WO2020259076A1 - 一种糖缀合物及其用途 - Google Patents

一种糖缀合物及其用途 Download PDF

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WO2020259076A1
WO2020259076A1 PCT/CN2020/087841 CN2020087841W WO2020259076A1 WO 2020259076 A1 WO2020259076 A1 WO 2020259076A1 CN 2020087841 W CN2020087841 W CN 2020087841W WO 2020259076 A1 WO2020259076 A1 WO 2020259076A1
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glycoconjugate
capsular polysaccharide
protein
streptococcus pneumoniae
carrier protein
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PCT/CN2020/087841
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English (en)
French (fr)
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王浩猛
张慢慢
严志红
李军强
朱涛
巢守柏
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康希诺生物股份公司
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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]
    • 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
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • A61K2039/6075—Viral proteins

Definitions

  • the invention relates to the technical field of vaccine development, in particular to a glycoconjugate prepared by reacting Streptococcus pneumoniae type 4 capsular polysaccharide with a carrier protein, an immunogenic composition containing the glycoconjugate, and Application of the glycoconjugate and immunogenic composition in the preparation of drugs or vaccines for preventing and/or treating individual Streptococcus pneumoniae infections and diseases related to Streptococcus pneumoniae.
  • Streptococcus pneumoniae is a gram-positive bacterium with a spear-like body, double or short chain-like diplococci, with opposite wide ends, opposite tips, and a thick capsule. Streptococcus pneumoniae infection is one of the main factors leading to morbidity and mortality worldwide. Pneumonia, febrile bacteremia and meningitis are the most common manifestations of invasive pneumococcal disease, and the spread of bacteria in the respiratory tract can lead to middle ear infections, sinusitis or recurrent bronchitis.
  • composition and structure of the capsular polysaccharide outside the cell wall of Streptococcus pneumoniae are often related to the pathogenicity and serotype of the bacteria.
  • Streptococcus pneumoniae conjugate vaccine (PCV) is a pneumococcal vaccine used to prevent diseases caused by Streptococcus pneumoniae.
  • the 13-valent vaccine PREBNAR is available worldwide .
  • the polysaccharide protein conjugate vaccine is prepared using the polysaccharide linked to the protein carrier.
  • the chemical bond between the polysaccharide and the protein carrier can be induced Immune response against bacteria exhibiting polysaccharides contained in vaccines on its surface, thereby preventing diseases, therefore, vaccination using polysaccharides derived from pathogenic bacteria is a potential strategy for enhancing host immunity.
  • the non-patent literature Deciphering Antigenic Determinants of Streptococcus pneumonia Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides discloses that the pyruvate base is a Streptococcus pneumoniae serotype 4 capsular polysaccharide conserveed antigenic sites.
  • the patent CN101180079A discloses the preparation method of the vaccine, including the weak acid hydrolysis of Streptococcus pneumoniae serotype 4 polysaccharide to remove part of the pyruvate protective group, and then oxidation by sodium periodate to obtain the pro-aldehyde group, which is then conjugated with the protein.
  • the polysaccharide protein conjugate is obtained.
  • the vaccine prepared by this method has achieved good results in terms of immune response and functional activity.
  • the method of oxidizing sugar chains by sodium periodate must require two adjacent free hydroxyl groups in the sugar chains.
  • the capsule There is still room for improvement in the immunogenicity, yield and bactericidal effect of polysaccharides.
  • Patent CN108524931A discloses activation of capsular polysaccharide and reaction with carrier protein to prepare glycoconjugates.
  • the capsular polysaccharide is S. pneumoniae (Pn)-serotypes 3, 10A, 12F and 33F Capsular polysaccharide, and verify the stability and immunogenicity of the glycoconjugate.
  • the immunogenic composition can also include Streptococcus pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, Glycoconjugates prepared by 19F, 22F and 23F capsular polysaccharides did not disclose the glycoconjugates of Streptococcus pneumoniae serotype 4 capsular polysaccharides coupled with protein carriers, nor did it disclose the activation of serotype 4 capsular polysaccharides. Oxidation site.
  • the present invention provides a glycoconjugate of Streptococcus pneumoniae serotype 4 capsular polysaccharide with multiple active sites coupled to a protein carrier, and discloses Streptococcus pneumoniae serotype 4 pod
  • the oxidation sites activated by membrane polysaccharides, the oxidation sites include acetylaminopyran-type mannosyl ( ⁇ -D-ManpNAc), acetylaminopyran-type galactosyl ( ⁇ -D-GalpNAc) and/or pyran Galactopyranosyl ( ⁇ -D-Galp).
  • the present invention also provides a method for preparing glycoconjugates.
  • the present invention further provides an immunogenic composition comprising the glycoconjugate.
  • the present invention also provides the application of the glycoconjugate and the immunogenic composition in the preparation of drugs or vaccines for preventing and/or treating individual Streptococcus pneumoniae infection and diseases related to Streptococcus pneumoniae.
  • the glycoconjugate of the present invention Compared with the conjugate of the connexin carrier after oxidation with periodate in the prior art, the glycoconjugate of the present invention has higher immunogenicity and significantly enhanced bactericidal effect.
  • the first aspect of the present invention provides a glycoconjugate, which is prepared by oxidizing the primary hydroxyl groups of bacterial capsular polysaccharides to aldehyde groups and reacting with the primary amino groups of lysine of the carrier protein. Obtained, wherein the bacterial capsular polysaccharide is Streptococcus pneumoniae type 4 capsular polysaccharide, and the primary hydroxyl group of Streptococcus pneumoniae type 4 capsular polysaccharide is oxidized to aldehyde groups including ⁇ -D-ManpNAc, ⁇ -D -GalpNAc and/or ⁇ -D-Galp.
  • the capsular polysaccharide of Streptococcus pneumoniae type 4 may be natural or artificially synthesized.
  • the carrier protein contains one or more primary amino groups.
  • the carrier protein can be a related protein antigen from a target pathogen that enhances the specific immune response to the pathogen, or a general immunogenic protein that is mainly used as an adjuvant or a general immune response stimulator.
  • the carrier protein is selected from CRM197, tetanus toxoid, outer membrane protein from gram-negative bacteria, Haemophilus influenzae surface lipoprotein (HiD), HiD protein gene from Haemophilus influenzae and A 1:1 fusion protein formed by the Hin47 protein gene of Haemophilus influenzae, pertussis toxin/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein or respiratory syncytial virus F and G protein or Its active part.
