WO2023202607A1 - 多价肺炎球菌多糖结合疫苗的成分及其应用 - Google Patents

多价肺炎球菌多糖结合疫苗的成分及其应用 Download PDF

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WO2023202607A1
WO2023202607A1 PCT/CN2023/089161 CN2023089161W WO2023202607A1 WO 2023202607 A1 WO2023202607 A1 WO 2023202607A1 CN 2023089161 W CN2023089161 W CN 2023089161W WO 2023202607 A1 WO2023202607 A1 WO 2023202607A1
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serotypes
polysaccharide
protein
ttd
capsular
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French (fr)
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祝先潮
陈华根
熊细双
刘畅
李颖
夏清风
何平
王娟娟
黄小敏
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上海瑞宙生物科技有限公司
上海微宙生物科技有限公司
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Publication of WO2023202607A1 publication Critical patent/WO2023202607A1/zh

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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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, 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
    • A61K39/02—Bacterial antigens
    • A61K39/116—Polyvalent bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the prevention of infection by bacterial pathogens through immune multivalent vaccines.
  • Streptococcus pneumoniae (S.pneumoniae) is a capsulated Gram-positive diplococcus. According to the composition difference of its capsular polysaccharide, it can be divided into nearly 100 serotypes, among which capsular polysaccharide is an important pathogenic factor. Streptococcus pneumoniae normally parasitizes the nasopharynx of healthy people.
  • Invasive infections occur through the mucosal defense system, such as entering the lower respiratory tract to cause pneumonia, crossing the blood-brain barrier to cause bacterial meningitis, crossing alveolar epithelial cells, invading vascular endothelial cells and entering the blood to cause bacteremia, and can also cause bacteremia from the nasopharynx It migrates into the sinuses, causing sinusitis, and enters the middle ear through the Eustachian tube, causing otitis media to non-invasively spread to other parts of the respiratory tract.
  • Pneumococcal disease is one of the serious public health problems worldwide.
  • the World Health Organization estimates that in 2005, approximately 1.6 million people died from pneumococcal disease each year worldwide, including 700,000 to 1 million children under the age of 5, most of whom lived in developing countries. It can be seen that pneumococcal bacteria have been seriously endangering children's health.
  • pneumococcal diseases mainly come from children under 2 years old and the elderly, and people with low immune function in all age groups.
  • pneumococcal diseases can be divided into two categories: invasive pneumococcal disease (IPD) and non-invasive pneumococcal disease (NIPD).
  • IPD invasive pneumococcal disease
  • NIPD non-invasive pneumococcal disease
  • the common treatment is antibiotic therapy, but pneumococcal resistance to commonly used antimicrobial drugs has become a growing problem globally. Years of clinical practice have proven that pneumococcal vaccination is the most cost-effective way to prevent pneumococcal disease.
  • Pneumonia vaccines currently on the market include pneumococcal polysaccharide vaccine (PPSV) and pneumococcal polysaccharide protein conjugate vaccine (PCV).
  • PPSV23 The 23-valent pneumonia polysaccharide vaccine (PPSV23) is not suitable for infants and people with low immunity due to its low immunogenicity and lack of immune memory and boosting effects.
  • Pneumonia polysaccharide conjugate vaccines that can be applied to infants and young children include PCV7, PCV10 and PCV13, but their serum coverage is small, their immune protection coverage is low, and they may cause immunosuppression.
  • the serotype coverage of existing polysaccharide-protein conjugate vaccines is not ideal. It is necessary to develop a multivalent pneumonia conjugate vaccine to cover a wider range of pathogenic serotypes of pneumococci, increase non-vaccine serotypes, and improve the effectiveness of new multivalent vaccines. Covering the protection rate and immunogenicity and reducing the risk of immunosuppressive effects of carrier proteins have important clinical value.
  • the present invention provides multivalent immunogenicity with wider application, stronger immunogenicity, and weakened immunosuppression. combination.
