule, an origin of replication, a selectable marker gene, a multiple cloning site, an enhancer, a promoter, and a terminator.
  • Expression vectors can be of many types and any suitable expression vector can be used. Generally, plasmid expression vectors, cosmid vectors and viral vectors are used.
  • the vectors may comprise prokaryotic and eukaryotic expression vectors, such as pET300, pET302, pBAD vectors of the E.
  • the vector may comprise pGEX-4T, pET21.
  • the present application provides cells that may comprise the nucleic acid molecule and/or the vector.
  • the nucleic acid molecule can comprise the nucleotide sequence of a codon-optimized TTD.
  • the cells described herein can express the correct TTD protein.
  • the cells can include cells from eukaryotes and prokaryotes, eg, Saccharomyces cerevisiae, Pichia, E. coli, Bacillus subtilis, sf9, plant cells, CHO cells, HEK293 cells, COS cells, BHK cells, SP2/0 cells , NIH3T3 cells, etc.
  • the cells may comprise E. coli cells.
  • the present application provides a carrier protein comprising the tetanus tetanus protein variant and the protein variant derivatives, homologues, and analogs.
  • the Bacillus tetanus protein variant can bind a polysaccharide from a bacterium or an oligosaccharide derived therefrom.
  • the bacterial polysaccharide includes, but is not limited to, Streptococcus pneumoniae polysaccharide, Tularemia endopolysaccharide, Bacillus anthracis polysaccharide, Haemophilus influenzae polysaccharide, Salmonella typhi polysaccharide, Salmonella species polysaccharide, and Shigella polysaccharide.
  • the present application provides a proteoglycan conjugate, which may comprise the polysaccharide from Streptococcus pneumoniae and the tetanus toxin protein truncate.
  • a proteoglycan conjugate which may comprise the polysaccharide from Streptococcus pneumoniae and the tetanus toxin protein truncate.
  • Most polysaccharides cannot induce thymus-dependent immune responses, and children under 2 years old or the elderly cannot acquire immunity, so it is necessary to chemically combine with protein carriers to make polysaccharides have thymus-dependent properties, which can induce relatively strong immunogens can induce both anti-polysaccharide and anti-protein antibodies.
  • the "polysaccharide from Streptococcus pneumoniae” may comprise a polysaccharide from a Streptococcus pneumoniae capsule and an oligosaccharide derived from a polysaccharide.
  • the Streptococcus pneumoniae can be derived from natural Streptococcus pneumoniae, or can be artificially modified Streptococcus pneumoniae.
  • Streptococcus pneumoniae can contain 91 serotypes, and the main pathogenic serotypes of pneumococcus can include the following 24 species, namely 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the polysaccharide of the proteoglycan conjugate has more than one S. pneumoniae serotype, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 serotypes , at least 7, at least 9, at least 12, at least 15, or at least 24.
  • the polysaccharide of the proteoglycan conjugate can be selected from any of the following S. pneumoniae serotypes: 1, 3, 5, 6A, 6B, 7F, 10A, 12F, 15B, 19A , 19F and 33F.
  • the saccharides from the S. pneumoniae capsule may also include oligosaccharides derived from capsular polysaccharides.
  • the "polysaccharide” generally refers to a polymeric carbohydrate consisting of at least 10 monosaccharides.
  • the "oligosaccharide” contains at least 2 sugar residues.
  • the capsular polysaccharide of S. pneumoniae may contain repeating oligosaccharide units of up to 8 saccharide residues.
  • the capsular saccharide serving as the antigen may be a full-length polysaccharide, may be an oligosaccharide unit, or may be a unit shorter than the natural length of the sugar chain of the repeating oligosaccharide unit.
  • all saccharides present in the proteoglycan conjugate are polysaccharides.
  • the polysaccharide and/or oligosaccharide may not be a naturally occurring polysaccharide and/or oligosaccharide, it may be synthesized by any technique known in the art, or it may be derived from or processed from a naturally occurring compound modified.
  • full-length polysaccharides can be "sized", eg, their size can be reduced by various methods, such as by acid hydrolysis treatment, peroxide treatment, by subsequent treatment with peroxide to generate oligosaccharide fragments to adjust their size .
  • modifications to polysaccharides and/or oligosaccharides may include purification, carboxylation, sulfonation, sulfation, depolymerization and/or deacetylation.
  • the modification of polysaccharides and/or oligosaccharides may be performed prior to their activation, or post-processing steps may be employed.
  • modified polysaccharides or oligosaccharides can increase antigenicity compared to wild type.
  • polysaccharides and/or oligosaccharides described herein may be derived from antigen-binding fragments of the S. pneumoniae.
  • polysaccharides and/or oligosaccharides described herein may be synthetically linked from fragments of polysaccharides and/or oligosaccharides from different types and/or strains to form polysaccharides and/or oligosaccharides comprising multiple epitopes or oligosaccharides.
  • the S. pneumoniae polysaccharide can be prepared by techniques known in the art.
  • the prepared Streptococcus pneumoniae strains are sterilized and inactivated after being amplified and cultured, a clear culture solution is collected, a polysaccharide precipitant is added, and a purified polysaccharide is obtained through a purification step.
  • the purification step may include adding NaCl solution to dissolve the complex sugar, centrifuging after the dissolution, taking the supernatant, adding alcohol to remove impurities and precipitation sugar.
  • the purified polysaccharide can be obtained by washing and precipitation for several times to remove impurities, and purifying by chromatography such as ion exchange column and composite packing.
  • the tetanus toxin protein truncate may comprise the C-fragment of the tetanus toxin protein and the tetanus toxin protein variant.
  • the tetanus toxin protein truncate may comprise the C fragment of the tetanus toxin protein.
  • the tetanus toxin protein truncate may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1-3.
  • the tetanus toxin protein truncate comprises at least about 70% (eg, at least about 75%, at least about 80%, at least about 85%) of the amino acid sequence shown in any one of SEQ ID NOs: 1-3. %, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% %, or at least about 100%) amino acid sequences of sequence identity.
  • the nucleic acid coding sequence expressing the truncate tetanus toxin protein is codon-optimized.
  • the nucleic acid coding sequence for a truncate tetanus toxin protein is set forth in any one of SEQ ID NOs: 4-6.
  • the nucleotide sequence of the codon-optimized tetanus toxin protein truncate may comprise at least about 70% of the nucleotide sequence set forth in any one of SEQ ID NOs: 4-6 (eg, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, nucleotide sequences of at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity.
  • SEQ ID NOs: 4-6 eg, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, nucleotide sequences of at least about 97%, at least about 98%, at least about 99%,
  • the proteoglycan conjugate comprises a truncate of the tetanus toxin protein and a capsular polysaccharide from any one of the serotypes of Streptococcus pneumoniae or an oligosaccharide derived therefrom.
  • the proteoglycan conjugate comprises a truncation of the tetanus toxin protein and any one pneumonia chain from the Streptococcus pneumoniae capsular polysaccharide or oligosaccharide derived therefrom selected from the group consisting of Coccus serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.
  • the proteoglycan conjugate comprises a truncation of the tetanus toxin protein and any one pneumonia chain from the Streptococcus pneumoniae capsular polysaccharide or oligosaccharide derived therefrom selected from the group consisting of Coccus serotypes: 1, 3, 5, 6A, 6B, 7F, 10A, 12F, 15B, 19A, 19F and 33F.
  • the proteoglycan conjugate comprises the C fragment of the tetanus toxin protein and any one of the Streptococcus pneumoniae serum from the Streptococcus pneumoniae capsular polysaccharide or oligosaccharide derived therefrom selected from the group consisting of Types: 1, 3, 5, 6A, 6B, 7F, 10A, 12F, 15B, 19A, 19F and 33F.
  • the proteoglycan conjugate comprises the C fragment of the tetanus toxin protein and any one of the Streptococcus pneumoniae serum from the Streptococcus pneumoniae capsular polysaccharide or oligosaccharide derived therefrom selected from the group consisting of Types: 1, 3, 6A and 15B.
  • the proteoglycan conjugate comprises the tetanus toxin protein variant TTD-1 and any one pneumonia chain from Streptococcus pneumoniae capsular polysaccharide or an oligosaccharide derived therefrom selected from the group consisting of Coccus serotypes: 3, 6B, 7F and 19A.
  • the proteoglycan conjugate comprises the tetanus toxin protein variant TTD-2 and S. pneumoniae type 10A capsular polysaccharide or oligosaccharide derived therefrom.
  • the protein may be combined with at least one (eg at least 2, at least 3, at least 4 or at least 5) of the polysaccharide, and the polysaccharide may comprise one or more than one (eg at least at least 5) of the polysaccharide 2, at least 3 or at least 4) polysaccharides of S. pneumoniae serotypes.
  • the polysaccharide may be associated with more than one of the protein molecules (eg, at least 2, at least 3, at least 4, or at least 5).
  • the "mass ratio” may include the ratio of the molecular mass of the polysaccharide to the molecular mass of the protein. In some cases, the mass ratio of the polysaccharide to the tetanus toxin protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae type 3 polysaccharide to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae type 5 polysaccharide to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6A polysaccharide to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae type 6B polysaccharide to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 12F to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae type 15B polysaccharide to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19A to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 19F to the protein truncate may be 0.4-2.5.
  • the mass ratio of S. pneumoniae polysaccharide type 33F to the protein truncate may be 0.4-2.5.
  • the proteoglycan conjugates can be prepared by any known coupling techniques.
  • the conjugation method may rely on activation of the sugar with 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) to form the cyanate ester.
  • CDAP 1-cyano-4-dimethylaminopyridine tetrafluoroborate
  • the activated sugar can thus be conjugated to amino groups on the carrier protein, either directly or through a spacer (linker) group.
  • the spacer can be cystamine or cysteamine to produce a thiolated polysaccharide that can be activated by a carrier protein with maleimide (eg using GMBS) or with a haloacetylated carrier protein (eg, the thioether bond obtained after the reaction of iodoacetimide (eg ethyl iodoacetimide) or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP is used to couple with the support.
  • a carrier protein with maleimide eg using GMBS
  • a haloacetylated carrier protein e.g, the thioether bond obtained after the reaction of iodoacetimide (eg ethyl iodoacetimide) or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP is used to couple with the support.
  • the present application provides a method for improving immunogenicity, which may include the steps of: providing a proteoglycan conjugate comprising a polysaccharide from Streptococcus pneumoniae and a truncate toxin protein truncate. Most polysaccharides belong to type 2 T cell-independent type 2 (TI-2). After entering the body, they can interact with the antigen-recognizing receptors on the surface of B cells, namely surface immunoglobulin molecules.
  • TI-2 T cell-independent type 2
  • the type of carrier protein has an influence on the immunogenicity of the polysaccharide conjugate.
  • the Hib conjugate vaccine with DT as the protein carrier compared with the conjugate vaccine with tetanus toxoid (TT), CRM197 and other proteins as the carrier, the Hib conjugate vaccine with DT as the carrier is in 18 months.
  • the following infants and young children have poor immunogenicity, so they were eliminated after listing; in addition, a clinical study of a foreign 11-valent pneumonia conjugate vaccine showed that when co-immunized with DTP combined vaccine, TT as a carrier
  • TT tetanus toxoid
  • carrier proteins used in the marketed conjugate vaccines include diphtheria toxoid (DT), diphtheria toxoid mutant (CRM197), tetanus toxoid (TT), group B meningococcal outer membrane protein complex (OMP), inseparable Haemophilus influenzae protein D (PD).
  • Carrier proteins currently under research and development include recombinant Pseudomonas aeruginosa exotoxin A (rEPA), recombinant Staphylococcus aureus enterotoxin C1 (rSEC), and cholera toxin B subunit (CTB).
  • CRM197 Diphtheria toxoid avirulent variant
  • C diphtheriae Corynebacterium diphtheriae
  • avirulent phage ⁇ 197tox which is produced by nitrosoguanidine mutagenesis of the toxin-producing carynephage b (Uchida et al. Nature New Biology (1971) 233; 8-11).
  • the CRM197 protein has a similar sequence and molecular weight to diphtheria toxin, but differs from diphtheria toxin by a single base change in the structural gene. This results in a conversion of the amino acid at position 52 from glycine to glutamine, which renders the fragment incapable of binding to NAD and thus non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983p560 -564).
  • the 13-valent pneumococcal conjugate vaccine Prevenar-13 developed by Pfizer used CRM197 (diphtheria toxoid avirulent variant) as a carrier protein and was first listed in the United States in 2010.
  • CRM197 diphtheria toxoid avirulent variant
  • the comparison of TTD as a novel carrier protein with CRM197 has important clinical significance and value.
  • the improvement of the immunogenicity means that the polysaccharide has the effect of improving the immunogenicity of the polysaccharide after it binds to the truncate tetanus.
  • the up-regulation of immunogenicity comprises that the conjugate of the polysaccharide and the truncated tetanus tetanus has no worse or higher immunogenicity than the conjugate of the polysaccharide CRM197.
  • the immunogenicity can be detected by performing any method well known to those skilled in the art.
  • the detection method may include the steps of: adding the polysaccharide-protein truncated body conjugate and the polysaccharide-CRM197 conjugate to an optional adjuvant, respectively, to prepare an immunizing antigen; the immunizing antigen may be introduced into Into the body of the subject, the subject's blood sample is drawn for testing within a certain period of time.
  • the adjuvant may comprise aluminium hydroxide gel, aluminium phosphate or aluminium salts of alum, but may also be other metal salts such as calcium, magnesium, iron or zinc salts; or may be acyl tyrosine, or acylated sugar, cationically derivatized or anionically derivatized sugar, or an insoluble mixture containing polyphosphazene.
  • the adjuvant may include aluminum hydroxide, aluminum phosphate, and/or Freund's adjuvant.
  • the introduction can include injection by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like.
  • the detection means may comprise by ELISA-bridge method, ELISA direct method, ELISA indirect method, radioimmunoassay method, electrochemiluminescence method, surface plasmon resonance, ELISA, immuno-PCR method Antibody titers in immune sera were detected.
  • the detection method may comprise detecting the production of specific antibodies by an opsonophagocytic bactericidal assay.
  • compositions for centuries, compositions, vaccines, uses and methods of prevention
  • the application provides a pharmaceutical composition comprising the proteoglycan conjugate and optionally a pharmaceutically acceptable adjuvant.
  • Suitable adjuvants may include: aluminium salts such as aluminium hydroxide gel, aluminium phosphate or alum, but may also be other metal salts such as calcium, magnesium, iron or zinc salts; or may be acylated tyrosine Acid, or acylated sugar, cationically derivatized or anionically derivatized sugar, or an insoluble mixture containing polyphosphazene.
  • the present application provides use of the tetanus toxin protein variant for preparing a medicament.
  • the use can include treatment of bacterial diseases, such as diseases caused by Gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, alpha-hemolytic streptococcus, beta-hemolytic streptococcus, Diseases caused by Streptococcus, Pneumococcus, Enterococcus, etc.