  • HiD Haemophilus influenzae surface lipoprotein
  • HiD HiD protein gene from Haemophilus influenzae
  • the carrier protein is CRM197.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8 to 1.7.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 1.0 to 1.4.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8-1.2.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8-1.0.
  • the carrier protein Preferably, for every 10 to 50 sugar repeating units of the bacterial capsular polysaccharide, there is at least one covalent bond between the carrier protein and the bacterial capsular polysaccharide.
  • the second aspect of the present invention provides a method for preparing glycoconjugates, including:
  • nitroxyl compounds and oxidants as activators to oxidize the primary hydroxyl groups of bacterial capsular polysaccharides to aldehyde groups
  • the aldehyde group-containing bacterial capsular polysaccharide is reacted with the primary lysine amino group of the carrier protein to prepare glycoconjugates;
  • the bacterial capsular polysaccharide is Streptococcus pneumoniae type 4 capsular polysaccharide
  • the primary hydroxyl group of Streptococcus pneumoniae type 4 capsular polysaccharide is oxidized to aldehyde groups.
  • the oxidation sites include ⁇ -D-ManpNAc and ⁇ -D-GalpNAc. And/or ⁇ -D-Galp.
  • the molar ratio of the nitroxyl compound to the capsular polysaccharide (0.02-0.1):1.
  • the molar ratio of the nitroxyl compound to the capsular polysaccharide (0.06 ⁇ 0.08):1.
  • the molar ratio of the oxidant to the capsular polysaccharide (0.1-5):1.
  • the molar ratio of the oxidant to the capsular polysaccharide (1.8-2.5):1.
  • the unreacted aldehyde group is reduced to a primary alcohol in a blocking reaction using sodium borohydride after conjugation with the carrier protein, so that the sugar epitope modification degree in the modification step involving oxidation and subsequent conjugation is minimize.
  • the nitroxyl compound has the ability to selectively oxidize primary hydroxyl groups in the presence of the oxidizing agent to generate aldehyde groups.
  • the oxidizing agent is an N-halogenated oxidizing agent capable of selectively oxidizing primary hydroxyl groups in the presence of nitroxyl compounds to generate aldehyde groups.
  • the carrier protein contains one or more primary amino groups.
  • the carrier protein can be a related protein antigen from a target pathogen that enhances the specific immune response to the pathogen, or a general immunogenic protein that is mainly used as an adjuvant or a general immune response stimulator.
  • the carrier protein is selected from CRM197, tetanus toxoid, outer membrane protein from gram-negative bacteria, Haemophilus influenzae surface lipoprotein (HiD), HiD protein gene from Haemophilus influenzae and A 1:1 fusion protein formed by the Hin47 protein gene of Haemophilus influenzae, pertussis toxin/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein or respiratory syncytial virus F and G protein or Its active part.
  • HiD Haemophilus influenzae surface lipoprotein
  • HiD HiD protein gene from Haemophilus influenzae
  • the carrier protein is CRM197.
  • the capsular polysaccharide of Streptococcus pneumoniae type 4 may be natural or artificially synthesized.
  • the nitroxyl compound is 2,2,6,6-tetramethyl-1-piperidinoxy radical (TEMPO) or a derivative thereof.
  • TEMPO 2,2,6,6-tetramethyl-1-piperidinoxy radical
  • the TEMPO or its derivatives are selected from: TEMPO, 2,2,6,6-tetramethylpiperidine oxide, 2,2,6,6-tetramethyl-4-(methyl Sulfonyloxy)-1-piperidinyloxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamido)-TEMPO Free radicals, 4-hydroxy-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxy, 4-cyano-TEMPO, 4-methoxy-TEMPO, 4-carboxyl -TEMPO, 4-(2-bromoacetamido)-TEMPO or 4-amino-TEMPO.
  • the nitroxyl compound is TEMPO.
  • the molar ratio of TEMPO to capsular polysaccharide (0.02-0.1):1.
  • the molar ratio of TEMPO to capsular polysaccharide (0.06 ⁇ 0.08):1.
  • the oxidant is an oxidant with N halogenation.
  • the oxidizing agent is selected from: N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, 1,3,5-trichloro-1,3 ,5-triazine-2,4,6-trione, 1,3,5-tribromo-1,3,5-triazine-2,4,6-trione or 1,3,5-triiodo -1,3,5-triazine-2,4,6-trione.
  • the oxidizing agent is N-iodosuccinimide (NIS).
  • the molar ratio of NIS to capsular polysaccharide (0.1-5):1.
  • the molar ratio of NIS to capsular polysaccharide (1.8-2.5):1.
  • the oxidation reaction of the primary hydroxyl group to an aldehyde group is carried out in an aqueous solvent or an aprotic solvent.
  • the aqueous solvent or aprotic solvent is selected from N-methyl-2-pyrrolidone (NMP), DMSO (dimethyl sulfoxide), sulfolane, dimethylacetamide (DMA), hexamethylphosphorus Amide (HMPA) or DMF (dimethylformamide).
  • NMP N-methyl-2-pyrrolidone
  • DMSO dimethyl sulfoxide
  • sulfolane dimethylacetamide
  • DMA dimethylacetamide
  • HMPA hexamethylphosphorus Amide
  • DMF dimethylformamide
  • the nitroxyl compound is used in a catalytic amount ⁇ 0.1 molar equivalent, and the desired degree of oxidation of the capsular polysaccharide can be achieved by changing the amount of the oxidant used.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.3 to 3.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8 to 1.7.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 1.0 to 1.4.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8-1.2.
  • the mass ratio of the bacterial capsular polysaccharide to the carrier protein is 0.8-1.0.
  • the carrier protein Preferably, for every 10 to 50 sugar repeating units of the bacterial capsular polysaccharide, there is at least one covalent bond between the carrier protein and the bacterial capsular polysaccharide.
  • the oxidation time is 1-4 hours.
  • the oxidation time is 2-4 hours.
  • the preparation method of the glycoconjugate includes:
  • the type 4 capsular polysaccharide of Streptococcus pneumoniae Hydrolyze the type 4 capsular polysaccharide of Streptococcus pneumoniae to a molecular weight of 50-900kDa, and use TEMPO and NIS as the activator to remove the ⁇ -D-ManpNAc, ⁇ -D-GalpNAc and ⁇ -
  • the primary hydroxyl at the D-Galp site is oxidized to an aldehyde group to obtain activated Streptococcus pneumoniae type 4 capsular polysaccharide; preferably, the degree of oxidation of the activated Streptococcus pneumoniae type 4 capsular polysaccharide is 10-30, and further Preferably, the degree of oxidation of the activated Streptococcus pneumoniae type 4 capsular polysaccharide is 12-20. More preferably, the degree of oxidation of the activated Streptococcus pneumoniae type 4 capsular polysaccharide is 13-18.