  • a first aspect of the present invention provides an immunogenic composition containing capsular polysaccharides from different serotypes of Streptococcus pneumoniae and a carrier, the serotypes at least include 2, 8, 9N, 10A, 11A, 12F, 15B , 17F, 20, 22F and 33F.
  • the serotypes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 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, and the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.
  • the serotypes include the following 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, or the following 24 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 carrier is selected from saline, Ringer's solution, and phosphate buffered saline One or more of the salt water species.
  • the immunogenic composition further contains an adjuvant.
  • the adjuvant is an aluminum-based adjuvant.
  • the adjuvant includes selected from the group consisting of aluminum phosphate, aluminum sulfate, aluminum hydroxide, monophosphoryl lipid A, QS21, CpG, MF59, stearoyltyrosine, Freund's adjuvant, and One or more other mucosal adjuvants.
  • the weight ratio of the capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide in the composition is from 10:1 to 1:10, for example 5:1 to 1:5, preferably 2:1.
  • the composition is a formulation in which the concentration of capsular polysaccharides from serotypes 3, 6B and 12F is each independently 1 to 8 ug/dose (preferably 4 ⁇ g/dose) and the remaining capsular polysaccharides are The concentrations are each independently 0.5 to 5ug/dose (preferably 2 ⁇ g/dose). Further, the concentration of aluminum phosphate adjuvant is 0.125 mg/dose to 0.5 mg/dose.
  • the composition further includes a surfactant, such as Tween 20 or Tween 80.
  • a surfactant such as Tween 20 or Tween 80.
  • the concentration of surfactant in the composition is 100-300 ⁇ g/dose, preferably 70-120 ⁇ g/dose.
  • the present invention also provides multivalent immunogenic compositions comprising a plurality of polysaccharide-protein conjugates and pharmaceutically acceptable excipients, wherein each polysaccharide-protein conjugate contains polysaccharide-protein conjugates from different serotypes conjugated to a carrier protein.
  • the carrier protein includes at least two carrier proteins.
  • the carrier protein includes (1) CRM197 and (2) TTD or a variant thereof.
  • the TTD is the C-terminal domain of TT.
  • the TTD has the sequence set forth in SEQ ID NO:2 and the TTD variant has a sequence that is at least 90% sequence identical to SEQ ID NO:2.
  • the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F.
  • the serotypes include the following 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, or the following 24 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 serotypes include the 20 serotypes from 8, 9, 10, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F.
  • Capsular polysaccharides of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 serotypes are respectively conjugated to the carrier protein TTD or a variant thereof.
  • the serotypes include the 20 serotypes, wherein capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F, and 23F are associated with the carrier protein TTD or Its variants are conjugated; capsular polysaccharides from serotypes 1, 4, 6A, 7F, 8, 9V, 10A, 11A, 14, 19A, 22F, 33F are respectively conjugated to the carrier protein CRM197.
  • the serotypes include the 24 serotypes from 8, 9, 10, including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F.
  • Capsular polysaccharides of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 serotypes are respectively conjugated to the carrier protein TTD or a variant thereof.
  • the serotypes include the 24 serotypes from serotypes 3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20
  • Capsular polysaccharides from serotypes 1, 2, 4, 7F, 8, 9V, 10A, 14, and 22F were conjugated to the carrier protein CRM197, respectively. combine.
  • the serotypes include the 24 serotypes, wherein capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are associated with the carrier protein TTD or Its variants are conjugated; capsular polysaccharides from serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F, and 33F are respectively conjugated to the carrier protein CRM197 combine.
  • the weight ratio of the capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide in the composition is from 10:1 to 1:10, for example 5:1 to 1:5, preferably 2:1.
  • the composition further comprises an adjuvant, such as an aluminum-based adjuvant.
  • the adjuvant is selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide, preferably aluminum phosphate.
  • the weight ratio of conjugate to adjuvant in the composition is from 1:10 to 1:2, preferably from 54:500 to 54:125.