  • the use may include treatment of diseases caused by Gram-negative bacteria, Meningococcus, Neisseria gonorrhoeae, Moraxella, Acinetobacter, Pseudomonas, Alcaligenes faecalis, Brucella, Pertussis, Legionella, Salmonella, Shigella, Klebsiella.
  • the bacterial disease can comprise Streptococcus pneumoniae disease.
  • the Streptococcus pneumoniae disease can include pneumonia, sepsis, meningitis, and/or otitis media.
  • the present application provides the use of the polysaccharide conjugate for preparing a medicine.
  • the use may include preventing and/or treating bacterial diseases.
  • the bacterial disease can comprise Streptococcus pneumoniae disease.
  • the Streptococcus pneumoniae disease can include pneumonia, sepsis, meningitis, and/or otitis media.
  • prevention generally refers to preventing the occurrence and onset, recurrence, and/or spread of a disease or one or more of its symptoms by taking certain measures in advance.
  • the “treating” generally refers to eliminating or ameliorating the disease, or alleviating one or more symptoms associated with the disease.
  • the use of the tetanus toxin protein variants described herein and/or medicaments prepared therefrom prevents the occurrence, onset, recurrence and/or spread of bacterial diseases.
  • the use of the polysaccharide conjugates described herein and/or medicaments prepared therefrom prevents the occurrence, onset, recurrence and/or spread of bacterial diseases.
  • the bacterial disease may be Streptococcus pneumoniae disease.
  • the Streptococcus pneumoniae disease may include pneumonia, sepsis, meningitis and/or otitis media.
  • the application provides a vaccine, which may comprise the proteoglycan conjugate, the pharmaceutical composition and/or optionally a pharmaceutically acceptable adjuvant.
  • the proteoglycan conjugate may comprise the tetanus toxin protein truncate, the tetanus toxin protein variant and/or the C-fragment of the tetanus toxin protein.
  • Each type of carrier protein can be used as a carrier for more than one saccharide, wherein the saccharides can be the same or different.
  • S. pneumoniae serotypes 3 and 5 can be conjugated to the same carrier protein, either to the same molecule of the carrier protein or to different molecules of the same carrier protein.
  • two or more different carbohydrates can be bound to the same carrier protein, either to the same molecule of the carrier protein or to different molecules of the same carrier protein.
  • Vaccine formulations containing the pharmaceutical compositions of the present application can be administered by systemic or mucosal routes for the protection or treatment of mammals susceptible to infection. These administrations may include injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary tract, respiratory tract, genitourinary tract.
  • the components of the pharmaceutical composition may also be co-administered at the same time or at different times (eg, S. pneumoniae may be administered separately at the same time or 1-2 weeks after administration of any bacterial protein component of the vaccine sugar conjugates, so that the immune response between the two can be optimally coordinated).
  • optional adjuvants may be present in any or all of the different administrations.
  • 2 different routes of administration can be used.
  • sugars or sugar conjugates can be administered intramuscularly (or intradermally), while bacterial proteins can be administered mucosally (or intradermally).
  • the application provides a method of preventing Streptococcus pneumoniae disease, which may comprise administering to a subject in need thereof the proteoglycan conjugate, the nucleic acid molecule, the vector, the cell, the The carrier protein, the pharmaceutical composition and/or the vaccine.
  • the Streptococcus pneumoniae disease can include pneumonia, sepsis, meningitis, and/or otitis media.
  • the proteoglycan conjugates, the pharmaceutical compositions and/or the pharmaceutically acceptable adjuvants provided herein can be used to prepare antiserum. In some cases, the proteoglycan conjugates, the pharmaceutical compositions and/or the pharmaceutically acceptable adjuvants provided herein can be used to prepare antibodies.
  • the proteoglycan conjugate may comprise the tetanus toxin protein truncate, the tetanus toxin protein variant and/or the C-fragment of the tetanus toxin protein.
  • Each type of carrier protein can be used as a carrier for more than one saccharide, wherein the saccharides can be the same or different.
  • the saccharide may be a Streptococcus pneumoniae capsular polysaccharide.
  • the proteoglycan conjugates, the pharmaceutical compositions and/or the pharmaceutically acceptable adjuvants provided herein can be used to detect antibodies in a sample. In some cases, the proteoglycan conjugates, the pharmaceutical compositions and/or the pharmaceutically acceptable adjuvants provided herein can be used to prepare a kit for detecting antibodies in a sample.
  • the antibody can be a bacterial antibody.
  • the bacterial antibody may be an antibody to Streptococcus pneumoniae.
  • S. pneumoniae serotype 3 antibody S. pneumoniae serotype 5 antibody, S. pneumoniae serotype 6A antibody, S. pneumoniae serotype 6B antibody, S. pneumoniae serotype 10A antibody, S. pneumoniae Antibodies to serotype 12F, antibodies to S. pneumoniae serotype 15B, antibodies to S. pneumoniae serotype 19A, and/or antibodies to S. pneumoniae serotype 33F.
  • the tetanus toxin protein variant, tetanus toxin protein truncate and/or the C fragment of the tetanus toxin protein provided in this application can also be used to prepare anti-tetanus bacteria Antibodies to toxins, antigens and/or vaccines for preparing anti-tetanus toxin, and/or kits for diagnosing tetanus toxin antibodies.
  • the present application also provides the use of the polysaccharide conjugate as an antigen to prepare an antibody.
  • the antibodies are used for diagnostic typing of isolated strains.
  • the present application also provides a kit comprising the polysaccharide conjugate.
  • the kit is used for diagnostic typing of isolated strains.
  • TTD tetanus toxin protein truncated body
  • Tetanus Toxin Domain excluding the tetanus toxin protein (Tetanus toxin, TT) N-terminal sequence, and contains its C-terminal sequence. Therefore, the TTD protein does not have the toxicity of the tetanus toxin protein, but retains its C-terminal domain and functional fragments. On the basis of maintaining the correct folding and stability of the protein, it provides T cell epitopes and structural flexibility, and more Favorable for chemical coupling.
  • the designed protein sequence 01 is a TTD protein sequence containing 487 amino acids obtained after the TT sequence is removed from the N-terminal 828 amino acids, that is, the N-terminal peptidase M27 and part of the translocation region are removed and retained.
  • the C-terminal functional domain (receptor binding domain), the amino acid sequence is shown in SEQ ID NO: 1.
  • the corresponding nucleic acid sequence (TTD-4) is shown in the sequence SEQ ID NO: 4 after codon optimization.
  • the designed protein sequence 02 (TTD-2) is a TTD protein sequence containing 476 amino acids obtained by removing 839 amino acids from the N-terminus of the TT sequence, that is, removing the N-terminal peptidase M27 and part of the translocation region, retaining The C-terminal functional domain (receptor binding domain), the amino acid sequence is shown in SEQ ID NO: 2.
  • the corresponding nucleic acid sequence (TTD-5) is shown in sequence SEQ ID NO: 5 after codon optimization.
  • the designed protein sequence 03 is a TTD protein sequence containing 451 amino acids obtained by removing 864 amino acids from the N-terminus of the TT sequence.
  • the C-terminal functional domain (receptor binding domain), the amino acid sequence is shown in SEQ ID NO: 3.
  • the corresponding nucleic acid sequence is shown in sequence SEQ ID NO:6 after codon optimization.
  • Example 2 Expression vector construction and protein expression
  • the designed protein TTD and its corresponding gene sequence were successively constructed into protein expression vectors (such as pGEX-4T, pET21) to obtain a recombinant expression plasmid.
  • protein expression vectors such as pGEX-4T, pET21
  • the recombinant protein expression plasmid was transformed by BL21 (DE3) competent cells (Thermo Fisher), the recombinant expression plasmid was obtained.
  • the results of PCR electrophoresis of TTD-4, TTD-5 and TTD-6 are shown in FIG. 1 .
  • the preserved strains were streaked and activated on Amp + -containing plates, and single clones were picked and inoculated into LB liquid medium containing Amp + , and cultured overnight at 37°C and 250 rpm.
  • the cultured strains were diluted in proportion to inoculate in LB liquid medium containing Amp + , shaken at 37°C until OD600 reached 0.5-1.5, and then induced by adding 1 mM IPTG. After the incubation, 1 mL was sampled for centrifugation, the supernatant was removed, and the lysate was added to conduct SDS-PAGE electrophoresis analysis to determine the expression of the recombinant protein.
  • the electrophoresis results are shown in Figure 2, and the target TTD protein was obtained.
  • a tube of prepared Streptococcus pneumoniae strain was inoculated into a shaker flask, and cultured overnight in a 37°C CO2 incubator. After the microscopic examination results were normal, the culture solution was inoculated into a 10-liter fermenter, and the fermentation culture was carried out for 6- 10 hours. After culturing, adding an inactivating agent for sterilization and inactivation, and then collecting the clarified culture solution by centrifugation, adding a polysaccharide precipitant to the clarified culture solution to obtain a crude precipitated complex sugar. Refined polysaccharides are then obtained after a series of purifications.
  • the purification process of refined polysaccharide includes, but is not limited to, adding NaCl solution to dissolve complex sugar, centrifuging after dissolution, taking supernatant, adding alcohol to remove impurities and precipitation sugar.
  • the purified polysaccharide can be obtained by washing and precipitation for several times to remove impurities, and purifying by chromatography such as ion exchange column and composite packing.
  • Streptococcus pneumoniae polysaccharide has chemical reactivity with carrier protein after activation.
  • Polysaccharide-protein chemical coupling can be carried out directly or through a linker, such as coupling using a linker adipic acid dihydrazide.
  • the usual methods of chemical coupling are by cyanogen bromide method, CDAP method or reductive amination method (US5952454, EP0720485, US4711779).
  • the polysaccharide-protein conjugate obtained by coupling can induce relatively strong immunogenicity, and can induce anti-polysaccharide and anti-protein specific antibodies at the same time.
  • the steps of using the cyanogen bromide method to prepare bacterial polysaccharide-protein conjugates are as follows: dissolve 200 mg of purified polysaccharide in 20 ml 0.9% NaCl solution, add cyanogen bromide, add 0.5N NaOH solution, adjust the pH of the solution to 10.5, and react for 10-15 After minutes, the solution was adjusted to pH 7.8 by adding 0.5N HCl. Then add adipic acid dihydrazide solution (ADH) to prepare polysaccharide-ADH derivatives.
  • ADH adipic acid dihydrazide solution
  • the carrier protein TTD or CRM97
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide hydrochloride
  • the reaction After mixing the polysaccharide-ADH derivative and the activated carrier protein in proportion, the reaction generates a polysaccharide-protein conjugate, which is then concentrated by ultrafiltration or chromatography to remove impurities and unreacted substances, and purify to obtain a polysaccharide-protein conjugate.
  • the steps for preparing bacterial polysaccharide-protein conjugates using the CDAP method are as follows: In a typical reaction, pneumococcal polysaccharide (50 mg) is dissolved in 10 ml of sodium borate buffer (100 mM, pH 9.0), followed by adding CDAP solution, and The pH of the reaction solution was adjusted to 9.0 with 0.2N NaOH solution. After stirring the reaction for 5-10 minutes at room temperature, 0.1N HCl solution was added to adjust the pH to 7.5. Then the reaction solution was desalted through a G25 column to collect activated polysaccharides. Meanwhile, the carrier protein was dissolved in 0.1M NaHCO3 pH8.0 buffer, then mixed with activated polysaccharide, and stirred at room temperature for 6-10 hours. After the reaction, the solution is concentrated and exchanged by ultrafiltration to remove impurities, and finally a polysaccharide-protein conjugate is obtained.
  • the steps for preparing bacterial polysaccharide-protein conjugates using the reductive amine method are as follows: In a typical reaction, lyophilized polysaccharides are dissolved in phosphate buffer (4 mg/ml), 100 mM sodium periodate solution is added, and after stirring overnight, The activated polysaccharide was obtained by desalting through a G25 column. At the same time, the carrier protein was dissolved in phosphate buffer, then mixed with the activated polysaccharide solution in proportion, a certain equivalent of sodium cyanoborohydride was added, and stirred overnight at room temperature or 37°C. After the reaction, the reaction solution was subjected to chromatography to remove impurities and purified to obtain a polysaccharide-protein conjugate.
  • mice The Streptococcus pneumoniae polysaccharide-protein conjugate was added to aluminum hydroxide or aluminum phosphate adjuvant to prepare an immune antigen, and Balb/c mice aged 6-8 weeks were selected for intraperitoneal immunization, each group of 5- Eight mice were immunized at a dose of 2 micrograms each time, and were immunized on day 0, day 14, and day 21, respectively, and blood was drawn on day 21 and day 35 to evaluate the immunogenicity of their polysaccharides.
  • Rabbit immunization experimental protocol The polysaccharide-protein conjugate was added to Freund's adjuvant to prepare an immunizing antigen, and New Zealand white rabbits of 2-2.5 kg were selected for immunization, with 2 rabbits in each group.
  • Freund's complete adjuvant was mixed with antigen emulsification, subcutaneously immunized, and the dose was 200 micrograms; on the 14th day, Freund's incomplete adjuvant was mixed with antigen emulsification, and the second subcutaneous immunization was performed with a dose of 200 micrograms; on the 28th day, the 100 micrograms of antigen for intravenous immunization.
  • blood was drawn to evaluate the immunogenicity of the polysaccharide.
  • the Streptococcus pneumoniae polysaccharide was diluted with coating buffer, coated on a 96-well microtiter plate, 100 ⁇ l/well, and the plate was washed after incubating at 37°C for 5 hours. After the mouse and rabbit antiserum was treated with the adsorbent, the mouse serum was first diluted 1:200 (rabbit serum was first diluted 1:10000), then 2.5-fold gradient dilution, diluted 8 gradients, and then the enzyme label was added. plate, 50 ⁇ l per well, and incubate overnight.
  • the ratio of the OD value of the measured well to the OD value of the negative well is greater than or equal to 2.1 to be determined as positive, and the dilution with the maximum dilution as positive is determined as the antibody titer of each serum, and the geometric mean of the antibody titer of each group of immunized animals is calculated. , using T-test to analyze the statistical significance between different groups.
  • Streptococcus pneumoniae capsular polysaccharide-specific antibody opsonophagocytic bactericidal test Dilute Streptococcus pneumoniae by 10 5 CFU/ml, and add 10 ⁇ l/well to a 96-well cell work plate. The inactivated serum samples were serially diluted and added to the above cell work plate at 20 ⁇ l/well, the cells and antiserum were incubated at 700rpm/min for 30min, and the HL-60 cells differentiated by DMF were washed with HBSS buffer and adjusted.
  • Example 6 The immune-enhancing effect of carrier protein TTD on type 3 polysaccharide
  • mice were immunized with polysaccharide type 3-CRM197 conjugates and polysaccharide type 3-TTD-1, 2, 3 conjugates, respectively, and antibody titers were evaluated at 21 and 35 days, respectively. The results are shown in Figure 5. At 35 days, the T3-TTD-1, T3-TTD-2, and T3-TTD-3 groups all induced higher antibody titers.