  • step 2) The activated Streptococcus pneumoniae type 4 capsular polysaccharide in step 1) is reacted with the primary lysine amino group of CRM197 to prepare a glycoconjugate. Among them, the unreacted aldehyde group is reduced to a primary alcohol in a blocking reaction using sodium borohydride after conjugation with the carrier protein.
  • the Streptococcus pneumoniae type 4 capsular polysaccharide is hydrolyzed to a molecular weight of 300-890 kDa.
  • the step 1) further includes the step of purifying the activated Streptococcus pneumoniae type 4 capsular polysaccharide.
  • the temperature of the sealing reaction in step 2) is 20-50°C.
  • the blocking reaction time in step 2) is 20-60h.
  • the third aspect of the present invention provides an immunogenic composition
  • an immunogenic composition comprising the glycoconjugate according to the first aspect of the present invention or the glycoconjugate prepared by the preparation method according to the second aspect of the present invention, and a pharmaceutically acceptable composition Accepted excipients, carriers and/or diluents.
  • the immunogenic composition further comprises glycoconjugates of other bacterial capsular polysaccharides, and the other bacterial capsular polysaccharides are selected from Streptococcus pneumoniae serotypes 1, 3, 3F, 5, 6A, 6B , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F capsular polysaccharides.
  • the dosage form of the immunogenic composition is selected from: tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, submicroemulsions, nanoparticles, gels, powders, Suspended emulsion, cream, jelly, spray, etc.
  • the method of administration of the immunogenic composition is selected from the group consisting of oral, enteral, subcutaneous, intramuscular, intravenous, nasal, transdermal, subconjunctival, and intraocular administration , Orbital administration, retro-ocular administration, retinal administration, choroidal administration, intrathecal injection, etc.
  • the immunogenic composition further includes an adjuvant.
  • the adjuvant is an aluminum adjuvant.
  • the aluminum-based adjuvant is selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide.
  • the fourth aspect of the present invention provides the glycoconjugate according to the first aspect of the present invention, the glycoconjugate prepared by the preparation method according to the second aspect of the present invention, or the immunogenicity according to the third aspect of the present invention.
  • glycoconjugates and immunogenic compositions provided by the present invention have high immunogenicity and induce therapeutic immune responses in individuals.
  • the disease related to Streptococcus pneumoniae is selected from the group consisting of pneumonia, meningitis, cellulitis, osteomyelitis, endocarditis, septic shock, febrile bacteremia, middle ear infection, sinusitis, recurrence Type bronchitis and other serious aggressive diseases.
  • the fifth aspect of the present invention provides a drug for preventing and/or treating individual Streptococcus pneumoniae infections and diseases related to Streptococcus pneumoniae.
  • the drug includes the glycoconjugate or immunogenicity of the present invention. combination.
  • the sixth aspect of the present invention provides a vaccine for preventing and/or treating individual Streptococcus pneumoniae infections and diseases related to Streptococcus pneumoniae.
  • the vaccine includes the glycoconjugate or immunogenicity of the present invention. combination.
  • the seventh aspect of the present invention provides a method for preventing and/or treating an individual's Streptococcus pneumoniae infection and a disease associated with Streptococcus pneumoniae, the method comprising administering to the individual an effective dose of the glycoconjugate of the present invention Compound or immunogenic composition.
  • glycoconjugate in the present invention refers to a sugar covalently conjugated to a carrier protein. Wherein, the glycoconjugate may contain a certain amount of free sugar.
  • the "degree of oxidation" in the present invention refers to the molar ratio of sugar repeating units per mole of aldehyde.
  • “Pharmaceutically acceptable” in the present invention means that it neither significantly stimulates the individual nor inhibits the biological activity and properties of the active substance of the administered product.
  • the "prevention" in the present invention refers to all behaviors that suppress symptoms or delay the tension of specific symptoms by administering the products of the present invention.
  • treatment refers to therapeutic intervention to improve the signs, symptoms, etc. of the disease or pathological state after the disease has begun to develop.
  • the "individual” in the present invention includes mammals and humans.
  • the "effective dose” in the present invention refers to the amount or dose of the composition of the present invention that provides the desired treatment or prevention after being administered to an individual or organ in a single or multiple doses.
  • Figures A and B respectively show the structural formula and expression of the capsular polysaccharide of Streptococcus pneumoniae serotype 4.
  • the three ellipses in Figure A indicate the oxidation sites of the capsular polysaccharide by TEMPO/NIS oxidation, respectively For ⁇ -D-ManpNAc, ⁇ -D-GalpNAc, and ⁇ -D-Galp, the position pointed by the arrow indicates the site of periodate-mediated oxidation, specifically ⁇ -D-Galp.
  • Figure 2 Comparison of the yield of S. pneumoniae serotype 4-CRM197 glycoconjugate prepared using periodate oxidation versus TEMPO/NIS oxidation.
  • Figure 3 Comparison of the immunogenicity of S. pneumoniae serotype 4-CRM197 glycoconjugate prepared using periodate oxidation and TEMPO/NIS oxidation.
  • Figure 4 Comparison of bactericidal effects of S. pneumoniae serotype 4-CRM197 glycoconjugate prepared using periodate oxidation and TEMPO/NIS oxidation.
  • Figure 5 Comparison of the nuclear magnetic structures of activated capsular polysaccharides and conjugates produced by periodate oxidation and activated capsular polysaccharides and conjugates produced by TEMPO/NIS oxidation.
  • F4 is the serotype of Streptococcus pneumoniae 4 Capsular polysaccharides.
  • TEMPO 2,2,6,6-tetramethylpiperidine oxide, Sinopharm Chemical Reagent Co., Ltd.;
  • NIS N-iodosuccinimide, Sinopharm Chemical Reagent Co., Ltd.;
  • Rabbit Beijing Beishengyan Biological Products Co., Ltd., ordinary rabbit, about 2.5kg;
  • Serotype 4 capsular polysaccharide homemade by Cansino Biotech Co., Ltd.;
  • CRM197 carrier protein self-made by Cansino Biotech Co., Ltd.;
  • serotype 4-CRM197 glycoconjugates 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) and N-iodosuccinyl as a co-oxidant were used Imine (NIS) is used to oxidize primary alcohols to aldehyde groups and react with the primary lysine amino groups of the carrier protein to prepare serotype 4-CRM197 glycoconjugates. NMR analysis indicated that the oxidation site is different from that of periodate-mediated oxidation.
  • TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy radical
  • N-iodosuccinyl N-iodosuccinyl as a co-oxidant
  • the oxidation sites are ⁇ -D-ManpNAc, ⁇ -D-GalpNAc, and ⁇ -D-Galp (see the elliptical circle part of Figure 1A), and when periodate is used, ⁇ - D-Galp is the main oxidation site (see arrow position in Figure 1A).
  • TEMPO/NIS Use TEMPO/NIS to activate serotype 4 capsular polysaccharide, specifically: 2g/L serotype 4 capsular polysaccharide polysaccharide concentration, the final concentration is 0.25M NaHCO 3 /0.025M Na 2 CO 3 pH8.6 carbonate Buffer solution, add 0.08Meq TEMPO, add NIS according to Table 1, avoid light with magnetic stirring, and perform activation reaction at room temperature for 2 hours.
  • the sugar content was measured by the anthrone method, the aldehyde group content was measured by the MBTH method, the molecular weight was measured by TSK, and the capsular polysaccharide structure was measured by NMR.
  • the reaction concentration of capsular polysaccharide is 10-20g/L, add CRM197 carrier protein, mix well, add 0.5-2g/L sodium cyanoborohydride, avoid light, and react at 37°C for 48h.
  • sodium borohydride is added to the reaction concentration to be 0.5 g/L, and the reaction is carried out for 1-3 h.
  • the capsular polysaccharide content was measured by anthrone method, protein content was measured by Lowry method, free polysaccharide content was measured by DOC precipitation method, free protein content was measured by SDS-PAGE method, and K D was measured by CL-4B.
  • Elisa method Dilute type 4 capsular polysaccharide with coating solution to the corresponding coating concentration, add 100 microliters to each well of a 96-well plate, coat overnight at 2 ⁇ 8°C, discard the coating solution, and wash the plate once , Add the serum to be tested, incubate at room temperature (20 ⁇ 25°C) for 1h, wash the plate 5 times, add 100 ⁇ l/well of 1:5000 diluted alkaline phosphatase labeled goat anti-mouse IgG, incubate at room temperature for 1h, wash the plate 5 Next, add 100 microliters of color-developing solution/well, avoid light for 30min, add 50 microliters of stop solution/well, and take the absorbance value at 405nm wavelength.
  • the sodium periodate test group used sodium periodate to oxidize the capsular polysaccharide of Streptococcus pneumoniae serotype 4 and prepare the conjugate.
  • the capsular polysaccharide of Streptococcus pneumoniae serotype 4 was hydrolyzed by weak acid to remove part of the pyruvate protective group. After oxidation by sodium periodate, the ortho aldehyde group is obtained, and then conjugated with the protein to obtain a polysaccharide protein conjugate (see patent CN101180079A for details).
  • the opsonophagocytic activity (OPA) titer of the serotype 4-CRM197 conjugate was determined in rabbits under standard conditions.
  • the conjugate produced by TEMPO/NIS batch TEMPO-1, TEMPO-2, TEMPO-3, TEMPO-4, respectively, see Table 2 for F4- 1.