  • the composition is a formulation in which the concentration of capsular polysaccharides from serotypes 3, 6B and 12F is each independently 1 to 8 ug/dose (preferably 4 ⁇ g/dose) and the remaining capsular polysaccharides are The concentrations are each independently 0.5 to 5ug/dose (preferably 2 ⁇ g/dose), and the concentration of the aluminum phosphate adjuvant is 0.125mg/dose to 0.5mg/dose.
  • the composition further includes a surfactant, such as Tween 20 or Tween 80.
  • a surfactant such as Tween 20 or Tween 80.
  • the concentration of surfactant in the composition is 100-300 ⁇ g/dose, preferably 70-120 ⁇ g/dose.
  • the pH of the composition is between 5.0 and 7.0, preferably between 5.0 and 6.2.
  • the present invention also provides the use of the immunogenic composition described in the first aspect herein in the preparation of a medicament for inducing an immune response to a pneumococcal capsular polysaccharide conjugate and/or an immune response to tetanus toxin.
  • the medicament is used to prevent or treat pneumococcal infection and/or tetanus toxin infection.
  • the invention also provides a method of inducing an immune response to a pneumococcal capsular polysaccharide conjugate and/or an immune response to tetanus toxin, comprising administering to a subject an immunologically effective amount of an immunogenic combination according to the first aspect of the invention. things.
  • the present invention also provides an immune composition that results in passive immunity, including a bactericidal antibody targeting pneumococci, the antibody being obtained by immunizing a mammal with the immunogenic composition according to any embodiment of this document.
  • the bactericidal antibodies are present in serum, gamma globulin fractions, or purified antibody preparations.
  • the immunogenic composition of the present invention can effectively prevent invasive infections of 24 different serotypes of pneumococcal bacteria, and can induce relatively balanced and high immunogenicity for 24 serotypes. Its immunogenicity is consistent with that of each serotype. It is related to the type of conjugate content ratio.
  • the immunogenic composition of the present invention contains different carrier proteins at the same time, and both carrier proteins are non-toxic, so there is no need for detoxification treatment. This dual-carrier design reduces potential safety and immunosuppressive risks.
  • the immunogenic composition is also protective against infection with tetanus toxin.
  • FIG. 1 Comparison of the immunogenicity of type 5 pneumococcal polysaccharide conjugates of different carriers.
  • FIG. 1 Comparison of the immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines in mice at different adjuvant doses (the same 13 serotypes as the positive vaccine).
  • FIG. 1 Comparison of the immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines in mice at different adjuvant doses (11 serotypes other than the 13 serotypes of the positive vaccine).
  • FIG. 1 Comparison of the immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccines at different pH in mice.
  • Figure 12 Survival curve of protective efficacy of 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 pneumococcal challenge in mice.
  • FIG. 14 Comparison of TT antibody titers after the second dose of immunization and before immunization.
  • Group A is normal saline;
  • Group B is diphtheria-tetanus pertussis vaccine;
  • Group C is 24-valent pneumococcal polysaccharide conjugate vaccine.
  • Figure 15 Survival rate curve after tetanus toxin challenge.
  • the present invention provides novel 20-valent or 24-valent pneumococcal capsular polysaccharide-protein conjugates and corresponding immunogenic compositions and vaccine preparations.
  • the vaccine preparation of the present invention induces higher antibody titers on some serotypes; it also shows better immunogenicity for serotypes other than 13 serotypes; compared with 13-valent polysaccharide conjugate vaccine and 23-valent polysaccharide vaccine can induce better immune protection in vivo.
  • the inventors found that using different carriers on different conjugates induced higher antibody titers than the single-carrier vaccine group.
  • the present invention first provides an immunogenic composition that induces the immune response of mammals to pneumococcal capsular polysaccharide conjugates and protects them from pneumococcal infection.
  • the immunogenic composition contains capsular polysaccharides from different serotypes of Streptococcus pneumoniae, including at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.