  • T3-CRM197 and T3-TTD-1 groups were subjected to OPA test. The results are shown in Figure 6.
  • the IC 50 (dilution of antiserum at 50% bactericidal rate) of T3-CRM197 and T3-TTD-1, T3-TTD-2 and T3-TTD-3 were 1599 and 9926, 7142, respectively. and 5728.
  • the results showed that TTD-1, TTD-2 and TTD-3 carrier protein conjugates induced significantly more type 3 polysaccharide-specific antibodies than CRM197 carrier protein conjugates.
  • Example 7 The immune-enhancing effect of carrier protein TTD on pneumonia 6B polysaccharide
  • T6B type 6B Streptococcus pneumoniae polysaccharide-TTD-1 conjugate
  • type 6B Streptococcus pneumoniae polysaccharide-CRM197 type 6B Streptococcus pneumoniae polysaccharide-CRM197
  • mice were immunized with type 6B polysaccharide-CRM197 conjugate and type 6B polysaccharide-TTD-1 conjugate, respectively, and the antibody titers were evaluated at 21 and 35 days, respectively. The results are shown in Figure 7. At 35 days, the 6B polysaccharide-TTD-1 conjugate could induce higher antibody titers.
  • the 21-day and 35-day immune sera of 6B-TTD-1 and 6B-CRM197 groups were used for OPA test. The results are shown in Figure 8.
  • the IC50 of sera from 6B-TTD-1 and 6B-CRM197 immunized for 21 days were 2851 and 746.3, respectively, 6B-TTD-1 and 6B-CRM197 immunized
  • the IC50 of serum at 35 days were 7677 and 1802, respectively, indicating that the TTD-1 carrier protein conjugate could induce higher 6B polysaccharide-specific functional antibodies than the CRM197 carrier protein conjugate. .
  • Example 8 The immune-enhancing effect of carrier protein TTD on pneumonia 15B polysaccharide
  • mice were immunized with 15B-type polysaccharide-CRM197 conjugate and 15B-type polysaccharide-TTD-3 conjugate, respectively, and the antibody titers were evaluated at 21 days and 35 days, respectively. The results are shown in Figure 9. At 35 days, polysaccharide- TTD-3 conjugates induced higher antibody titers.
  • 15B-CRM197 and 15B-TTD-3 two groups of 21 days and 35 days of immune sera were used for OPA test. The results are shown in Figure 10.
  • the 21-day immune serum IC 50 of 15B-CRM197 and 15B-TTD-3 were 1392 and 2633, respectively.
  • the 35-day immune serum IC 50 of 15B-CRM197 and 15B-TTD-3 were 9005 and 14997, respectively. It indicated that the TTD-3 carrier protein conjugates induced significantly more 15B-type polysaccharide-specific antibodies than the CRM197 carrier protein conjugates.
  • Example 9 The immune-enhancing effect of carrier protein TTD on pneumonia type 6A polysaccharide
  • mice were immunized with type 6A (T6A) S. pneumoniae polysaccharide-CRM197 conjugate and type 6A polysaccharide-TTD-3 conjugate, and antibody titers were assessed at 21 and 35 days, respectively (as shown in Figure 11). At 35 days, the polysaccharide-TTD conjugate induced antibody titers not lower than that of polysaccharide-CRM197.
  • the T6A-CRM197 and T6A-TTD-3 two groups of 21-day and 35-day immune sera were used for OPA test.
  • the 21-day immune serum IC50 of T6A-CRM197 and T6A-TTD-3 were 458 and 1606, respectively.
  • the 35-day immune serum IC 50 of T6A-CRM197 and T6A-TTD-3 were 9103 and 20850, respectively.
  • the TTD-3 carrier protein conjugate induced 6A polysaccharide-specific antibodies significantly higher than the CRM197 carrier protein conjugate.
  • Example 10 The immune-enhancing effect of carrier protein TTD on pneumonia 7F polysaccharide
  • Type 7F Streptococcus pneumoniae polysaccharide-TTD-1 conjugates with polysaccharide-CRM197 conjugates. Mice were immunized with the polysaccharide-CRM197 conjugate and the polysaccharide-TTD-1 conjugate, respectively, and antibody titers were evaluated at 21 and 35 days, respectively. The results are shown in Figure 13. At 35 days, the 7F polysaccharide-TTD-1 conjugate induced antibody titers comparable to that of polysaccharide-CRM197.
  • the immune sera of T7F-CRM197 and T7F-TTD-1 groups at 21 days and 35 days were used for OPA test.
  • the results are shown in Figure 14: the 21-day immune serum IC50 of T7F-CRM197 and T7F-TTD-1 were 1213 and 1925, respectively; the 35-day immune serum IC50 of T7F-CRM197 and T7F-TTD-1 were 5362 and 7975, respectively.
  • the results showed that the TTD-1 carrier protein conjugate induced 7F-type polysaccharide-specific antibody was not lower than the CRM197 carrier protein conjugate.
  • Example 11 The immune-enhancing effect of carrier protein TTD on pneumonia type 10A polysaccharide
  • Type 10A Streptococcus pneumoniae polysaccharide-TTD-2 conjugates with polysaccharide-CRM197 conjugates.
  • the mice were immunized with polysaccharide-CRM197 conjugate and polysaccharide-TTD-2 conjugate, respectively, and the antibody titers at 21 days and 35 days were evaluated respectively. The results are shown in Figure 15. At 35 days, 10A polysaccharide-TTD The -2 conjugate induced higher antibody titers than the polysaccharide-CRM197 conjugate.
  • the 35-day immune sera of T10A-CRM197 and T10A-TTD-2 groups were used for OPA test.
  • the results are shown in Figure 16: the 35-day immune serum IC 50 of T10A-CRM197 and T10A-TTD-2 were 23264 and 30709, respectively.
  • the results showed that the TTD-2 carrier protein conjugate induced 10A polysaccharide-specific antibodies comparable to the CRM197 carrier protein conjugate.
  • Example 12 The immune-enhancing effect of carrier protein TTD on pneumonia 19A polysaccharide
  • New Zealand white rabbits were immunized with polysaccharide type 19A-CRM197 conjugate and polysaccharide type 19A-TTD-1 conjugate, respectively, and antibody titers were evaluated at 21 and 42 days, respectively. The results are shown in Figure 17. At 42 days, the polysaccharide-TTD conjugates induced higher antibody titers.
  • the 42-day immune sera of T19A-CRM197 and T19A-TTD-1 groups were used for OPA test. The results are shown in Figure 18.
  • the 42-day immune serum IC50 of 19A-CRM197 and 19A-TTD-1 were 8617 and 35780, respectively.
  • the results showed that TTD-1 carrier protein conjugates induced significantly more 19A-type polysaccharide-specific antibodies than CRM197 carrier protein conjugates.
  • Example 13 The immune-enhancing effect of carrier protein TTD on pneumonia type 1 polysaccharide
  • T1 Streptococcus pneumoniae polysaccharide-TTD-3 conjugates Comparison of type 1 (T1) Streptococcus pneumoniae polysaccharide-TTD-3 conjugates with polysaccharide-CRM197 conjugates. Rabbits were immunized with the polysaccharide-CRM197 conjugate and the polysaccharide-TTD-3 conjugate, respectively, and the antibody titers were evaluated at 21 and 42 days, respectively. The results are shown in 19. At 42 days, the polysaccharide-TTD conjugate induced even higher antibody titers.
  • T1-CRM197 and T1-TTD-3 two groups of 21-day and 42-day immune sera were used for OPA test.
  • the results are shown in Figure 20: T1-CRM197 and T1-TTD-3 21-day immune serum IC 50 were 6523 and 35571, respectively; T1-CRM197 and T1-TTD-3 42-day immune serum IC 50 were 193732 and 493114, respectively.
  • the results showed that the type 1 polysaccharide-specific antibody induced by the TTD-3 carrier protein conjugate was significantly higher than that of the CRM197 carrier protein conjugate.
  • Example 14 The immune-enhancing effect of carrier protein TTD on other types of Streptococcus pneumoniae polysaccharides