  • Characterization data of F4-3, F4-4, F4-5 conjugates higher yield (see Figure 2), higher immunogenicity (see Figure 3), and significantly enhanced bactericidal effect (see Figure 4) .
  • the yields of 4 batches of glycoconjugates generated by TEMPO/NIS were 55%, 60%, 62%, 57%, and the highest yield was 62%.
  • the immunogenicity and bactericidal effect of the TEMPO/NIS test group were significantly higher than that of the periodate test group, and the difference was significant.

Abstract

提供了一种糖缀合物，和将细菌荚膜多糖的伯羟基氧化为醛基并与载体蛋白赖氨酸的伯氨基反应制备糖缀合物的具体制备方法及一种包含所述糖缀合物的免疫原性组合物。还提供了所述糖缀合物、免疫原性组合物在制备预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物或疫苗中的应用。

Description

一种糖缀合物及其用途 技术领域
本发明涉及疫苗研制技术领域，具体涉及一种肺炎链球菌4型荚膜多糖与载体蛋白反应制备获得的糖缀合物，一种包含所述糖缀合物的免疫原性组合物，以及所述的糖缀合物、免疫原性组合物在制备预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物或疫苗中的应用。
背景技术
肺炎链球菌为革兰氏阳性菌，菌体似矛头状，成双或成短链状排列的双球菌，宽端相对，尖端相背，有较厚的荚膜。肺炎链球菌感染致病在世界范围内是导致发病率和死亡率的主要因素之一。肺炎、发热性菌血症和脑膜炎是侵袭性肺炎链球菌疾病的最常见表现，而细菌在呼吸道内散播可导致中耳感染、鼻窦炎或者复发型支气管炎。
肺炎链球菌细胞壁外的荚膜多糖成分与结构常常与细菌的病原性和血清型有关，目前发现的肺炎链球菌荚膜多糖的变化具有超过90多种不同的血清型，其中，20多种用于制备由肺炎链球菌感染所导致疾病的疫苗。肺炎链球菌缀合物疫苗(PCV)是用于预防肺炎链球菌所致疾病的肺炎球菌疫苗，目前在全球上可以获得十三价疫苗PREBNAR
Figure PCTCN2020087841-appb-000001
。
尽管荚膜多糖自身是免疫原性的，但多糖与载体蛋白的缀合已用于改善免疫原性，使用与蛋白载体连接的多糖制备多糖蛋白缀合物疫苗，多糖与蛋白载体的化学键可诱导针对在其表面上展现疫苗所包含的多糖的细菌的免疫应答，由此预防疾病，因此，使用来自致病细菌的多糖的疫苗接种是增强宿主免疫力的潜在策略。例如：
非专利文献Deciphering Antigenic Determinants of Streptococcus pneumonia Serotype 4 Capsular Polysaccharide using Synthetic Oligosaccharides(ACS Chem.Biol.，2016，11(2)，pp 335–344)公开了丙酮酸基是肺炎链球菌血清型4荚膜多糖的保守抗原位点。同时，专利CN101180079A公开了疫苗的制备方法，包括肺炎链球菌血清型4多糖经过弱酸水解，脱除部分丙酮酸保护基，后经高碘酸钠氧化得到临位醛基，再与蛋白缀合，得到多糖蛋白缀合物。该方法制备的疫苗在免疫应答和功能活性上均取得了很好的效果，但是高碘酸钠 氧化糖链的方法必须要求糖链中具有2个相邻的游离羟基基团，同时，荚膜多糖的免疫原性、产率、杀菌作用等依然有提高的空间。
专利CN108524931A公开了活化荚膜多糖并与载体蛋白反应制备糖缀合物，具体的，所述的荚膜多糖为肺炎链球菌(S.pneumoniae)(Pn)-血清型3、10A、12F和33F荚膜多糖，并对该糖缀合物的稳定性、免疫原性程度进行验证。但是该专利只是泛泛的提到了在免疫原性组合物中还可以包括通过肺炎链球菌血清型1、4、5、6A、6B、7F、8、9V、11A、14、15B、18C、19A、19F、22F和23F荚膜多糖制备的糖缀合物，并没有公开肺炎链球菌血清型4荚膜多糖与蛋白载体偶联的糖缀合物，更没有披露血清型4荚膜多糖活化时的氧化位点。
发明内容
基于上述现有技术的缺陷，本发明提供了一种具有多个活性部位的肺炎链球菌血清型4荚膜多糖与蛋白载体偶联的糖缀合物，并且公开了肺炎链球菌血清型4荚膜多糖活化的氧化位点，所述的氧化位点包括乙酰氨基吡喃型甘露糖基(β-D-ManpNAc)、乙酰氨基吡喃型半乳糖基(α-D-GalpNAc)和/或吡喃型半乳糖基(α-D-Galp)。本发明还提供了一种糖缀合物的制备方法，该方法适用于任何包含伯羟基的多糖，这是优于高碘酸盐氧化方法的明显优点，高碘酸钠氧化方法需要在糖链中具有两个相邻的(即邻位的)游离羟基(-OH)基团，而本发明采用的氧化方法只要求荚膜多糖含有伯羟基。本发明进一步提供了一种包含所述糖缀合物的免疫原性组合物。本发明还提供了所述糖缀合物、所述免疫原性组合物在制备预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物或疫苗中的应用。
本发明所述的糖缀合物与现有技术中用高碘酸盐氧化后连接蛋白载体的缀合物相比，免疫原性更高，杀菌作用也显著增强。
具体地，本发明的第一方面，提供了一种糖缀合物，所述的糖缀合物为将细菌荚膜多糖的伯羟基氧化为醛基与载体蛋白的赖氨酸伯氨基反应制备获得，其中，所述的细菌荚膜多糖为肺炎链球菌4型荚膜多糖，肺炎链球菌4型荚膜多糖的伯羟基氧化为醛基的氧化位 点包括β-D-ManpNAc、α-D-GalpNAc和/或α-D-Galp。
优选的，所述的肺炎链球菌4型荚膜多糖可以为天然的或人工合成的。
优选的，所述的载体蛋白含有一个或多个伯氨基。所述的载体蛋白可以是来自靶标病原体的增强对该病原体的特异性免疫应答的相关蛋白抗原，或是主要作为佐剂或一般免疫应答刺激剂的一般免疫原性蛋白。
进一步优选的，所述的载体蛋白选自CRM197、破伤风类毒素、得自革兰氏阴性菌的外膜蛋白质、流感嗜血杆菌表面脂蛋白(HiD)、由流感嗜血杆菌HiD蛋白基因和流感嗜血杆菌Hin47蛋白基因以1：1方式形成的融合蛋白、百日咳毒素/类毒素、乙型肝炎表面抗原、乙型肝炎核心抗原、轮状病毒VP7蛋白质或呼吸道合胞病毒F和G蛋白或其活性部分。
在本发明的一个具体实施方式中，所述的载体蛋白为CRM197。