  • the serotypes include: 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 pneumococcal capsular polysaccharide present in the immunogenic composition may be in the form of the free polysaccharide or as a component of a conjugate in which the polysaccharide is covalently linked to a protein.
  • the immunogenic composition may also include a pharmaceutically acceptable carrier, such as saline, Ringer's solution, or phosphate buffered saline.
  • the capsular polysaccharide is covalently linked to the protein to form a conjugate.
  • any protein or fragment thereof that is acceptable to an individual and is capable of inducing an immune cell (eg, T-cell) dependent response is suitable for conjugation to the pneumococcal capsular polysaccharide.
  • any protein can serve as a conjugated protein.
  • the selected protein must have at least one free amino group for conjugation to the polysaccharide.
  • Preferred proteins are any native or recombinant bacterial proteins that are themselves immunogens that induce T-cell dependent responses in young and adult mammals.
  • Such proteins include, but are not limited to, tetanus toxoid, cholera toxoid, diphtheria toxoid, and CRM197 or variants thereof.
  • Other candidates for conjugation proteins include toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including E. coli.
  • the pneumococcal capsular polysaccharide is covalently linked to the protein to form a conjugate.
  • an agent that is acceptable to the individual and capable of inducing an immune cell (e.g., T-cell) dependent response Any protein or fragment thereof is suitable for conjugation to the pneumococcal capsular polysaccharide.
  • any protein can serve as a conjugated protein.
  • the selected protein must have at least one free amino group for conjugation to the polysaccharide.
  • Preferred proteins are any native or recombinant bacterial proteins that are themselves immunogens that induce T-cell dependent responses in young and adult mammals.
  • Such proteins include, but are not limited to, tetanus toxoid, cholera toxoid, and diphtheria toxoid.
  • Other candidates for conjugation proteins include toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including E. coli.
  • the protein conjugated to the pneumococcal capsular polysaccharide may be any protein or fragment thereof that is acceptable to the individual and capable of inducing an immune cell (eg, T-cell) dependent response.
  • any protein can serve as a conjugated protein.
  • the selected protein must have at least one free amino group for conjugation to the polysaccharide.
  • Preferred proteins are any native or recombinant bacterial proteins that are themselves immunogens that induce T-cell dependent responses in young and adult mammals. Examples of such proteins include, but are not limited to, tetanus toxoid, cholera toxoid, and diphtheria toxoid.
  • Other candidates for conjugation proteins include toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including E. coli.
  • non-toxic variants of the protein toxin can be used, such as CRM197.
  • Such mutations retain epitopes of the native toxin.
  • These mutated toxins are called "cross-reactive substances," or CRMs.
  • the CRM197 carrier protein (NCBI: AMV91693.1, SEQ ID NO: 3) is a non-toxic variant of diphtheria toxin.
  • the protein is not toxic but retains the immunogenicity of diphtheria toxoid (DT). Fermentation and purification methods are available in published articles and patents (US5614382).
  • the nontoxic variant protein of diphtheria toxoid is a carrier protein that has been clinically proven to be safe and effective, and has been widely used in the marketed pneumococcal polysaccharide conjugate vaccine.
  • Other diphtheria toxin variants are also suitable for use as carrier proteins.
  • Pneumococcal polysaccharides can also be conjugated with fragments of these proteins, provided that these fragments are long enough, ie preferably at least 10 amino acids, to identify T-cell epitopes.
  • Tetanus toxoid has many reports in the open literature.
  • the inventors discovered a variant of the TT protein, the TTD protein.
  • This protein is not toxic but retains the immunogenicity of TT protein.
  • TTD protein is located at the C-terminus of the heavy chain of TT protein, with a relative molecular weight of 50KDa. It is the receptor binding region of the toxin. It is non-toxic and has good immunogenicity. Its allergenicity is lower than that of tetanus toxoid and it is a potential tetanus vaccine. Antigenic components and carrier proteins.