  • serotype Streptococcus pneumoniae polysaccharide conjugates such as serotypes 5, 12F, 33F, 19F. Mice were immunized with the polysaccharide-CRM197 conjugate and the polysaccharide-TTD conjugate, respectively, and the antibody titers were assessed at 21 and 35 days, respectively (as shown in Figure 21). At 35 days, the polysaccharide-TTD conjugate induced higher antibody titers.
  • the present invention also compares the immune enhancement effect of TTD protein on other serotypes of pneumococcal polysaccharide, and finds that TTD protein can induce higher immunogenicity than CRM197.
  • mice were immunized with polysaccharide-CRM197 conjugates and polysaccharide-TTD conjugates, respectively, and the antibody titers at 21 and 35 days were evaluated, respectively.
  • the antibody titer ratio of -CRM197 conjugates can reach 2-20 times, which further illustrates the advantage of TTD as a carrier protein.
  • TTD variants Based on protein sequences 01, 02, and 03, amino acids of different lengths at the N-terminus were truncated, and their C-terminus sequences were included.
  • TX-CRM197 conjugate TX-CRM197 group
  • TX-TTD type X polysaccharide-TTD conjugate
  • TTD carrier protein conjugate induced significantly more X-type polysaccharide-specific antibodies than the CRM197 carrier protein conjugate.

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Abstract

提供了一种破伤风杆菌毒素蛋白变体，所述破伤风杆菌毒素蛋白变体包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段；还提供蛋白多糖结合物，所述蛋白多糖结合物包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体；还提供了一种可以提高免疫原性的方法，所述方法包括以下的步骤：提供包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体的蛋白多糖结合物。

Description

一种蛋白多糖结合物及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种蛋白多糖结合物及其应用。所述蛋白多糖结合物包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体。所述蛋白多糖结合物可以提高肺炎链球菌荚膜多糖的免疫原性。
背景技术
肺炎链球菌(Streptococcus pneumoniae，肺炎球菌)，旧名“肺炎双球菌”，是一种具有荚膜的革兰阳性双球菌，根据荚膜多糖的组成差异，区分为91多种血清型，其中荚膜多糖为重要致病因子。肺炎球菌性疾病是全球严重的公共卫生问题之一。据世界卫生组织估计,2005年全球每年有160万人死于肺炎球菌疾病，包括70-100万5岁以下的儿童，其中多数生活在发展中国家。可见肺炎球菌一直在严重危害着儿童身体健康。在发达国家，肺炎球菌的疾病主要来自于2岁以下儿童和老年人，以及各年龄组免疫功能低下者。
肺炎球菌多糖结合疫苗通过肺炎球菌多糖与载体蛋白偶联而成，载体蛋白对于提高肺炎多糖的免疫原性起到非常重要的作用。在国内外多项对肺炎链球菌疫苗的临床研究中，未达到保护效果而终止的情况并不少见(Marilla G Lucero et al,The Pediatric Infectious Disease Journal.28(6):455 462,JUN 2009)(Jan Poolman et al，Vaccine.2009 May 21；27(24):3213-22)。因此，亟需开发能够诱导免疫原性更强的肺炎链球菌疫苗的方案。
发明内容
本申请提供了一种破伤风杆菌蛋白变体，所述破伤风杆菌蛋白变体包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段，所述蛋白变体可作为载体蛋白与肺炎链球菌多糖结合，并具有提高肺炎链球菌多糖免疫原性的效果。本申请还提供蛋白多糖结合物，所述蛋白多糖结合物包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体，所述多糖结合物具有显著更好的免疫原性。本申请还提供了一种可以提高免疫原性的方法，所述方法包括以下的步骤：提供包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体的蛋白多糖结合物，所述方法可以提高肺炎链球菌免疫原性。
一方面，本申请提供了一种破伤风杆菌毒素蛋白变体，其包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段。
在某些实施方式中，所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的T细胞表位P2。
在某些实施方式中，所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
在某些实施方式中，所述易位区域部分片段包含SEQ ID NO：7-8中任一项所示的氨基酸序列。
在某些实施方式中，所述破伤风杆菌毒素蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
在某些实施方式中，所述的破伤风杆菌毒素蛋白变体包含SEQ ID NO：1-2中任一项所示的氨基酸序列。
另一方面，本申请提供了蛋白多糖结合物，其包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体(TTD)。
在某些实施方式中，所述多糖源自肺炎链球菌荚膜多糖。
在某些实施方式中，所述多糖具备一种以上肺炎链球菌血清型。
在某些实施方式中，所述多糖选自下组中的任一种肺炎链球菌血清型：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C片段。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C片段，其中所述多糖选择下组中的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。
在某些实施方式中，所述破伤风杆菌毒素蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段。
在某些实施方式中，所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的广谱的T细胞表位P2。
在某些实施方式中，所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
在某些实施方式中，所述易位区域部分片段包含SEQ ID NO：7-8中任一项所示的氨基酸序列。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含SEQ ID NO：1-3中任一项所示的氨基酸序列。
在某些实施方式中，所述多糖与所述破伤风杆菌毒素蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，1型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，3型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，5型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，6A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，6B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，7F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，10A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，12F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，15B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，19A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，19F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，33F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，所述的蛋白多糖结合物，可以采用偶联的方法将所述多糖和所述蛋白变体偶联起来。
在某些实施方式中，所述偶联的方法包含以下任一种方法：溴化氢法、CDAP法、还原胺法。
另一方面，本申请提供了一种提高免疫原性的方法，其包括以下的步骤：提供包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体的蛋白多糖结合物。
在某些实施方式中，所述多糖源自肺炎链球菌荚膜多糖。
在某些实施方式中，所述多糖具备一种以上肺炎链球菌血清型。
在某些实施方式中，所述多糖选自下组中的任一种肺炎链球菌血清型：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C 片段，其中所述多糖选择下组中的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。
在某些实施方式中，所述破伤风杆菌蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段和破伤风杆菌毒素蛋白易位区域部分片段。
在某些实施方式中，所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的广谱的T细胞表位P2。
在某些实施方式中，所述的破伤风杆菌毒素蛋白的易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
在某些实施方式中，所述破伤风杆菌毒素蛋白截短体包含SEQ ID NO：1-3中任一项所示的氨基酸序列。
在某些实施方式中，所述肺炎链球菌多糖与所述破伤风杆菌毒素蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，1型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，3型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，5型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，6A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，6B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，7F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，10A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，12F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，15B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，19A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，19F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，33F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
在某些实施方式中，所述方法包含采用偶联的方法将所述多糖和所述蛋白变体偶联起来。
在某些实施方式中，所述偶联的方法包含以下任一种方法：溴化氢法、CDAP法、还原胺法。
在某些实施方式中，所述提高细菌多糖免疫原性包含与多糖CRM197结合物相比，所述 蛋白多糖结合物的免疫原性更高。
在某些实施方式中，所述免疫原性更高是在动物免疫实验中检测到的。
在某些实施方式中，所述动物免疫试验包含以下步骤：将所述的蛋白多糖结合物与佐剂配制成免疫抗原。
在某些实施方式中，所述的免疫抗原的注射方法包含腹腔注射、皮下注射、肌肉注射和/或静脉注射。
在某些实施方式中，所述动物免疫试验包含以下步骤：对获得的免疫动物的血清中的抗体进行ELISA检测。
在某些实施方式中，所述动物免疫试验包含以下步骤：对获得的免疫动物的血清进行调理吞噬杀菌试验。
在某些实施方式中，所述动物包含小鼠、大鼠和/或兔。
在某些实施方式中，所述佐剂包含氢氧化铝、磷酸铝和/或弗氏佐剂等。
另一方面，本申请提供了一种核酸分子，其包含编码所述的破伤风杆菌毒素蛋白变体。
在某些实施方式中，所述的核酸分子包含如SEQ ID NO：4-6所示的核苷酸序列。
另一方面，本申请提供了载体，其包含所述的核酸分子。
另一方面，本申请还提供了细胞，其包含所述的核酸分子或所述的载体。
另一方面，本申请还提供了一种载体蛋白，其包含所述的破伤风杆菌毒素蛋白变体。
另一方面，本申请还提供了一种药物组合物，其包含所述的蛋白多糖结合物和任选地药学上可接受的佐剂。
另一方面，本申请还提供了所述破伤风杆菌毒素蛋白变体用于制备药物的用途。
另一方面，本申请还提供了所述多糖结合物用于制备药物的用途。
在某些实施方式中，所述药物用于预防和/或治疗肺炎链球菌性疾病。
在某些实施方式中，所述肺炎链球菌性疾病包括肺炎、败血症、脑膜炎和/或中耳炎。
另一方面，本申请还提供了疫苗，其包含所述的蛋白多糖结合物、所述的药物组合物和/或任选地药学上可接受的佐剂。
另一方面，本申请还提供了预防肺炎链球菌性疾病的方法，其包含向有需要的受试者施用所述蛋白多糖结合物、所述核酸分子、所述载体、所述细胞、所述载体蛋白、所述的药物组合物和/或所述的疫苗。
在某些实施方式中，所述肺炎链球菌性疾病包括肺炎、败血症、脑膜炎和/或中耳炎。
另一方面，本申请还提供了所述多糖结合物作为抗原制备抗体的用途。
在某些实施方式中，所述抗体用于分离菌株的诊断分型。
另一方面，本申请还提供了试剂盒，其包含所述多糖结合物。
在某些实施方式中，所述试剂盒用于分离菌株的诊断分型。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述破伤风杆菌毒素蛋白截短体(TTD)表达质粒的电泳图。
图2显示的是本申请所述TTD在大肠杆菌中表达后的电泳图。
图3显示的是本申请所述TTD纯化后的电泳图。
图4显示的是本申请所述TTD纯化后的SEC-HPLC图谱。
图5显示的是本申请所述3型多糖-TTD-1、2、3和3型多糖-CRM197结合物免疫后小鼠血清中抗体滴度。
图6显示的是本申请所述3型多糖-TTD-1和3型多糖-CRM197结合物免疫后小鼠血清中特异性抗体的调理吞噬功能。
图7显示的是本申请所述6B型多糖-TTD-1和6B型多糖-CRM197结合物免疫后小鼠血清中的抗体滴度。
图8显示的是本申请所述6B型多糖-TTD-1和6B型多糖-CRM197结合物免疫后小鼠血清中特异性抗体的调理吞噬功能。
图9显示的是本申请所述15B型多糖-TTD-3和15B型多糖-CRM197结合物免疫后小鼠血清中的抗体滴度。
图10显示的是本申请所述15B型多糖-TTD-3和15B型多糖-CRM197结合物免疫后小鼠血清中特异性抗体的调理吞噬功能。
图11显示的是本申请所述6A型多糖-TTD-3和6A型多糖-CRM197结合物免疫后小鼠血清中的抗体滴度。
图12显示的是本申请所述6A型多糖-TTD-3和6A型多糖-CRM197结合物免疫后小鼠血清中特异性抗体的调理吞噬功能。
图13显示的是本申请所述7F型多糖-TTD-1结合物和7F型多糖-CRM197结合物免疫后大兔血清中的抗体滴度。