优选的，所述的细菌荚膜多糖与载体蛋白的质量比为0.3至3。
进一步优选的，所述的细菌荚膜多糖与载体蛋白的质量比为0.8至1.7。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为1.0至1.4。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为0.8-1.2。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为0.8-1.0。
优选的，所述的细菌荚膜多糖的每10至50个糖重复单元，在所述载体蛋白与所述细菌荚膜多糖之间存在至少一个共价键。
本发明的第二方面，提供了一种糖缀合物的制备方法，包括：
以硝酰基化合物和氧化剂作为活化剂将细菌荚膜多糖的伯羟基氧化为醛基；
将含有醛基的细菌荚膜多糖与载体蛋白的赖氨酸伯氨基反应制备获得糖缀合物；
其中，所述的细菌荚膜多糖为肺炎链球菌4型荚膜多糖，肺炎链球菌4型荚膜多糖的伯羟基氧化为醛基的氧化位点包括β-D-ManpNAc、α-D-GalpNAc和/或α-D-Galp。
优选的，所述的硝酰基化合物与荚膜多糖的摩尔比＝(0.02～0.1)：1。
进一步优选的，所述的硝酰基化合物与荚膜多糖的摩尔比＝(0.06～0.08):1。
优选的，所述的氧化剂与荚膜多糖的摩尔比＝(0.1～5)：1。
进一步优选的，所述的氧化剂与荚膜多糖的摩尔比＝(1.8～2.5)：1。
优选的，未反应的醛基在与载体蛋白缀合之后使用硼氢化钠在封闭反应中还原为伯醇，由此使在涉及氧化及随后的缀合的修饰步骤中，糖表位修饰程度为最小化。
优选的，所述的硝酰基化合物为具有所述氧化剂的存在下选择性氧化伯羟基以产生醛基的能力。
优选的，所述的氧化剂为具有硝酰基化合物的存在下选择性氧化伯羟基以产生醛基的能力的带有N卤代的氧化剂。
优选的，所述的载体蛋白含有一个或多个伯氨基。所述的载体蛋白可以是来自靶标病原体的增强对该病原体的特异性免疫应答的相关蛋白抗原，或是主要作为佐剂或一般免疫应答刺激剂的一般免疫原性蛋白。
进一步优选的，所述的载体蛋白选自CRM197、破伤风类毒素、得自革兰氏阴性菌的外膜蛋白质、流感嗜血杆菌表面脂蛋白(HiD)、由流感嗜血杆菌HiD蛋白基因和流感嗜血杆菌Hin47蛋白基因以1：1方式形成的融合蛋白、百日咳毒素/类毒素、乙型肝炎表面抗原、乙型肝炎核心抗原、轮状病毒VP7蛋白质或呼吸道合胞病毒F和G蛋白或其活性部分。
在本发明的一个具体实施方式中，所述的载体蛋白为CRM197。
优选的，所述的肺炎链球菌4型荚膜多糖可以为天然的或人工合成的。
优选的，所述的硝酰基化合物为2,2,6,6-四甲基-1-哌啶氧基自由基(TEMPO)或其衍生物。
进一步优选的，所述的TEMPO或其衍生物选自：TEMPO、2,2,6,6-四甲基哌啶氧化物、2,2,6,6-四甲基-4-(甲基磺酰氧基)-1-哌啶氧基、4-膦酰氧基-TEMPO、4-氧代-TEMPO、4-异硫氰酸基-TEMPO、4-(2-碘乙酰氨基)-TEMPO自由基、4-羟基-TEMPO、4-乙酰氨基-2,2,6,6-四甲基哌啶1-氧基、4-氰基-TEMPO、4-甲氧基-TEMPO、4-羧基-TEMPO、4-(2-溴乙酰氨基)-TEMPO或4-氨基-TEMPO。
在本发明的一个具体实施方式中，所述的硝酰基化合物为TEMPO。
优选的，所述的TEMPO与荚膜多糖的摩尔比＝(0.02～0.1)：1。
进一步优选的，所述的TEMPO与荚膜多糖的摩尔比＝(0.06～0.08):1。
优选的，所述的氧化剂为带N卤代的氧化剂。进一步优选的，所述的氧化剂选自：N-氯代琥珀酰亚胺、N-溴代琥珀酰亚胺、N-碘代琥珀酰亚胺、1,3,5-三氯-1,3,5-三嗪-2,4,6-三酮、1,3,5-三溴-1,3,5-三嗪-2,4,6-三酮或1,3,5-三碘-1,3,5-三嗪-2,4,6-三酮。
在本发明的一个具体实施方式中，所述氧化剂为N-碘代琥珀酰亚胺(NIS)。
优选的，NIS与荚膜多糖的摩尔比＝(0.1～5)：1。
进一步优选的，所述的NIS与荚膜多糖的摩尔比＝(1.8～2.5)：1。
优选的，所述的伯羟基氧化为醛基得反应是在水性溶剂或非质子溶剂中进行。
更优选的，所述的水性溶剂或非质子溶剂选自N-甲基-2-吡咯烷酮(NMP)、DMSO(二甲亚砜)、环丁砜、二甲基乙酰胺(DMA)、六甲基磷酰胺(HMPA)或DMF(二甲基甲酰胺)。
优选的，所述的硝酰基化合物以催化量≤0.1摩尔当量使用，并且通过改变所用的氧化剂的量来实现期望的荚膜多糖的氧化度。
优选的，所述的细菌荚膜多糖与载体蛋白的质量比为0.3至3。
进一步优选的，所述的细菌荚膜多糖与载体蛋白的质量比为0.8至1.7。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为1.0至1.4。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为0.8-1.2。
在本发明的一个具体实施方式，所述的细菌荚膜多糖与载体蛋白的质量比为0.8-1.0。
优选的，所述的细菌荚膜多糖的每10至50个糖重复单元，在所述载体蛋白与所述细菌荚膜多糖之间存在至少一个共价键。
优选的，所述氧化的时间为1-4h。
在本发明的一个具体实施方式中，所述的氧化时间为2-4h。
在本发明的一个具体实施方式中，所述的糖缀合物的制备方法，包括：
1)将肺炎链球菌4型荚膜多糖水解至50-900kDa的分子量，以TEMPO和NIS作为 活化剂将肺炎链球菌4型荚膜多糖的β-D-ManpNAc、α-D-GalpNAc和α-D-Galp位点的伯羟基氧化为醛基，获得活化的肺炎链球菌4型荚膜多糖；优选的，所述的活化的肺炎链球菌4型荚膜多糖的氧化度为10-30，进一步优选的，所述的活化的肺炎链球菌4型荚膜多糖的氧化度为12-20，更进一步优选的，所述的活化的肺炎链球菌4型荚膜多糖的氧化度为13-18。优选的，所述的活化反应时间为1-5h。进一步优选的，所述的活化反应时间为2-3h。
2)将步骤1)活化的肺炎链球菌4型荚膜多糖与CRM197的赖氨酸伯氨基反应制备获得糖缀合物。其中，未反应的醛基在与载体蛋白缀合之后使用硼氢化钠在封闭反应中还原为伯醇。
3)纯化步骤2)获得的糖缀合物。
优选的，所述步骤1)中将肺炎链球菌4型荚膜多糖水解至300-890kDa的分子量。
优选的，所述的步骤1)还包括纯化活化的肺炎链球菌4型荚膜多糖的步骤。
优选的，所述步骤2)中封闭反应的温度为20-50℃。
优选的，所述步骤2)中封闭反应时间为20-60h。
本发明的第三方面，提供了免疫原性组合物，包含本发明第一方面所述的糖缀合物或本发明第二方面所述的制备方法制备的糖缀合物，以及药学上可接受的赋形剂、载体和/或稀释剂。