  • TTD can be obtained through recombinant expression and purification (Immunobiology, Vol.216, Issue 4, 2011, P 485-490).
  • Exemplary TTD expression and purification methods include: cloning the DNA sequence expressing TTD (SEQ ID NO: 1) into the protein expression plasmid pET21, and constructing an engineering strain (such as E. coli BL21) that recombinantly expresses the TTD protein (SEQ ID NO: 2). (DE3)).
  • the process of isolating proteins from culture is well known in the art, for example, the following steps: centrifuge the culture medium at 8000 rpm, 4°C, then resuspend in PBS and disrupt (for example, ultrasonic disruption) the cells, centrifuge the disrupted liquid at 8000 rpm, 4°C, and collect TTD. Protein supernatant.
  • the method for purifying TTD protein from the supernatant can use methods commonly used in the art for purifying proteins from liquids, such as ammonium sulfate precipitation, clarification filtration, combined chromatography such as ion exchange chromatography, composite media chromatography, and/or affinity. It is obtained by chromatography and other chromatography methods, and the purity is above 95%.
  • the molecular weight determined by mass spectrometry was consistent with the theoretical molecular weight.
  • Suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (see, for example, WO2004/083251), E. coli LT, E. coli ST, and polypeptides from Pseudomonas aeruginosa aeruginosa) exotoxins.
  • inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (see, for example, WO2004/083251), E. coli LT, E. coli ST, and polypeptides from Pseudomonas aeruginosa aeruginosa) exotoxins.
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D.
  • Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivatives of tuberculin (PPD) can also be used as carrier proteins.
  • TTD activity such as toxin binding activity
  • those skilled in the art can change one or more of the TTD of the present invention (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of TTD.
  • These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. Amino acid residues that are subject to conservative substitutions are well known in the art.
  • TTD variants described herein include TTD variants that have at least 90% (eg, at least 95%, at least 98%, at least 99%) sequence identity to the TTD set forth in SEQ ID NO: 2 and retain its toxin-binding activity.
  • the immunogenic composition formed after mixing has better immunogenicity and lower risk of immunosuppression.
  • at least two carrier proteins such as CRM197 and TTD or variants thereof, are included in the immunogenic composition.
  • 20 or 24 pneumococcal polysaccharides of the present invention are chemically coupled with CRM197 and TTD carrier proteins to prepare polysaccharide-protein conjugates.
  • the obtained polysaccharide-protein conjugates of different serotypes have good physical and chemical properties. For example, the polysaccharide-to-protein ratio is between 0.5 and 3.0, the free sugar content is below 20%, and other residual impurities are controlled within a very low range.
  • At least one or more or all capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F, and 23F, respectively, are combined with a carrier protein TTD or its variant conjugation.
  • 21, 22, 23 or 24 serotypes are respectively conjugated to the carrier protein TTD or a variant thereof.
  • capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are conjugated to the carrier protein TTD or a variant thereof, respectively; from serotypes 1, 2, 4, 6A, 7F,
  • the capsular polysaccharides of 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F, and 33F were conjugated to the carrier protein CRM197 respectively.
  • the ratio of capsular polysaccharide and carrier protein from different serotypes is between 0.2-3.0, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0 or any two of the above
  • the range between numerical values is preferably 0.5-2.5, 0.5-2.0, 0.5-1.5, more preferably 0.6-2.5, 0.6-2.0, 0.6-1.5.
  • the weight ratio of the capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is from 10:1 to 1:10, for example 5:1 to 1:5, preferably 2:1.
  • the composition may be a formulation in which the concentration of capsular polysaccharides from serotypes 3, 6B and 12F is each independently 1 to 8 ug/dose (preferably 4, preferably u dose), and the remaining capsular polysaccharides are The concentrations are each independently 0.5 to 5ug/dose (preferably 2, preferably 5 doses), and the concentration of the aluminum phosphate adjuvant is 0.125mg/dose to 0.5mg/dose.