图14显示的是本申请所述7F型多糖-TTD-3和7F型多糖-CRM197结合物免疫后小鼠血 清中特异性抗体的调理吞噬功能。
图15显示的是本申请所述10A型多糖-TTD-2结合物和10A型多糖-CRM197结合物免疫后大兔血清中的抗体滴度。
图16显示的是本申请所述10A型多糖-TTD-2和10A型多糖-CRM197结合物免疫后小鼠血清中特异性抗体的调理吞噬功能。
图17显示的是本申请所述19A型多糖-TTD-1结合物和19A型多糖-CRM197结合物免疫后大兔血清中的抗体滴度。
图18显示的是本申请所述19A型多糖-TTD-1结合物和19A型多糖-CRM197结合物免疫后大兔血清中特异性抗体的调理吞噬功能。
图19显示的是本申请所述1型多糖-TTD-3结合物和1型多糖-CRM197结合物免疫后大兔血清中的抗体滴度。
图20显示的是本申请所述1型多糖-TTD-3结合物和1型多糖-CRM197结合物免疫后大兔血清中特异性抗体的调理吞噬功能。
图21显示的是本申请所述5、12F、33F、19F型多糖-TTD及相应多糖-CRM197结合物免疫后小鼠血清中的抗体滴度。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“破伤风杆菌”又名“破伤风梭菌”(clostridium tetani)，通常指能够引起感染性、中毒性疾病的细菌。其可以通过合成一种毒素蛋白影响被感染者的神经系统。所述破伤风杆菌产生的毒素蛋白，可称为“破伤风杆菌毒素蛋白”、“破伤风杆菌类毒素”或“破伤风痉挛毒素”。所述破伤风杆菌毒素蛋白可以包含A、B和C三个片段，每个片段的分子量可以在50kDa左右。例如，A片段可以是具有多肽内切酶活性的催化区域。例如，B片段可以是易位结构域。例如，C片段可以是具有与受体结合能力的结构域(Ana C.Calvo,Int.J.Mol.Sci.2012,13,6883-6901；doi:10.3390/ijms13066883)。例如，B片段可以包含SEQ ID NO：7中所示的氨基酸序列。例如，B片段可以包含SEQ ID NO：8中所示的氨基酸序列。例如，C片段可以包含SEQ ID NO：3所示的氨基酸序列。在本申请中，术语“破伤风杆菌毒素蛋白易位区域”可以指所述破伤风杆菌毒素蛋白的B片段。在本申请中，术语“破伤风杆菌毒素蛋白 C片段”可以指所述破伤风杆菌毒素蛋白的C片段。
在本申请中，术语“蛋白变体”“变体”通常指与天然生物活性蛋白质或多肽具有序列同源性的化合物。本申请所述的蛋白变体可以包括通过添加(包含插入)、缺失、修饰和/或取代一个或多个氨基酸残基而具有改变的氨基酸序列的蛋白，其同时保留亲本序列的至少一种生物活性。例如，变体可与亲本蛋白具有至少约0％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的序列同一性。变体可以天然存在或者是非天然存在的。可以使用本领域已知的技术来生成非天然存在的变体。蛋白变体可以包含保守的或非保守的氨基酸替代、删除或添加。在本申请中，所述“破伤风杆菌毒素蛋白变体”可以指所述破伤风杆菌毒素蛋白部分氨基酸序列缺失的蛋白。
在本申请中，术语“截短体”通常是指少于整体的任何事物。在本申请中，所述“破伤风杆菌毒素蛋白截短体(TTD)”可以指所述氨基酸序列少于破伤风杆菌毒素蛋白全部序列的化合物。例如，截短体少于亲本蛋白至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的氨基酸序列。截短体可以天然存在或者是非天然存在的。可以使用本领域已知的技术来生成非天然存在的截短体。
在本申请中，术语“T细胞表位”通常是指T淋巴细胞可以识别的多肽或者蛋白片段。所述“T细胞表位P2”通常是指破伤风杆菌毒素蛋白能够被T细胞识别的多肽，其可包含p2、p4和/或p30。例如，p2可具有识别所有MHC(组织相容性复合物分子)的DR分子。所述T细胞表位p2可以包含破伤风杆菌毒素蛋白830-844位氨基酸序列。例如，p30可以和大量不同的MHCⅡ结合，显示能够被T细胞识别，具有免疫原性的特性。所述T细胞表位p30可以包含破伤风杆菌毒素蛋白947-967位氨基酸序列。所述T细胞表位p4可以包含破伤风杆菌毒素蛋白1273-1284位氨基酸序列。在本申请中，例如，蛋白变体可以包含所述“T细胞表位p2”区域。
在本申请中，术语“肺炎链球菌荚膜多糖”、“肺炎链球菌多糖”和“肺炎球菌多糖”可以互换使用，通常是指肺炎链球菌表面松散的粘液物质。肺炎链球菌多糖主要诱发机体的不依赖T细胞的免疫应答。在本申请中，所述免疫应答可以包含机体受抗原刺激后，免疫细胞对抗原分子识别、活化、增殖和分化，产生免疫物质发生特异性免疫效应的过程。所述免疫应答可包括抗原递呈、淋巴细胞活化、免疫分子形成及免疫效应发生等一系列的生理反应。肺炎链球菌可以根据荚膜多糖抗原性的不同分为不同的血清型。在某些情形中，根据荚膜多糖 抗原的不同，肺炎链球菌可以包含91种血清型。肺炎链球菌的主要致病性的血清型可以包括以下24种，分别是：1，2，3，4，5，6A，6B，7F，8，9N，9V，10A，11A，12F，14，15B，17F，18C，19A，19F，20，22F，23F和33F。
在某些情形中，肺炎链球菌的荚膜多糖可以包含重复的寡糖单元，所述寡糖单元可以包含多达8个糖残基。在某些情形中，荚膜多糖可以是全长多糖。在某些情形中，荚膜多糖可以是一个寡糖单元或短于天然长度的重复寡糖单元的糖链。
在本申请中，术语“肺炎链球菌性疾病”通常指因肺炎链球菌感染机体后造成的疾病，包含但不限于儿童肺炎、脑膜炎、菌血症、急性中耳炎和鼻窦炎等。
在本申请中，术语“载体蛋白”通常是指可以与来自微生物的糖或多糖结合，将微生物的糖或多糖携带到受试者体内并引起免疫应答的蛋白质、蛋白质同源物或多肽。糖与载体蛋白结合后可增强糖的免疫原性，因为这将糖由非胸腺依赖性抗原(thymus independent antigen)转变为胸腺依赖性抗原(thymus dependent antigen)，由此能够引发免疫记忆。胸腺依赖性抗原，也可以称为T细胞依赖性抗原；非胸腺依赖性抗原，也可以称非T细胞依赖性抗原。对于儿童、老年人、免疫缺陷者，T细胞依赖性抗原可以产生有效的免疫应答，产生更能维持免疫效果的抗体。在本申请中，术语“蛋白多糖结合物”，即将所述载体蛋白与来自细菌的糖或多糖结合后产生的单一结构的物质。
在本申请中，术语“偶联”是指将两个部分的物质以共价或者非共价的方式结合以形成单一结构，其中第一部分是抗原，尤其是多糖，而第二部分是免疫原性载体，例如载体蛋白。所述结合可通过分子之间的共价化学键或通过采用连接基团来实现，例如己二酸二酰肼。
在本申请中，术语“免疫原性”通常是指能够引起免疫应答的性能，包括但不限于能够刺激细胞活化、增殖、分化，产生免疫效应物质抗体和致敏淋巴细胞的特性。在本申请中，所述免疫原性更高包含但不限于在对受试者进行免疫后，受试者血清中抗体滴度更高、血清型特异性抗体更多和杀菌效率更高。
在本申请中，术语“多糖与蛋白质量比”通常可以用来衡量结合物中蛋白质被糖基化修饰程度，不同的质量比影响结合物的免疫原性，游离多糖、游离蛋白质均属于结合反应的底物，结合物中游离的荚膜多糖会降低结合物的免疫反应，过量的游离蛋白质也会对免疫反应有抑制作用。在某些情形下，结合物分子大小分布直接与产品免疫原性相关，也是衡量结合工艺和结合物稳定性的重要指标。
在本申请中，术语“载体”通常是指含有克隆的一个或多个核酸分子转录和翻译所必需的调节序列、且从而可以转录和克隆核酸分子的载体。所述载体可含有与核酸分子可操作地 连接的一个或多个调节序列，可以根据使用的宿主细胞类型选择此调节序列。调节序列包括启动子、增强子和其他表达控制元件，例如聚腺苷酸化(poly(A)+)序列。其他载体组分可包括但不限于下列一个或多个：信号序列、复制起点、一个或多个选择基因和转录终止序列。
在本申请中，术语“药物组合物”通常是指适合于向有需要的受试者施用的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的蛋白多糖结合物以及药学上可接受的载剂。
在本申请中，术语“疫苗”是指含有有效诱导受试者的抗特定病原体或疾病的治疗程度的免疫性的活性组分的试剂或组合物。在本申请中，所述疫苗可以包含细菌多糖和载体蛋白。在本申请中，所述疫苗还可以包含其他免疫原活性组分。在本申请中，所述细菌多糖和蛋白的结合物还可以作为多组分疫苗中的一种活性组分。在本申请中，所述“疫苗”还可以包含医药组合物，且因此通常包括医药学上可接受的稀释剂、运载剂或赋形剂。其可能包含或可能不包含其他活性成分。在某些情形下，其可为另外包含诱导免疫反应(例如抗细菌多糖的其他蛋白质和/或抗其他感染物)的其他组分的组合疫苗。
在本申请中，术语“ELISA”通常是指酶联免疫吸附测定。其可以包含用于免疫检测的多种方案。例如，ELISA方法可以包括夹心ELISA、桥连ELISA、ELISA直接法和ELISA间接法等。在某些情形中，ELISA免疫测定可以是手动测定，也可以通过自动方式来实施。例如，在本申请中，可以用ELISA法评价肺炎链球菌多糖免疫原性。
在本申请中，术语“调理吞噬杀菌试验”也称“OPA”，通常是指通过抗体、补体促进吞噬细胞吞噬细菌等颗粒性抗原的试验。所述抗体包含来自受试者经抗原免疫后产生的抗体。所述抗体包含来自于血清中。所述吞噬细胞包含吞噬细胞或由具有分化能力的细胞(例如HL(Hela细胞)-60)分化而来的吞噬细胞。所述细菌包含肺炎链球菌。
在本申请中，术语“受试者”或“个体”或“动物”或“患者”可互换用于指施用本申请的药物组合物需要的受试者，例如哺乳动物受试者。动物受试者包括人类、非人类灵长类、狗、猫、豚鼠、兔子、大鼠、小鼠、马、黄牛、乳牛等等，例如小鼠，例如大鼠，例如兔。其中所述兔可以是大兔，例如，新西兰大白兔。
在本申请中，术语“同源性”通常是指与比较的氨基酸序列和比较的核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相 同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”或“包括”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
蛋白变体
一方面，本申请提供一种破伤风杆菌毒素蛋白(tetanus toxin)变体(Tetanus Toxin Domain，TTD)，与野生型破伤风杆菌毒素蛋白(NBCI登录号WP011100836)的氨基酸序列相比，所述破伤风杆菌毒素蛋白变体包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段。
在某些情形下，所述破伤风杆菌蛋白变体是通过基因工程重组蛋白的技术，去除了破伤风杆菌毒素蛋白的N端的氨基酸序列，获得不含有N端部分氨基酸序列的新的蛋白片段。破伤风杆菌毒素蛋白N端的A片段可以通过抑制神经递质释放发挥毒性作用，因此在本申请中，有毒性的N段的片段通过本领域的常用的技术去除了。
在某些情形下，所述破伤风杆菌毒素蛋白的C片段包含如SEQ ID NO：3所示的氨基酸序列。在某些情形下，所述破伤风杆菌毒素蛋白的C片段可包含与SEQ ID NO：3所示的氨基酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的氨基酸序列。
在某些情形下，所述的破伤风杆菌毒素蛋白易位区域部分片段包含所述破伤风杆菌毒素 蛋白的易位区域的T细胞表位P2。
在某些情形下，例如，所述破伤风杆菌毒素蛋白易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
在某些情形下，所述破伤风杆菌毒素蛋白易位区域部分片段可以包含所述破伤风杆菌毒素蛋白易位区域的1-36个氨基酸，例如：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸、5个氨基酸、6个氨基酸、7个氨基酸、8个氨基酸、9个氨基酸、10个氨基酸、11个氨基酸、12个氨基酸、13个氨基酸、14个氨基酸、15个氨基酸、16个氨基酸、17个氨基酸、18个氨基酸、19个氨基酸、20个氨基酸、21个氨基酸、22个氨基酸、23个氨基酸、24个氨基酸、25个氨基酸、26个氨基酸、27个氨基酸、28个氨基酸、29个氨基酸、30个氨基酸、31个氨基酸、32个氨基酸、33个氨基酸、34个氨基酸、35个氨基酸或36个氨基酸。
在某些情形下，所述第829-864位氨基酸是SEQ ID NO：1所示氨基酸序列的第1-36位氨基酸残基。
在某些情形下，例如，所述破伤风杆菌毒素蛋白易位区域部分片段包含所述破伤风杆菌毒素蛋白的：第830-864位氨基酸、第831-864位氨基酸、第832-864位氨基酸、第833-864位氨基酸、第834-864位氨基酸、第835-864位氨基酸、第836-864位氨基酸、第837-864位氨基酸、第838-864位氨基酸、第839-864位氨基酸、第840-864位氨基酸、第841-864位氨基酸、第842-864位氨基酸、第843-864位氨基酸、第844-864位氨基酸、第845-864位氨基酸、第846-864位氨基酸、第847-864位氨基酸、第848-864位氨基酸、第849-864位氨基酸、第850-864位氨基酸、第851-864位氨基酸、第852-864位氨基酸、第853-864位氨基酸、第854-864位氨基酸、第855-864位氨基酸、第856-864位氨基酸、第857-864位氨基酸、第858-864位氨基酸、第859-864位氨基酸、第860-864位氨基酸、第862-864位氨基酸或第863-864位氨基酸。
在某些情形中，所述破伤风杆菌毒素蛋白的易位区域部分片段可以包含SEQ ID NO：7中所示的氨基酸序列。
在某些情形中，所述破伤风杆菌毒素蛋白的易位区域部分片段可以包含SEQ ID NO:8中所示的氨基酸序列。
在某些情形下，所述的破伤风杆菌毒素蛋白可包含与SEQ ID NO：1-3中任一项所示的氨基酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的氨基酸序列。
在某些情形下，所述蛋白变体可以包含所述破伤风杆菌C片段的至少约70％(如约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)的部分和所述易位的区域片段的至少约70％(如约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)的部分。
在某些情形中，所述的蛋白变体包含如SEQ ID NO：1-2中任一项所示的氨基酸序列。在某些情形中，所述的蛋白变体包含如与SEQ ID NO：1-2中任一项所示的氨基酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的氨基酸序列。
在某些情形下，可以对所述蛋白变体氨基酸序列进行各种添加，删除和替代，以产生变体，而不会不利地影响分子的载体能力。例如，可以进行保守和半保守氨基酸取代。示例性的保守氨基酸取代包括但不限于以下变化：丙氨酸变为丝氨酸；精氨酸变为赖氨酸；天冬酰胺变为谷氨酰胺或组氨酸；半胱氨酸到丝氨酸；谷氨酰胺转化为天冬酰胺；谷氨酸到天冬氨酸；甘氨酸到脯氨酸；组氨酸为天冬酰胺或谷氨酰胺；异亮氨酸为亮氨酸或缬氨酸；亮氨酸为缬氨酸或异亮氨酸；赖氨酸为精氨酸、谷氨酰胺或谷氨酸；甲硫氨酸转化为亮氨酸或异亮氨酸；苯丙氨酸转化为酪氨酸、亮氨酸或蛋氨酸；丝氨酸至苏氨酸；苏氨酸到丝氨酸；色氨酸为酪氨酸；酪氨酸变成色氨酸或苯丙氨酸；缬氨酸为异亮氨酸或亮氨酸。如蛋白质工程领域的普通技术人员将理解的，本文提到的保守和半保守突变不影响TTD的载体功能。例如，用于各种目的突变的合适区域(例如，添加多肽“标签”以简化蛋白质的纯化)包括C端和N端区域。
另一方面，本申请提供了核酸分子，其包含编码所述破伤风杆菌毒素蛋白变体。在某些情形下，所述编码TTD的核酸序列包含野生型的TTD的核酸序列、序列片段、互补序列和/或其同源物。在某些情形下，所述编码TTD的核酸序列包含野生型TTD核酸序列的突变、截短、置换突变或易位。
在某些情形下，所述TTD蛋白表达的核酸编码序列经过密码子优化得到。例如，所述TTD蛋白的核酸编码序列如SEQ ID NO:4-6中任一项所示。例如，所述密码子优化的TTD核苷酸序列可以包含与SEQ ID NO:4-6中任一项所示的核苷酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约 93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的核苷酸序列。在某些情形中所述破伤风杆菌毒素蛋白的C片段核酸编码序列经过密码子优化得到。例如，所述破伤风杆菌毒素蛋白的C片段的核酸编码序列如SEQ ID NO:6所示。例如，所述密码子优化的破伤风杆菌毒素蛋白的C片段可以包含与SEQ ID NO:6所示的核苷酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的核苷酸序列。所述密码子优化可提高所述TTD的蛋白表达量。例如，经过所述密码子优化后，所述TTD蛋白表达的核酸序列导入细胞后，蛋白表达量提高、序列完整程度提高、序列正确程度提高和/或扩增情况提高至少约10％(例如，至少约15％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％或更高)。