优选的，所述的免疫原性组合物还包含其他细菌荚膜多糖的糖缀合物，所述的其他细菌荚膜多糖选自肺炎链球菌血清型1、3、3F、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F荚膜多糖。
优选的，所述免疫原性组合物的剂型选自：片剂、胶囊、丸剂、注射剂、吸入剂、含片、栓剂、乳剂、微乳剂、亚微乳剂、纳米颗粒、凝胶剂、粉剂、悬乳液、乳膏剂、胶冻剂、喷雾剂等。
优选的，所述免疫原性组合物的给药方式选自：口服、肠给药、皮下注射、肌肉注射、静脉注射、鼻腔给药、透皮给药、结膜下给药、眼球内给药、眼眶给药、眼球后给药、视 网膜给药、脉络膜给药、鞘内注射等。
优选的，所述的免疫原性组合物还包括佐剂。更优选的，所述的佐剂为铝系佐剂。最优选的，所述铝系佐剂选自磷酸铝、硫酸铝和氢氧化铝。
本发明的第四方面，提供了本发明第一方面所述的糖缀合物、本发明第二方面所述的制备方法制备的糖缀合物或本发明第三方面所述的免疫原性组合物在制备预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物或疫苗中的应用。
本发明提供的糖缀合物、免疫原性组合物具有较高的免疫原性，并在个体中诱导治疗性免疫应答。
优选的，所述的与肺炎链球菌相关的疾病选自肺炎、脑膜炎、蜂窝组织炎、骨髓炎、心内膜炎、败血性休克、发热性菌血症、中耳感染、鼻窦炎、复发型支气管炎及其他严重的侵袭性疾病。
本发明的第五方面，提供了一种预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物，所述的药物包括本发明所述的糖缀合物或免疫原性组合物。
本发明的第六方面，提供了一种预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的疫苗，所述的疫苗包括本发明所述的糖缀合物或免疫原性组合物。
本发明的第七方面，提供了一种预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的方法，所述的方法包括向个体施用有效剂量的本发明所述的糖缀合物或免疫原性组合物。
本发明所述的“糖缀合物”是指与载体蛋白共价缀合的糖。其中，所述的糖缀合物中可以包含一定量的游离糖。
本发明所述的“氧化度”是指每摩尔醛的糖重复单元摩尔比。
本发明所述的“药学上可接受的”是指既不显著刺激个体也不抑制所施用的产品的活性物质的生物学活性及特性。
本发明所述的“预防”是指通过施用本发明所述的产品来抑制症状或者延缓特定症状紧张的所有行为。
本发明所述的“治疗”是指在疾病已开始发展后改善疾病或病理状态的体征、症状等等的治疗干预。
本发明所述的“个体”包括哺乳动物和人。
本发明所述的“有效剂量”是指在以单个或多个剂量给予至个体或器官之后提供所希望的治疗或预防的本发明的组合物的量或剂量。
本发明所述的“和/或”包括择一列出的项目以及任何数量的项目组合。
本发明所述的“包括”是开放式的描述，含有所描述的指定成分或步骤，以及不会实质上影响的其他指定成分或步骤。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：图A、B分别表示肺炎链球菌血清型4的荚膜多糖的结构式及表达式，其中，图A中3个椭圆圈位置表示通过TEMPO/NIS氧化荚膜多糖的氧化位点，分别为β-D-ManpNAc、α-D-GalpNAc和α-D-Galp，箭头指向的位置表示高碘酸盐介导的氧化的位点，具体为α-D-Galp。
图2：使用高碘酸盐氧化相对于使用TEMPO/NIS氧化所制备的肺炎链球菌血清型4-CRM197糖缀合物的产率比较。
图3：使用高碘酸盐氧化相对于使用TEMPO/NIS氧化所制备的肺炎链球菌血清型4-CRM197糖缀合物的免疫原性比较。
图4：使用高碘酸盐氧化相对于使用TEMPO/NIS氧化所制备的肺炎链球菌血清型4-CRM197糖缀合物的杀菌作用比较。
图5：由高碘酸盐氧化生成的活化荚膜多糖、缀合物与由TEMPO/NIS氧化生成的活化荚膜多糖、缀合物的核磁结构的比较，其中，F4为肺炎链球菌血清型4荚膜多糖。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实 施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明具体实施例中所用试剂的购买厂家及配方：
TEMPO：2,2,6,6-四甲基哌啶氧化物，国药集团化学试剂有限公司；
NIS：N-碘代琥珀酰亚胺，国药集团化学试剂有限公司；
家兔：北京北生研生物制品有限公司，普通家兔，2.5kg左右；
血清型4荚膜多糖：康希诺生物股份公司自制；
CRM197载体蛋白：康希诺生物股份公司自制；
高碘酸盐：天津市科密欧化学试剂有限公司，纯度99.0％；
NMR：布鲁克(北京)科技有限公司，600MHz；
PREBNAR
Figure PCTCN2020087841-appb-000002
：辉瑞投资公司，进口药品注册证号：S20160042。
实施例1 使用TEMPO/NIS氧化荚膜多糖的肺炎链球菌血清型4-CRM197糖缀合物制备
为了改善血清型4-CRM197糖缀合物的产率，使用2,2,6,6-四甲基-1-哌啶氧基自由基(TEMPO)和作为共氧化剂的N-碘代琥珀酰亚胺(NIS)来将伯醇氧化成醛基，并与载体蛋白的赖氨酸伯氨基反应，制备获得血清型4-CRM197糖缀合物。NMR分析表明氧化位点与高碘酸盐介导的氧化的位点不同。在TEMPO-NIS氧化的情况下，氧化位点为β-D-ManpNAc、α-D-GalpNAc和α-D-Galp(见图1A椭圆圈部分)，而当使用高碘酸盐时，α-D-Galp是主要的氧化位点(见图1A箭头位置)。
制备血清型4-CRM197糖缀合物的步骤如下：
(1)采用酸性条件下加热来将血清型4荚膜多糖水解至50kDa至900kDa的分子量；
(2)用TEMPO/NIS活化血清型4荚膜多糖，具体为：2g/L血清型4荚膜多糖多糖浓度，终浓度为0.25M NaHCO 3/0.025M Na 2CO 3pH8.6碳酸盐缓冲液，加入0.08Meq TEMPO，按表1加入NIS，避光磁力搅拌，室温进行活化反应2小时。
表1 加入的TEMPO与NIS的量
Figure PCTCN2020087841-appb-000003
50kD膜包，纯化水超滤。蒽酮法测糖含量，MBTH法测醛基含量，TSK测分子量，核磁检测荚膜多糖结构。
(3)将活化的血清型4荚膜多糖与CRM197载体蛋白缀合反应：
荚膜多糖反应浓度10-20g/L，加入CRM197载体蛋白，混匀，加入0.5-2g/L氰基硼氢化钠，避光，37℃反应48h。其中，加入硼氢化钠至反应浓度0.5g/L，反应1-3h。
(4)将步骤(3)获得的缀合物纯化：
100kD膜包，0.9％NaCl超滤40次。
(5)缀合物检测：
蒽酮法测荚膜多糖含量，Lowry法测蛋白含量，DOC沉淀法测游离多糖含量，SDS-PAGE法测游离蛋白含量，CL-4B测定K D。
其中，使用TEMPO/NIS氧化的荚膜多糖生成条件并表征多个血清型4-CRM197缀合物，具体结果见表2：
表2 TEMPO/NIS氧化的荚膜多糖生成条件及表征情况
Figure PCTCN2020087841-appb-000004
Figure PCTCN2020087841-appb-000005
实施例2 使用TEMPO/NIS氧化法的肺炎链球菌血清型4-CRM197缀合物的免疫原性
按照实施例1所述制备血清型4-CRM197糖缀合物的步骤制备试验组血清型4-CRM197缀合物，并验证缀合物的应用效果，动物实验采用，普通家兔，2.5kg左右，雌雄各半，实验共设10组，8组实验组、1组阳性对照、1组阴性对照，每组4只家兔，皮下注射，分别于0、14、28天进行免疫，注射剂量为人用二分之一剂量，免疫后42天颈部静脉采集全血4℃条件下8000rpm离心6min，分离血清，放置于-80℃保存，具体试验组别见表3：
Elisa方法：用包被液将4型荚膜多糖稀释到相应的包被浓度，于96孔板中每孔加入100微升，2～8℃包被过夜，弃包被液，洗板1次，加入待测血清，室温(20～25℃)孵育1h，洗板5次，加入100微升/孔1:5000稀释的碱性磷酸酶标记的羊抗鼠IgG，室温孵育1h，洗板5次，加入显色液100微升/孔，避光显色30min，加入终止液50微升/孔，于405nm波长取出吸光度值。
表3 不同氧化方法制备缀合物的试验组别
Figure PCTCN2020087841-appb-000006
Figure PCTCN2020087841-appb-000007
高碘酸钠试验组采用高碘酸钠氧化肺炎链球菌血清型4荚膜多糖及制备缀合物的方法为肺炎链球菌血清型4荚膜多糖经过弱酸水解，脱除部分丙酮酸保护基，后经高碘酸钠氧化得到邻位醛基，再与蛋白缀合，得到多糖蛋白缀合物(具体参见专利CN101180079A)。
在标准条件下的家兔中测定血清型4-CRM197缀合物在家兔中的调理吞噬活性(OPA)效价。与由高碘酸盐氧化生成的缀合物相比，由TEMPO/NIS生成的缀合物(批次TEMPO-1、TEMPO-2、TEMPO-3、TEMPO-4分别参见表2中对F4-1、F4-3、F4-4、F4-5缀合物的表征数据)产率更高(参见图2)，免疫原性更高(参见图3)，杀菌作用显著增强(参见图4)。其中，由TEMPO/NIS生成4个批次的糖缀合物产率分别为55％、60％、62％、57％，最高产率为62％。TEMPO/NIS试验组比高碘酸盐试验组的免疫原性、杀菌作用明显更高，差异具有显著性。
实施例3 核磁结构比较
由高碘酸盐氧化生成的活化荚膜多糖、缀合物与由TEMPO/NIS氧化生成的活化荚膜多糖、缀合物的核磁结构的比较(参见图5)，表明由TEMPO/NIS氧化生成肺炎链球菌4活化荚膜多糖、缀合物中荚膜多糖结构与纯化荚膜多糖结构更接近，无显著差异，保守抗原α-D-Galp中2,3位丙酮酸基无明显变化，对端基质子与丙酮酸甲基进行积分面积统计，计算摩尔比，具体数据参见表4，结果表明TEMPO/NIS氧化生成的活化荚膜多糖丙酮酸保护基得到了很好保留。
表4 不同氧化方法得到活化多糖丙酮酸甲基与端基质子摩尔比
名称 丙酮酸甲基与端基质子摩尔比
F4 TEMPO法氧化所得活化多糖 4.59
F4高碘酸钠法氧化所得活化多糖 1.43
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (12)

  1. 一种糖缀合物，其特征在于，所述的糖缀合物为将细菌荚膜多糖的伯羟基氧化为醛基与载体蛋白的赖氨酸伯氨基反应制备获得，其中，所述的细菌荚膜多糖为肺炎链球菌4型荚膜多糖，肺炎链球菌4型荚膜多糖的伯羟基氧化为醛基的氧化位点包括β-D-ManpNAc、α-D-GalpNAc和/或α-D-Galp。
  2. 根据权利要求1所述的糖缀合物，其特征在于，所述的载体蛋白选自CRM197、破伤风类毒素、得自革兰氏阴性菌的外膜蛋白质、流感嗜血杆菌表面脂蛋白(HiD)、由流感嗜血杆菌HiD蛋白基因和流感嗜血杆菌Hin47蛋白基因以1：1方式形成的融合蛋白、百日咳毒素/类毒素、乙型肝炎表面抗原、乙型肝炎核心抗原、轮状病毒VP7蛋白质或呼吸道合胞病毒F和G蛋白或其活性部分。
  3. 根据权利要求1或2所述的糖缀合物，其特征在于，所述的细菌荚膜多糖与载体蛋白的质量比为0.3至3。
  4. 根据权利要求1-3任一所述的糖缀合物，其特征在于，所述的细菌荚膜多糖的每10至50个糖重复单元，在所述载体蛋白与所述细菌荚膜多糖之间存在至少一个共价键。
  5. 一种糖缀合物的制备方法，其特征在于，包括：
    以硝酰基化合物和氧化剂作为活化剂将细菌荚膜多糖的伯羟基氧化为醛基；
    将含有醛基的细菌荚膜多糖与载体蛋白的赖氨酸伯氨基反应制备获得糖缀合物；
    其中，所述的细菌荚膜多糖为肺炎链球菌4型荚膜多糖，肺炎链球菌4型荚膜多糖的伯羟基氧化为醛基的氧化位点包括β-D-ManpNAc、α-D-GalpNAc和/或α-D-Galp。
  6. 根据权利要求5所述的一种糖缀合物的制备方法，其特征在于，所述的硝酰基化合物为2,2,6,6-四甲基-1-哌啶氧基自由基或其衍生物。
  7. 根据权利要求5或6所述的一种糖缀合物的制备方法，其特征在于，所述的氧化剂为N-碘代琥珀酰亚胺。
  8. 根据权利要求5-7任一所述的一种糖缀合物的制备方法，其特征在于，所述的载体蛋白选自CRM197、破伤风类毒素、得自革兰氏阴性菌的外膜蛋白质、流感嗜血杆菌表面脂蛋白(HiD)、由流感嗜血杆菌HiD蛋白基因和流感嗜血杆菌Hin47蛋白基因以1：1 方式形成的融合蛋白、百日咳毒素/类毒素、乙型肝炎表面抗原、乙型肝炎核心抗原、轮状病毒VP7蛋白质或呼吸道合胞病毒F和G蛋白或其活性部分。
  9. 根据权利要求5-8任一所述的一种糖缀合物的制备方法，其特征在于，所述的细菌荚膜多糖与载体蛋白的质量比为0.3至3。
  10. 免疫原性组合物，其特征在于，包含权利要求1-4任一所述的糖缀合物或权利要求5-9任一所述的制备方法制备的糖缀合物，以及药学上可接受的赋形剂、载体和/或稀释剂。
  11. 根据权利要求10所述的免疫原性组合物，其特征在于，所述的免疫原性组合物还包含其他细菌荚膜多糖的糖缀合物，所述的其他细菌荚膜多糖选自肺炎链球菌血清型1、3、3F、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F荚膜多糖。
  12. 权利要求1-4任一所述的糖缀合物、权利要求5-9任一所述的制备方法制备的糖缀合物或权利要求10-11任一所述的免疫原性组合物在制备预防和/或治疗个体肺炎链球菌感染、与肺炎链球菌相关的疾病的药物或疫苗中的应用。
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