在某些情形下，所述TTD蛋白可以包含所述T细胞表位p2。在某些情形下，所述T细胞表位p2可以包含破伤风杆菌毒素蛋白830位到844位氨基酸。在某些情形下，所述TTD蛋白可以包含所述T细胞表位p2的氨基酸序列至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的核苷酸序列。
在某些情形中，所述TTD蛋白可以通过将其表达序列克隆到蛋白表达载体中，实现高产量蛋白表达。在某些情形中，所述蛋白表达载体的宿主可以是本领域常用的表达系统，例如，大肠杆菌表达系统、毕赤酵母表达系统、或枯草芽孢杆菌表达系统、昆虫细胞表达系统、植物细胞表达系统和/或哺乳动物表达系统。例如，所述蛋白表达系统可以包括大肠杆菌表达系统。
另一方面，本申请提供了载体，其包含所述核酸分子。在某些情形下，所述载体可以包含所述核酸分子、复制起始位点、选择标记基因、多克隆位点、增强子、启动子以及终止子。表达载体可以有很多类型，可以使用任何合适的表达载体。通常使用的是质粒表达载体、cosmid载体还有病毒载体。在某些情形下，所述载体可以包含原核表达载体和真核表达载体，例如大肠杆菌表达系统的pET300、pET302、pBAD载体；噬菌体DNA的衍生物，例如M 13和其他丝状单链DNA噬菌体；毕赤酵母表达系统的pPIC9、pPIC9K、pPIC3K、pPICZ；枯草芽孢杆菌表达系统的pGPST、pEB10、pEB20、pUB18；哺乳动物表达系统的SV40，腺病毒，逆转录病毒衍生的DNA序列的众所周知的衍生物和衍生自功能性哺乳动物载体(例如 上述那些)的组合的穿梭载体，以及功能性质粒和噬菌体DNA。在某些情形下，所述载体可以包含pGEX-4T、pET21。
另一方面，本申请提供了细胞，所述细胞可以包含所述核酸分子和/或所述载体。在某些情形下，所述核酸分子可以包含密码子优化的TTD的核苷酸序列。本申请所述的细胞可以表达正确的所述TTD蛋白。所述细胞可以包括来自真核生物和原核生物，例如，酿酒酵母、毕赤酵母、大肠杆菌、枯草芽孢杆菌、sf9、植物细胞、CHO细胞、HEK293细胞、COS细胞、BHK细胞、SP2/0细胞、NIH3T3细胞等。在本申请中，所述细胞可以包含大肠杆菌细胞。
另一方面，本申请提供了一种载体蛋白，其包含所述破伤风杆菌蛋白变体及所述蛋白变体衍生物、同源物、类似物。在某些情形下，所述破伤风杆菌蛋白变体可以结合来自细菌的多糖或其衍生的寡糖。在某些情形下，所述细菌多糖包含但不限于肺炎链球菌多糖、土拉杆菌内多糖、炭疽杆菌多糖、流感嗜血杆菌多糖、伤寒沙门氏菌多糖、沙门氏菌种多糖和志贺氏杆菌多糖。
蛋白多糖复合物及制备方法
另一方面，本申请提供了蛋白多糖结合物，所述蛋白多糖结合物可以包含所述来自肺炎链球菌的多糖和所述破伤风杆菌毒素蛋白截短体。大多数多糖不能诱导胸腺依赖的免疫应答反应，不足2岁的儿童或老年人无法获得免疫能力，所以有必要通过与蛋白载体化学结合而使多糖具有胸腺依赖性特性，能诱导比较强的免疫原性，可同时诱导抗多糖和抗蛋白的抗体。
在某些情形中，所述“来自肺炎链球菌的多糖”可以包含来自肺炎链球菌荚膜的多糖和衍生自多糖的寡糖。所述肺炎链球菌可以是来自天然的肺炎链球菌，也可以是经过人为改造的肺炎链球菌。根据荚膜多糖的抗原性，肺炎链球菌可以包含91种血清型，而肺炎球菌主要致病性的血清型可以包括以下24种，分别是1，2，3，4，5，6A，6B，7F，8，9N，9V，10A，11A，12F，14，15B，17F，18C，19A，19F，20，22F，23F和33F。在某些情形下，所述的蛋白多糖结合物的多糖具备一种以上肺炎链球菌血清型，例如，至少1种、至少2种、至少3种、至少4种、至少5种、至少6种、至少7种、至少9种、至少12种、至少15种或至少24种。
在某些情形下，所述的蛋白多糖结合物的多糖可以选自下组中的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。
在本申请中，所述来自肺炎链球菌荚膜的糖还可以包括衍生自荚膜多糖的寡糖。所述“多糖”通常指含有至少10个的单糖组成的聚合糖高分子碳水化合物。所述“寡糖”含有至少2个糖残基。肺炎链球菌的荚膜多糖可以包含多至8个糖残基的重复寡糖单元。在某些情形下， 作为抗原的荚膜糖可以是全长多糖，也可以是一个寡糖单元，或者可以是比重复寡糖单元的天然长度的糖链更短的单元。在某些情形下，所述蛋白多糖结合物中存在的所有糖都是多糖。
在某些情形下，所述多糖和/或寡糖可以不是天然存在的多糖和/或寡糖，其可以通过本领域已知的任何技术合成，也可以衍生自天然存在的化合物或对其进行修饰得到。例如，全长多糖可以经“大小调整”，例如它们的大小可以通过各种方法缩减，例如通过酸解处理、过氧化物处理、通过随后通过过氧化物处理以产生寡糖片段从而调整其大小。例如对多糖和/或寡糖的修饰可以包括纯化、羧化、磺化、硫酸化、解聚和/或脱乙酰基。
在某些情形下，对多糖和/或寡糖的修饰可以在其活化前，也可以采用后处理的步骤。
在某些情形下，修饰的多糖或寡糖与野生型相比可以增加抗原性。
在某些情形下，本申请所述的多糖和/或寡糖可以来自所述肺炎链球菌的抗原结合片段。
在某些情形下，本申请所述的多糖和/或寡糖可以由来自不同类型和/或菌株的多糖和/或寡糖的片段通过合成手段连接，形成包含多个表位的多糖和/或寡糖。
在某些情形下，肺炎链球菌多糖可以通过本领域已知技术制备。例如，将制备好的肺炎链球菌菌种扩增培养后杀菌灭活，收集澄清培养液，加入多糖沉淀剂，再通过纯化步骤得到精制多糖。纯化的步骤可以包括加入NaCl溶液溶解复合糖，溶解后离心，取上清，加入酒精去除杂质和沉糖。可通过多次洗涤沉淀去杂，利用层析法例如离子交换柱、复合型填料纯化得到精制多糖。
在某些情形下，所述的破伤风杆菌毒素蛋白截短体可以包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白变体。
在某些情形下，所述的破伤风杆菌毒素蛋白截短体可以包含所述破伤风杆菌毒素蛋白的C片段。
在某些情形下，所述破伤风杆菌毒素蛋白截短体可以包含SEQ ID NO：1-3中任一项所示的氨基酸序列。所述的破伤风杆菌毒素蛋白截短体包含与SEQ ID NO：1-3中任一项所示的氨基酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的氨基酸序列。在某些情形中，表达所述破伤风杆菌毒素蛋白截短体的核酸编码序列通过密码子优化得到。例如，破伤风杆菌毒素蛋白截短体的核酸编码序列如SEQ ID NO:4-6中任一项所示。例如，所述密码子优化的破伤风杆菌毒素蛋白截短体的核苷酸序列可以包含与SEQ ID NO:4-6中任一项所示的核苷酸序列具有至少约70％(例如，至少约75％，至少约80％，至少约85％，至少约90％，至少 约91％，至少约92％，至少约93％，至少约94％，至少约95％，至少约96％，至少约97％，至少约98％，至少约99％，或至少约100％)序列同一性的核苷酸序列。
在某些情形下，所述蛋白多糖结合物包含所述破伤风风杆菌毒素蛋白的截短体和来自所述任一种血清型肺炎链球菌的荚膜多糖或其衍生的寡糖。在某些情形下，所述蛋白多糖结合物包含所述破伤风杆菌毒素蛋白的截短体和来自所述肺炎链球菌荚膜多糖或其衍生的寡糖选自下组的任一种肺炎链球菌血清型：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。在某些情形下，所述蛋白多糖结合物包含所述破伤风杆菌毒素蛋白的截短体和来自所述肺炎链球菌荚膜多糖或其衍生的寡糖选自下组的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。在某些情形下，所述蛋白多糖结合物包含所述破伤风风杆菌毒素蛋白的C片段和来自肺炎链球菌荚膜多糖或其衍生的寡糖选自下组的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。在某些情形下，所述蛋白多糖结合物包含所述破伤风风杆菌毒素蛋白的C片段和来自肺炎链球菌荚膜多糖或其衍生的寡糖选自下组的任一种肺炎链球菌血清型：1、3、6A和15B。在某些情形下，所述蛋白多糖结合物包含所述破伤风风杆菌毒素蛋白变体TTD-1和来自肺炎链球菌荚膜多糖或其衍生的寡糖选自下组的任一种肺炎链球菌血清型：3、6B、7F和19A。在某些情形下，所述蛋白多糖结合物包含所述破伤风风杆菌毒素蛋白变体TTD-2和10A型肺炎链球菌荚膜多糖或其衍生的寡糖。
在本申请中，所述蛋白可以和至少1个(例如至少2个、至少3个、至少4个或至少5个)所述多糖结合，所述多糖可以包含1种或1种以上(例如至少2种，至少3种或至少4种)肺炎链球菌血清型的多糖。在本申请中，所述多糖可以和不止1个所述蛋白分子结合(例如至少2个、至少3个、至少4个或至少5个)。
在本申请中，所述“质量比”可以包含所述多糖的分子质量和所述蛋白的分子质量的比值。在某些情形下，所述多糖与所述破伤风杆菌毒素蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，3型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，5型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，6A型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，6B型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，12F型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，15B型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，19A型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，19F型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在某些情形下，33F型肺炎链球菌多糖与所述蛋白截短体的质量比可以为0.4-2.5。
在本申请中，所述蛋白多糖结合物可以通过任何已知的偶联技术制备。结合方法可以依靠用四氟硼酸1-氰基-4-二甲基氨基吡啶(CDAP)来活化糖从而形成氰酸酯。因此活化的糖可以直接或通过间隔物(接头)基团与载体蛋白上的氨基缀合。例如，间隔物可以是胱胺或半胱胺，以产生硫醇化(thiolated)多糖，后者可以通过与马来酰亚胺活化的载体蛋白(例如使用GMBS)或与卤代乙酰化载体蛋白(例如使用碘乙酰亚胺(例如乙基碘乙酰亚胺)或溴乙酸N-丁二酰亚胺基酯或SIAB、或SIA、或SBAP)反应后获得的硫醚键，与载体偶联。在某些情形下，根据多糖的特点，可以选择有溴化氰法(US5952454)、1-氰基-4-二甲氨基砒啶四氟硼酸酯(CDAP)(EP0720485)、高碘酸氧化法(US4711779)。
免疫原性提高的方法
另一方面，本申请提供了一种提高免疫原性的方法，其可以包括以下的步骤：提供包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体的蛋白多糖结合物。大部分多糖属于2型T细胞非依赖抗原(T cell-independent type 2，TI-2)，进入机体后，可以通过与B细胞表面的抗原识别受体即表面免疫球蛋白分子(surface immunoglobulin molecules，sIg)交联而激活B细胞，引起一系列下游变化，使B细胞分化为可分泌抗体的浆细胞，在整个免疫过程中并没有T细胞的参与，因此不会形成免疫记忆，产生的抗体主要是亲和力较低的IgM和IgG2。在结合糖蛋白后，蛋白多糖结合物可以经抗体提呈细胞提呈，由T辅助细胞活化成熟的B细胞，诱导产生lgG类抗体，可以引起免疫记忆性应答。因此载体蛋白类型对多糖结合物免疫原性存在影响。最典型案例就是以DT为蛋白载体的Hib结合疫苗的撤市:与以破伤风类毒素(TT)，CRM197等蛋白为载体的结合疫苗相比，以DT为载体的Hib结合疫苗在18个月以下的婴幼儿中免疫原性较差，因此，在上市后被淘汰；此外，一项国外的11价肺炎结合疫苗的临床研究显示，与百白破联合疫苗共同免疫时，以TT为载体的多糖血清抗体受到明显抑制，该项研究由于该结果终止研发，但这种抑制效应没有出现在以DT为载体的结合物中。
目前，已上市结合疫苗中使用的载体蛋白质包括白喉类毒素(DT)、白喉类毒素突变体(CRM197)、破伤风类毒素(TT)、B群流脑外膜蛋白复合物(OMP)、不可分型流感嗜血杆菌蛋白D(PD)。目前在研究开发的载体蛋白质还有重组铜绿假单胞菌外毒素A(rEPA)、重组金黄色葡萄球菌肠毒素C1(rSEC)、霍乱毒素B亚单位(CTB)等。白喉类毒素无毒变异体(CRM197)是一种常用的，在临床已证明安全、有效的载体蛋白，已在上市的肺炎球菌多糖结合疫苗中广泛使用。CRM197由感染不产毒噬菌体β197tox的白喉棒杆菌(C diphtheriae) 产生，噬菌体β197tox通过亚硝基胍诱变产毒素carynephage b而产生(Uchida等Nature New Biology(1971)233；8-11)。CRM197蛋白具有与白喉毒素类似的序列和分子量，但与白喉毒素不同的是结构基因中单个碱基改变。这导致第52位的氨基酸由甘氨酸转变为谷氨酰胺，该转变致使片段不能与NAD结合并因此而不具毒性(Pappenheimer 1977，Ann Rev，Biochem.46；69-94，Rappuoli Applied and Environmental Microbiology Sept 1983p560-564)。
辉瑞公司开发的13价肺炎球菌结合疫苗Prevenar-13利用CRM197(白喉类毒素无毒变异体)为载体蛋白在2010年首次在美国上市，然而在临床应用中发现不同血清型多糖的免疫原性存在比较大的差别。TTD作为一种新型的载体蛋白与CRM197的比较具有重要的临床意义和价值。在本申请中，所述免疫原性的提高，是指所述多糖在结合破伤风杆菌截短体后，具有可以提高多糖免疫原性的作用。在某些情形下，所述免疫原性的调高，包含与多糖CRM197结合物相比，所述多糖与破伤风杆菌截短体的结合物具有不差于或者更高的免疫原性。
在本申请中，所述免疫原性可以通过进行任一本领域技术人员熟知的方法进行检测。在某些情形下，所述检测方法可以包括以下步骤：将多糖-蛋白截短体结合物和多糖-CRM197结合物分别加入任选的佐剂，配制成免疫抗原；所述免疫抗原可以被导入到受试者体内，在一定期限内抽取受试者的血样进行检测。在本申请中，所述佐剂可以包括氢氧化铝凝胶、磷酸铝或明矾的铝盐，但也可以是诸如钙盐、镁盐、铁盐或锌盐的其他金属盐；或者可以是酰化酪氨酸、或酰化糖、阳离子衍生化或阴离子衍生化的糖或含聚磷腈的不溶性混合液。在某些情形下，所述佐剂可以包括氢氧化铝、磷酸铝和/或弗氏佐剂。在某些情形下，所述导入可以包括通过静脉注射、腹腔注射、肌肉注射、皮下注射等注射方式。
在某些情形下，所述检测方式可以包含通过ELISA-桥法、ELISA直接法、ELISA间接法、放射免疫分析法、电化学发光法、表面等离子体共振、酶联免疫斑点法、免疫PCR法检测免疫血清中的抗体滴度。在某些情形下，所述检测方法可以包含通过调理吞噬杀菌试验检测特异性抗体的产生。
药物组合物、疫苗、用途和预防方法
另一方面，本申请提供了药物组合物，其包含所述蛋白多糖结合物和任选地药学上可接受的佐剂。合适的佐剂可以包括：诸如氢氧化铝凝胶、磷酸铝或明矾的铝盐，但也可以是诸如钙盐、镁盐、铁盐或锌盐的其他金属盐；或者可以是酰化酪氨酸、或酰化糖、阳离子衍生化或阴离子衍生化的糖或含聚磷腈的不溶性混合液。
另一方面，本申请提供了所述破伤风杆菌毒素蛋白变体用于制备药物的用途。在某些情 形下，所述用途可以包括治疗细菌性疾病，例如革兰氏阳性菌引起的疾病，如金黄色葡萄球菌、表皮葡萄球菌、α型溶血链球菌、β型溶血链球菌、非溶血链球菌、肺炎球菌、肠球菌等引起的疾病。在某些情形下，所述用途可以包括治疗革兰氏阴性菌引起的疾病，脑膜炎球菌、淋球菌、摩拉卡他菌、不动杆菌属、假单胞菌属、粪产碱杆菌、布鲁菌属、百日咳杆菌、军团菌、沙门菌属、志贺菌属、克雷伯菌属。在某些情形下，所述细菌性疾病可以包含肺炎链球菌性疾病。在某些情形下，所述肺炎链球菌性疾病可以包括肺炎、败血症、脑膜炎和/或中耳炎。
另一方面，本申请提供了所述多糖结合物用于制备药物的用途。在某些情形下，所述用途可以包括预防和/或治疗细菌性疾病。在某些情形下，所述细菌性疾病可以包含肺炎链球菌性疾病。在某些情形下，所述肺炎链球菌性疾病可以包括肺炎、败血症、脑膜炎和/或中耳炎。
在本申请中，所述“预防”通常是指通过预先采取某些措施而防止疾病或其一种或多种症状的产生和发作，复发，和/或扩散。所述“治疗”通常是指消除或改善疾病，或缓解与疾病相关的一种或多种症状。例如，使用本申请所述破伤风杆菌毒素蛋白变体和/或其制备的药物防止细菌性疾病产生，发作，复发和/或扩散。例如，使用本申请所述多糖结合物和/或其制备的药物防止细菌性疾病的产生，发作，复发和/或扩散。例如，所述细菌性疾病可以是肺炎链球菌性疾病。例如，所述肺炎链球菌性疾病可以包括肺炎、败血症、脑膜炎和/或中耳炎。
另一方面，本申请提供了疫苗，其可以包含所述蛋白多糖结合物、所述药物组合物和/或任选地药学上接受的佐剂。所述蛋白多糖结合物可以包含所述破伤风杆菌毒素蛋白截短体、所述破伤风杆菌毒素蛋白变体和/或所述破伤风杆菌毒素蛋白的C片段。每种类型的载体蛋白可以用作不止一种糖的载体，其中所述糖可以相同或不同。例如，肺炎链球菌血清型3和5可以与相同的载体蛋白缀合，或者与载体蛋白的相同分子或与相同载体蛋白的不同分子结合。在某些情形下，两种或更多种不同的糖可以与相同的载体蛋白结合，或者与载体蛋白的相同分子或与相同载体蛋白的不同分子结合。
含有本申请药物组合物的疫苗制剂可以通过系统途径或粘膜途径施用所述疫苗，用于保护或治疗易于感染的哺乳动物。这些施用可以包括通过肌内、腹膜内、皮内或皮下途径注射；或通过粘膜施用到口腔/消化道、呼吸道、泌尿生殖道。在某些情形下，所述药物组合物的组分也可以于同一时间或不同时间共同施用(例如可以同时地或在施用疫苗的任何细菌蛋白组分1-2周后，单独施用肺炎链球菌糖结合物，使两者相互之间的免疫应答达到最佳的配合)。对于共同施用，可以在任何或所有不同的施用中存在任选的佐剂。除了单一的施用途径以外，还可以使用2种不同的施用途径。例如，糖或糖结合物可以肌内(或皮内)施用，而细菌蛋 白可以粘膜(或皮内)施用。
另一方面，本申请提供了预防肺炎链球菌性疾病的方法，其可以包含向有需要的受试者施用所述蛋白多糖结合物、所述核酸分子、所述载体、所述细胞、所述载体蛋白、所述药物组合物和/或所述疫苗。在某些情形下，所述肺炎链球菌性疾病可以包括肺炎、败血症、脑膜炎和/或中耳炎。
在某些情形中，本申请提供的所述蛋白多糖结合物、所述药物组合物和/或药学上可接受的佐剂可以用于制备抗血清。在某些情形中，本申请提供的所述蛋白多糖结合物、所述药物组合物和/或药学上可接受的佐剂可以用于制备抗体。所述蛋白多糖结合物可以包含所述破伤风杆菌毒素蛋白截短体、所述破伤风杆菌毒素蛋白变体和/或所述破伤风杆菌毒素蛋白的C片段。每种类型的载体蛋白可以用作不止一种糖的载体，其中所述糖可以相同或不同。所述糖可以是肺炎链球菌荚膜多糖。
在某些情形中，本申请提供的所述蛋白多糖结合物、所述药物组合物和/或药学上可接受的佐剂可以用于检测样品中的抗体。在某些情形中，本申请提供的所述蛋白多糖结合物、所述药物组合物和/或药学上可接受的佐剂可以用于制备检测样品中的抗体的试剂盒。
例如，所述抗体可以是细菌的抗体。例如，所述细菌的抗体可以是肺炎链球菌的抗体。例如，肺炎链球菌血清型3的抗体、肺炎链球菌血清型5的抗体、肺炎链球菌血清型6A的抗体、肺炎链球菌血清型6B的抗体、肺炎链球菌血清型10A的抗体、肺炎链球菌血清型12F的抗体、肺炎链球菌血清型15B的抗体、肺炎链球菌血清型19A的抗体和/或肺炎链球菌血清型33F的抗体。
在某些情形中，本申请提供的所述破伤风杆菌毒素蛋白变体、破伤风杆菌毒素蛋白截短体和/或所述破伤风杆菌毒素蛋白的C片段还可以用于制备抗破伤风杆菌毒素的抗体、用于制备抗破伤风杆菌毒素的抗原和/或疫苗、和/或用于诊断破伤风杆菌毒素抗体的试剂盒。
另一方面，本申请还提供了所述多糖结合物作为抗原制备抗体的用途。
在某些实施方式中，所述抗体用于分离菌株的诊断分型。
另一方面，本申请还提供了试剂盒，其包含所述多糖结合物。
在某些实施方式中，所述试剂盒用于分离菌株的诊断分型。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的蛋白变体、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1：TTD重组蛋白序列设计
基于已公开Clostridium tetani的tetanus neurotoxin序列(NBCI WP011100836)及其晶体结构(PDB:5N0B),本申请设计了破伤风杆菌毒素蛋白截短体(Tetanus Toxin Domain，TTD)，排除了破伤风杆菌毒素蛋白(Tetanus toxin，TT)的N端序列，而包含其C端序列。所以该TTD蛋白没有破伤风杆菌毒素蛋白的毒性，但保留了其C端的结构域及功能片断，在保持蛋白的正确的折叠、稳定性基础上，提供T细胞表位和结构灵活性，更有利于化学偶联。
所设计的蛋白序列01(TTD-1)是将TT序列去除了N端828个氨基酸后，获得的含有487个氨基酸的TTD蛋白序列，即去除了N端的多肽酶M27及部分转位区域，保留了C端的功能域(受体结合域)，氨基酸序列如SEQ ID NO:1所示。相应的核酸序列(TTD-4)经过密码子优化后如序列SEQ ID NO:4所示。
所设计的蛋白序列02(TTD-2)是将TT序列去除了N端839个氨基酸后，获得的含有476个氨基酸的TTD蛋白序列，即去除了N端的多肽酶M27及部分转位区域，保留了C端的功能域(受体结合域)，氨基酸序列如SEQ ID NO:2所示。相应的核酸序列(TTD-5)经过密码子优化后如序列SEQ ID NO:5所示。
所设计的蛋白序列03(TTD-3)是将TT序列去除了N端864个氨基酸后，获得的含有451个氨基酸的TTD蛋白序列，即去除了N端的多肽酶M27及部分易位区域，保留了C端的功能域(受体结合域)，氨基酸序列如SEQ ID NO:3所示。相应的核酸序列(TTD-6)经过密码子优化后如序列SEQ ID NO:6所示。
实施例2：表达载体构建及蛋白表达
为利用大肠杆菌蛋白表达系统，将所设计的蛋白TTD和其相应的基因序列先后通过基因序列合成、引物设计、PCR扩增、分子克隆等常用技术构建到蛋白表达载体中(如pGEX-4T，pET21)，得到重组表达质粒。将重组蛋白表达质粒通过BL21(DE3)感受态细胞(Thermo Fisher)转化后，获得重组表达质粒。TTD-4、TTD-5和TTD-6的PCR电泳的结果如图1所示。取保存的菌种在含Amp +的平板上划线活化，挑取单克隆接种到含有Amp +的LB液体培养基中，于37℃，250rpm培养过夜。将培养的菌种按比例稀释接种于含有Amp +的LB液体培养基中，37℃震荡培养至OD600达到0.5-1.5时，加入1mM IPTG诱导。培养结束后，取样1mL离心，去上清后加入裂解液，进行SDS-PAGE电泳分析，以确定重组蛋白表达。电泳结果如图2所示，均获得了目的TTD蛋白。
实施例3：TTD蛋白的纯化
将重组TTD菌种通过发酵得到的菌体25g，用10倍50mM Tris-HCl缓冲液溶解为250mL的体系，用高压均质机破碎二次，通过高速离心后去沉淀，保留上清。在上清中加入硫酸铵沉淀，低温搅拌2小时，再通过高速离心1小时收集沉淀，将收集得到的沉淀用缓冲液溶解后，通过超滤换液，然后分别通过复合型层析、离子交换层析，并采用不同NaCl浓度梯度洗脱，去除杂质，最后得到纯化度超过95％的TTD蛋白。纯化结果如图3所示。纯化的TTD蛋白通过SEC-HPLC分析显示，蛋白有良好的均一性。TTD-1的SEC-HPLC的结果如图4所示。
实施例4：肺炎链球菌多糖制备
将一管制备好的肺炎链球菌菌种(ATCC)接种到摇瓶中，在37℃CO 2培养箱培养过夜，镜检结果正常后，将培养液接种到10升发酵罐，发酵培养6-10小时。培养结束后，加入灭活剂杀菌灭活，然后通过离心收集澄清培养液，在澄清培养液中加入多糖沉淀剂，得到粗制沉淀复合糖。然后通过一系列纯化后得到精制多糖。通常精制多糖纯化工艺包括，但不限于，加入NaCl溶液溶解复合糖，溶解后离心，取上清，加入酒精去除杂质和沉糖。可通过多次洗涤沉淀去杂，利用层析法例如离子交换柱、复合型填料纯化得到精制多糖。制备的不同类型多糖通过质量分析，各个指标符合欧州药典标准，例如蛋白及核酸等杂质含量均低于1％。
实施例5：制备肺炎链球菌多糖-蛋白结合物
肺炎链球菌多糖经过活化后具有与载体蛋白化学反应活性。多糖-蛋白化学偶联可以直接偶联或通过连接子实施，如利用连接子己二酸二酰肼进行偶联。化学偶联通常方法有通过溴化氰法、CDAP法或还原胺化法(US5952454,EP0720485,US4711779)。通过偶联得到的多糖-蛋白缀合物能诱导比较强的免疫原性，可同时诱导抗多糖和抗蛋白的特异性抗体。
使用溴化氰法制备细菌多糖-蛋白结合物的步骤如下：将纯化多糖200mg溶解在20ml 0.9％NaCl溶液中,加入溴化氰，加入0.5N NaOH溶液,调整溶液pH到10.5,反应10-15分钟后，加入0.5N HCl将溶液调整到pH 7.8。然后加入己二酸二酰肼溶液(ADH)制备成多糖-ADH衍生物。同时将载体蛋白(TTD或CRM97)与EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodimide hydrochloride)反应，制备载体蛋白衍生物。最后将多糖-ADH衍生物与活化载体蛋白按比例混合后，反应生成多糖-蛋白结合物，再通过超滤浓缩或层析，去除杂质及未反应物质，纯化得到多糖-蛋白结合物。
使用CDAP法制备细菌多糖-蛋白结合物的步骤如下：在一个典型的反应中，将肺炎多糖 (50mg)溶解于10ml的硼酸钠缓冲液中(100mM,pH 9.0)后，依次加入CDAP溶液，和0.2N NaOH溶液调整反应液pH到9.0。室温搅拌反应5-10分钟后，加入0.1N HCl溶液调整pH到7.5。然后将反应液通过G25柱脱盐,收集活化多糖。同时将载体蛋白溶解于0.1MNaHCO3 pH8.0缓冲液中,然后与活化多糖混合，在室温条件下，搅拌6-10小时。反应结束后，通过超滤浓缩换液，去除杂质，最终得到多糖-蛋白结合物。
使用还原胺法制备细菌多糖-蛋白结合物的步骤如下：在一个典型的反应中，将冻干多糖溶解于磷酸缓冲液中(4mg/ml)，加入100mM高碘酸钠溶液，搅拌过夜后，通过G25柱脱盐，得到活化多糖。同时将载体蛋白溶解在磷酸缓冲液中，然后按比例与活化多糖溶液混合，加入一定当量氰基硼氢化钠，在室温或37℃度搅拌过夜。反应结束后，将反应液通过层析，去除杂质，纯化得到多糖-蛋白结合物。
实施例6-15：蛋白多糖结合物的免疫原性评价
小鼠免疫实验方案：将肺炎链球菌多糖-蛋白结合物加入氢氧化铝或磷酸铝佐剂，配制成免疫抗原，选择6-8周龄的Balb/c小鼠进行腹腔免疫，每组5-8个小鼠，每次免疫剂量为2微克，分别在第0天、第14天、第21天进行免疫，并在第21天和第35天抽血评价其多糖的免疫原性。
大兔免疫实验方案：将多糖-蛋白结合物加入弗氏佐剂，配制成免疫抗原，选择2～2.5kg的新西兰大白兔进行免疫，每组2只兔。首次以弗氏完全佐剂与抗原乳化混合，皮下免疫，剂量为200微克；第14天以弗氏不完全佐剂与抗原乳化混合，皮下第二次免疫，剂量为200微克；第28天以100微克抗原静脉免疫。并在第21天与42天抽血评价其多糖的免疫原性。
ELISA法评价肺炎链球菌多糖免疫原性：将肺炎链球菌多糖用包被缓冲液稀释，包被到96孔酶标板，100μl/孔，并在37℃孵育5小时后洗板。将小鼠、大兔抗血清通过吸附剂处理后，小鼠血清先以1：200稀释(大兔血清先以1：10000稀释)，然后2.5倍梯度稀释，稀释8个梯度，再加入酶标板中，每孔50μl，孵育过夜。洗板后，将二抗以1：20000稀释后加入酶标板中，每孔100μl，孵育2小时后洗板，然后加入1mg/ml的PNPP-Na显色底物,每孔100μl，孵育2小时后,以50μl/孔加入3M NaOH中止反应，上机读取波长405nm处吸收值。测定孔OD值与阴性孔OD值比值大于或等于2.1判定为阳性，以稀释最大倍数为阳性的稀释度定为每个血清的抗体滴度，并计算每组免疫动物的抗体滴度几何平均值，利用T-test分析不同组别间的统计学意义。
肺炎链球菌荚膜多糖特异性抗体调理吞噬杀菌试验：将肺炎链球菌稀释10 5CFU/ml，以10μl/孔加入到96孔细胞工作板中。将灭活的血清样品梯度稀释后以20μl/孔加入到上述细胞 工作板中，使菌体与抗血清在700rpm/min孵育30min，将经DMF分化的HL-60细胞经HBSS缓冲液洗涤后调整到浓度1×10 7个/ml，再将1×10 7个/ml的细胞液与稀释好的补体按1：4体积比混合，混合液以50μl/孔加入到96孔细胞工作中。将96孔细胞工作板置混匀仪上，放入5％CO 2、温度为37℃的CO 2培养箱中，振荡培养45min。经中止调理吞噬后，点样到血平板中，于CO 2培养箱中培养过夜。计算各稀释血清下的杀菌率。
实施例6：载体蛋白TTD对3型多糖的免疫增强效应
通过比较3型(T3)肺炎链球菌多糖-TTD结合物与3型肺炎链球菌多糖-CRM197的免疫效果，分析该型肺炎链球菌多糖-TTD在小鼠中的免疫增强效应。
分别以3型多糖-CRM197结合物和3型多糖-TTD-1、2、3结合物免疫小鼠，并分别评价在21天和35天的抗体滴度。结果如图5所示，在35天，T3-TTD-1、T3-TTD-2、T3-TTD-3组均可诱导出更加高的抗体滴度。
对35天免疫血清的抗体滴度进行组间分析，经ANOVA单因素方差分析，T3-TTD组与T3-CRM197组比较，P＝0.001<0.01，统计差异有高度统计学意义，说明TTD-1、TTD-2和TTD-3载体蛋白均比CRM197具有更加明显的免疫加强效应。
将T3-CRM197和T3-TTD-1二组35天的免疫血清进行OPA试验。结果如图6所示，T3-CRM197与T3-TTD-1、T3-TTD-2和T3-TTD-3的IC 50(50％杀菌率时抗血清的稀释倍数)分别为1599与9926、7142和5728。结果表明TTD-1、TTD-2和TTD-3载体蛋白结合物诱导产生的3型多糖特异性抗体均明显多于CRM197载体蛋白结合物。
实施例7：载体蛋白TTD对肺炎6B型多糖的免疫增强效应
通过比较6B型(T6B)肺炎链球菌多糖-TTD-1结合物与6B型肺炎链球菌多糖-CRM197的免疫效果，分析该型肺炎链球菌多糖-TTD-1在小鼠中的免疫增强效应。
分别以6B型多糖-CRM197结合物、6B型多糖-TTD-1结合物进行免疫小鼠，并分别评价在21天和35天的抗体滴度。结果如图7所示，在35天，6B多糖-TTD-1结合物可诱导出更加高的抗体滴度。
将6B-TTD-1、6B-CRM197二组21天和35天的免疫血清进行OPA试验。结果如图8所示，6B-TTD-1与6B-CRM197免疫21天血清的IC50(50％杀菌率时抗血清的稀释倍数)分别为2851与746.3，6B-TTD-1与6B-CRM197免疫35天血清的IC50(50％杀菌率时抗血清的稀释倍数)分别为7677与1802，表明TTD-1载体蛋白结合物比CRM197载体蛋白结合物能诱导产生更高的6B型多糖特异性功能抗体。
实施例8：载体蛋白TTD对肺炎15B型多糖的免疫增强效应
通过比较15B型(T15B)肺炎链球菌多糖-TTD-3结合物与15B型肺炎链球菌多糖-CRM197的免疫效果，分析该型肺炎链球菌多糖-TTD在小鼠中的免疫增强效应。
分别以15B型多糖-CRM197结合物和15B型多糖-TTD-3结合物免疫小鼠，并分别评价在21天和35天的抗体滴度，结果如图9所示，在35天，多糖-TTD-3结合物可诱导出更加高的抗体滴度。
将15B-CRM197和15B-TTD-3二组21天与35天的免疫血清进行OPA试验。结果如图10所示，15B-CRM197与15B-TTD-3 21天免疫血清IC 50分别为1392与2633。15B-CRM197与15B-TTD-3 35天免疫血清IC 50分别为9005与14997。表明TTD-3载体蛋白结合物诱导产生的15B型多糖特异性抗体明显多于CRM197载体蛋白结合物。
实施例9：载体蛋白TTD对肺炎6A型多糖的免疫增强效应
分别以6A型(T6A)肺炎链球菌多糖-CRM197结合物和6A型多糖-TTD-3结合物免疫小鼠，并分别评价在21天和35天的抗体滴度(如图11所示)。在35天，多糖-TTD结合物可诱导出不低于多糖-CRM197的抗体滴度。
将T6A-CRM197和T6A-TTD-3二组21天与35天的免疫血清进行OPA试验。结果，如图12所示，T6A-CRM197与T6A-TTD-3 21天免疫血清IC 50分别为458与1606。T6A-CRM197与T6A-TTD-3 35天免疫血清IC 50分别为9103与20850，TTD-3载体蛋白结合物诱导产生的6A型多糖特异性抗体明显高于CRM197载体蛋白结合物。
实施例10：载体蛋白TTD对肺炎7F型多糖的免疫增强效应
7F型(T7F)肺炎链球菌多糖-TTD-1结合物与多糖-CRM197结合物的比较。分别对以多糖-CRM197结合物和多糖-TTD-1结合物进行免疫小鼠，并分别评价在21天和35天的抗体滴度。结果如图13所示，在35天，7F多糖-TTD-1结合物可诱导出与多糖-CRM197的抗体滴度相当。
将T7F-CRM197和T7F-TTD-1二组21天与35天的免疫血清进行OPA试验。结果如图14所示：T7F-CRM197与T7F-TTD-1 21天免疫血清IC 50分别为1213与1925；T7F-CRM197与T7F-TTD-1 35天免疫血清IC50分别为5362与7975。结果表明TTD-1载体蛋白结合物诱导产生的7F型多糖特异性抗体不低于CRM197载体蛋白结合物。
实施例11：载体蛋白TTD对肺炎10A型多糖的免疫增强效应
10A型(T10A)肺炎链球菌多糖-TTD-2结合物与多糖-CRM197结合物的比较。分别对以多糖-CRM197结合物和多糖-TTD-2结合物进行免疫小鼠，并分别评价在21天和35天的抗体滴度，结果如图15所示，在35天，10A多糖-TTD-2结合物可诱导出比多糖-CRM197结合物更高的抗体滴度。
将T10A-CRM197和T10A-TTD-2二组35天的免疫血清进行OPA试验。结果如图16所示：T10A-CRM197与T10A-TTD-2 35天免疫血清IC 50分别为23264与30709。结果表明TTD-2载体蛋白结合物诱导产生的10A型多糖特异性抗体与CRM197载体蛋白结合物相当。
实施例12：载体蛋白TTD对肺炎19A型多糖的免疫增强效应
通过比较19A型(T19A)肺炎链球菌多糖-TTD结合物与19A型肺炎链球菌多糖-CRM197的免疫效果，分析该型肺炎链球菌多糖-TTD在新西兰大白兔中的免疫增强效应。
分别以19A型多糖-CRM197结合物和19A型多糖-TTD-1结合物进行免疫新西兰大白兔，并分别评价在21天和42天的抗体滴度。结果如图17所示，在42天，多糖-TTD结合物可诱导出更加高的抗体滴度。
将T19A-CRM197和T19A-TTD-1二组42天的免疫血清进行OPA试验。结果如图18所示，19A-CRM197与19A-TTD-1 42天免疫血清IC 50分别为8617与35780。结果表明TTD-1载体蛋白结合物诱导产生的19A型多糖特异性抗体明显多于CRM197载体蛋白结合物。
实施例13：载体蛋白TTD对肺炎1型多糖的免疫增强效应
1型(T1)肺炎链球菌多糖-TTD-3结合物与多糖-CRM197结合物的比较。分别对以多糖-CRM197结合物和多糖-TTD-3结合物进行免疫大兔，并分别评价在21天和42天的抗体滴度。结果如19所示，在42天，多糖-TTD结合物可诱导出更加高的抗体滴度。
将T1-CRM197和T1-TTD-3二组21天与42天的免疫血清进行OPA试验。结果如图20所示：T1-CRM197与T1-TTD-3 21天免疫血清IC 50分别为6523与35571；T1-CRM197与T1-TTD-3 42天免疫血清IC 50分别为193732与493114。结果表明TTD-3载体蛋白结合物诱导产生的1型多糖特异性抗体明显高于CRM197载体蛋白结合物。
实施例14：载体蛋白TTD对其他型肺炎链球菌多糖的免疫增强效应
其它血清型肺炎链球菌多糖结合物，如血清型5、12F、33F、19F。分别以多糖-CRM197结合物和多糖-TTD结合物免疫小鼠，并分别评价在21天和35天的抗体滴度(如图21所示)。 在35天，多糖-TTD结合物可诱导出更加高的抗体滴度。
本发明同时比较了TTD蛋白对其他血清型肺炎多糖的免疫增强效应，发现TTD蛋白比CRM197能诱导更高的免疫原性。例如，以多糖-CRM197结合物和多糖-TTD结合物分别进行免疫小鼠，并分别评价在21天和35天的抗体滴度，在35天，多糖-TTD结合物诱导出抗体滴度与多糖-CRM197结合物的抗体滴度比值可达到2-20倍，进一步说明TTD作为载体蛋白的优势。
实施例15：多糖-与TTD变异体结合物的免疫原性
TTD变异体的设计：以蛋白序列01、02、03为基础，截断N端不同长度的氨基酸，而包含其C端序列。
多糖-TTD变异体结合物的免疫原性：分别以X型多糖-CRM197结合物(TX-CRM197组)和X型多糖-TTD结合物(TX-TTD)免疫小鼠，并分别评价在21天和35天的抗体滴度。在35天，多糖-TTD结合物可诱导出更加高的抗体滴度。X为任一种肺炎链球菌多糖糖型。
将TX-CRM197和TX-TTD二组35天的免疫血清进行OPA试验。结果表明TTD载体蛋白结合物诱导产生的X型多糖特异性抗体明显多于CRM197载体蛋白结合物。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (84)

  1. 一种破伤风杆菌毒素蛋白变体，其包含所述破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段。
  2. 根据权利要求1所述的破伤风杆菌毒素蛋白变体，其中所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的T细胞表位P2。
  3. 根据权利要求1-2中任一项所述的破伤风杆菌毒素蛋白变体，其中所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
  4. 根据权利要求1-3中任一项所述的破伤风杆菌毒素蛋白变体，其中所述易位区域部分片段包含SEQ ID NO：7-8中任一项所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的破伤风杆菌毒素蛋白变体，其中所述破伤风杆菌毒素蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的破伤风杆菌毒素蛋白变体，其包含SEQ ID NO：1-2中任一项所示的氨基酸序列。
  7. 蛋白多糖结合物，其包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体(TTD)。
  8. 根据权利要求7所述的蛋白多糖结合物，其中所述多糖源自肺炎链球菌荚膜多糖。
  9. 根据权利要求7-8中任一项所述的蛋白多糖结合物，其中所述多糖具备一种以上肺炎链球菌血清型。
  10. 根据权利要求7-9中任一项所述的蛋白多糖结合物，其中所述多糖选自下组中的任一种肺炎链球菌血清型：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。
  11. 根据权利要求7-10中任一项所述的蛋白多糖结合物，其中所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C片段。
  12. 根据权利要求7-11中任一项所述的蛋白多糖结合物，其中所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C片段，其中所述多糖选择下组中的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。
  13. 根据权利要求7-12中任一项所述的蛋白多糖结合物，其中所述破伤风杆菌毒素蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
  14. 根据权利要求7-13中任一项所述的蛋白多糖结合物，其中所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段和所述破伤风杆菌毒素蛋白的易位区域部分片段。
  15. 根据权利要求14所述的蛋白多糖结合物，其中所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的广谱的T细胞表位P2。
  16. 根据权利要求14-15中任一项所述的蛋白多糖结合物，其中所述易位区域部分片段包含所 述破伤风杆菌毒素蛋白的第829-864位氨基酸。
  17. 根据权利要求14-16中任一项所述的蛋白多糖结合物，其中所述易位区域部分片段包含SEQ ID NO：7-8中任一项所示的氨基酸序列。
  18. 根据权利要求7-17中任一项所述的蛋白多糖结合物，其中所述破伤风杆菌毒素蛋白截短体包含SEQ ID NO：1-3中任一项所示的氨基酸序列。
  19. 根据权利要求7-18中任一项所述的蛋白多糖结合物，其中所述多糖与所述破伤风杆菌毒素蛋白截短体的质量比为0.4-2.5。
  20. 根据权利要求10-19中任一项所述的蛋白多糖结合物，其中所述1型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  21. 根据权利要求10-20中任一项所述的蛋白多糖结合物，其中所述3型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  22. 根据权利要求10-21中任一项所述的蛋白多糖结合物，其中所述5型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  23. 根据权利要求10-22中任一项所述的蛋白多糖结合物，其中所述6A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  24. 根据权利要求10-23中任一项所述的蛋白多糖结合物，其中所述6B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  25. 根据权利要求10-24中任一项所述的蛋白多糖结合物，其中所述7F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  26. 根据权利要求10-25中任一项所述的蛋白多糖结合物，其中所述10A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  27. 根据权利要求10-26中任一项所述的蛋白多糖结合物，其中所述12F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  28. 根据权利要求10-27中任一项所述的蛋白多糖结合物，其中所述15B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  29. 根据权利要求10-28中任一项所述的蛋白多糖结合物，其中所述19A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  30. 根据权利要求10-29中任一项所述的蛋白多糖结合物，其中所述19F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  31. 根据权利要求10-30中任一项所述的蛋白多糖结合物，其中所述33F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  32. 根据权利要求7-31中任一项所述的蛋白多糖结合物，可以采用偶联的方法将所述多糖和所述蛋白变体偶联起来。
  33. 根据权利要求32中所述的蛋白多糖结合物，其中所述偶联的方法包含以下任一种方法：溴化氢法、CDAP法、还原胺法。
  34. 一种提高免疫原性的方法，其包括以下的步骤：提供包含来自肺炎链球菌的多糖和破伤风杆菌毒素蛋白截短体的蛋白多糖结合物。
  35. 根据权利要求34所述的方法，其中所述多糖源自肺炎链球菌荚膜多糖。
  36. 根据权利要求34-35中任一项所述的方法，其中所述多糖具备一种以上肺炎链球菌血清型。
  37. 根据权利要求34-36中任一项所述的蛋白多糖结合物，其中所述多糖选自下组中的任一种肺炎链球菌血清型：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。
  38. 根据权利要求34-37中任一项所述的方法，其中所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段。
  39. 根据权利要求34-38所述的方法，其中所述破伤风杆菌毒素蛋白截短体包含所述破伤风杆菌毒素蛋白的C片段，其中所述多糖选择下组中的任一种肺炎链球菌血清型：1、3、5、6A、6B、7F、10A、12F、15B、19A、19F和33F。
  40. 根据权利要求38-39中任一项所述的方法，其中所述破伤风杆菌蛋白的C片段包含SEQ ID NO：3所示的氨基酸序列。
  41. 根据权利要求34-40中任一项所述的方法，其中所述破伤风杆菌毒素蛋白截短体包含破伤风杆菌毒素蛋白的C片段和破伤风杆菌毒素蛋白易位区域部分片段。
  42. 根据权利要求41中所述的方法，其中所述易位区域部分片段包含所述破伤风杆菌毒素蛋白的易位区域的广谱的T细胞表位P2。
  43. 根据权利要求41-42中任一项所述的方法，其中所述的破伤风杆菌毒素蛋白的易位区域部分片段包含所述破伤风杆菌毒素蛋白的第829-864位氨基酸。
  44. 根据权利要求34-43中任一项所述的方法，其中所述破伤风杆菌毒素蛋白截短体包含SEQ ID NO：1-3中任一项所示的氨基酸序列。
  45. 根据权利要求34-44中任一项所述的方法，其中所述肺炎链球菌多糖与所述破伤风杆菌毒素蛋白截短体的质量比为0.4-2.5。
  46. 根据权利要求37-45中任一项所述的方法，其中所述1型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  47. 根据权利要求37-46中任一项所述的方法，其中所述3型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  48. 根据权利要求37-47中任一项所述的方法，其中所述5型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  49. 根据权利要求37-48中任一项所述的方法，其中所述6A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  50. 根据权利要求37-49中任一项所述的方法，其中所述6B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  51. 根据权利要求37-50中任一项所述的方法，其中所述7F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  52. 根据权利要求37-51中任一项所述的方法，其中所述10A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  53. 根据权利要求37-52中任一项所述的方法，其中所述12F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  54. 根据权利要求37-53中任一项所述的方法，其中所述15B型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  55. 根据权利要求37-54中任一项所述的方法，其中所述19A型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  56. 根据权利要求37-55中任一项所述的方法，其中所述19F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  57. 根据权利要求37-56中任一项所述的方法，其中所述33F型肺炎链球菌多糖与所述蛋白截短体的质量比为0.4-2.5。
  58. 根据权利要求34-57中任一项所述的方法，其中所述方法包含采用偶联的方法将所述多糖和所述蛋白变体偶联起来。
  59. 根据权利要求58所述的方法，其中所述偶联的方法包含以下任一种方法：溴化氢法、CDAP法、还原胺法。
  60. 根据权利要求34-59中任一项所述的方法，其中所述提高细菌多糖免疫原性包含与多糖CRM197结合物相比，所述蛋白多糖结合物的免疫原性更高。
  61. 根据权利要求34-60中任一项所述的方法，其中所述免疫原性更高是在动物免疫实验中检测到的。
  62. 根据权利要求61中所述的方法，其中所述动物免疫试验包含以下步骤：将权利要求7-33所述的蛋白多糖结合物与佐剂配制成免疫抗原。
  63. 根据权利要求62中所述的方法，其中所述的免疫抗原的注射方法包含腹腔注射、皮下注射、肌肉注射和/或静脉注射。
  64. 根据权利要求61-63中任一项所述的方法，其中所述动物免疫试验包含以下步骤：对获得的免疫动物的血清中的抗体进行ELISA检测。
  65. 根据权利要求61-64中任一项所述的方法，其中所述动物免疫试验包含以下步骤：对获得的免疫动物的血清进行调理吞噬杀菌试验。
  66. 根据权利要求61-65中任一项所述的方法，其中所述动物包含小鼠、大鼠和/或兔。
  67. 根据权利要求62-66中任一项所述的方法，其中所述佐剂包含氢氧化铝、磷酸铝和/或弗氏佐剂等。
  68. 核酸分子，其包含编码权利要求1-6中任一项所述的破伤风杆菌毒素蛋白变体。
  69. 根据权利要求68所述的核酸分子，其包含如SEQ ID NO：4-6所示的核苷酸序列。
  70. 载体，其包含权利要求68-69中任一项所述的核酸分子。
  71. 细胞，其包含权利要求68-69中任一项所述的核酸分子或权利要求70所述的载体。
  72. 一种载体蛋白，其包含权利要求1-6中任一项所述的破伤风杆菌毒素蛋白变体。
  73. 药物组合物，其包含权利要求7-33中任一项所述的蛋白多糖结合物和任选地药学上可接受的佐剂。
  74. 权利要求1-6中任一项所述的破伤风杆菌毒素蛋白变体用于制备药物的用途。
  75. 权利要求7-33中任一项所述的多糖结合物用于制备药物的用途。
  76. 根据权利要求74-75中任一项所述的用途，其中所述药物用于预防和/或治疗肺炎链球菌性疾病。
  77. 根据权利要求76所述的用途，其中所述肺炎链球菌性疾病包括肺炎、败血症、脑膜炎和/或中耳炎。
  78. 疫苗，其包含权利要求7-33中任一项所述的蛋白多糖结合物、权利要求73所述的药物组合物和/或任选地药学上可接受的佐剂。
  79. 预防肺炎链球菌性疾病的方法，其包含向有需要的受试者施用权利要求7-33中任一项所述蛋白多糖结合物、权利要求68-69中任一项所述的核酸分子、权利要求70所述的载体、权利要求71所述的细胞、权利要求72所述的载体蛋白、权利要求73所述的药物组合物和/或权利要求78所述的疫苗。
  80. 根据权利要求79所述的方法，其中所述肺炎链球菌性疾病包括肺炎、败血症、脑膜炎 和/或中耳炎。
  81. 权利要求7-33中任一项所述的多糖结合物作为抗原制备抗体的用途。
  82. 根据权利要求81所述的用途，其中所述抗体用于分离菌株的诊断分型。
  83. 试剂盒，其包含权利要求7-33中任一项所述的多糖结合物。
  84. 根据权利要求83所述的试剂盒，其用于分离菌株的诊断分型。
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