  • the immunogenic composition can be used as a vaccine, further containing an adjuvant such as an aluminum-based adjuvant (aluminum hydroxide, aluminum phosphate or aluminum sulfate). Additionally, the immunogenic compositions described herein may also include surfactants, such as Tween 20 or Tween 80.
  • the immunogenic compositions of the present invention are capable of inducing active and passive protection against pneumococcal infection. For passive protection, immunizing antibodies are produced by immunizing a mammal with a vaccine prepared from the immunogenic composition of the invention and then recovering the immunizing antibodies from the mammal.
  • the invention also provides methods of immunizing a subject against pneumococcal infection by administering an immunologically effective amount of a composition of the invention.
  • Capsular polysaccharides are prepared by standard techniques known to those skilled in the art.
  • Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 20, 22F, 23F and 33F prepared capsular polysaccharide.
  • These pneumococcal conjugates are prepared by separate methods and formulated into single dose formulations.
  • Bacterial capsule polysaccharide is an important component of bacterial cells and an important virulence factor of pathogenic bacteria.
  • Pneumococcal bacterial capsular polysaccharides are released into the culture medium during bacterial culture.
  • the pneumococcal capsular polysaccharide in the present invention is obtained by culturing, fermenting and purifying pneumococcal bacteria of different serotypes. This type of technology is mature and has been used in the industry for many years, with multiple published articles or patent reports (US4242501).
  • each pneumococcal polysaccharide serotype is cultured in soy-based medium.
  • Various polysaccharides are then purified by centrifugation, precipitation, ultrafiltration and column chromatography (US4686102).
  • the purification method of pneumococcal capsular polysaccharide is also a common technique in the art, for example, it is accomplished by an organic solvent-free method (US Pat. No. 5,714,354).
  • the purification process of refined polysaccharides includes, but is not limited to, adding NaCl solution to dissolve the complex sugar, dissolving with NaCl solution, centrifuging, taking the supernatant, and adding wine. Refining to remove impurities and precipitated sugar, washing multiple times to precipitate and remove impurities, or purifying polysaccharides using chromatography methods such as ion exchange columns and composite packing according to different sugar types to obtain refined polysaccharides.
  • the purification process mainly includes: centrifuging the bacterial inactivated liquid to separate the bacterial cells, harvesting the centrifugal supernatant, and then filtering through a 0.22 ⁇ m filter membrane and concentrating through 100KD membrane cassette ultrafiltration to obtain the pneumonia polysaccharide fermentation concentrate. .
  • Add CTAB solution to the pneumonia polysaccharide fermentation concentrate stir at room temperature, centrifuge at 8000 rpm, and collect the polysaccharide complex precipitate (for 7F, 14, and 33F polysaccharides, collect the supernatant).
  • the polysaccharide complex is then dissociated with 0.25-0.5M NaCl solution, and then purified through precipitation, column chromatography, ultrafiltration and other processes to obtain a purified polysaccharide solution.
  • the polysaccharide solution was filtered through a 0.22 ⁇ m membrane and stored at low temperature.
  • Methods for mass analysis of the resulting polysaccharides are known in the art, for example by nuclear magnetic resonance.
  • the prepared different types of pneumococcal capsular polysaccharides are identified by specific serum, the chemical structure of the pneumococcal polysaccharide is analyzed by nuclear magnetic resonance (NMR), and the polysaccharide can be analyzed by chemical color development methods.
  • the functional groups contained (such as rhamnose, uronic acid, O-acetyl and other components) are measured for content.
  • a number of conjugation methods can be used to produce the polysaccharide-protein conjugates of the invention.
  • polysaccharide activation and protein conjugation steps are usually included.
  • Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein is achieved by conventional methods. See, for example, U.S. Patent Nos. 4,673,574 and 4,902,506.
  • parameters such as the molecular weight of polysaccharide, polysaccharide activation reaction, polysaccharide-protein reaction ratio, reaction temperature, reaction time, and reaction solution pH value.
  • the obtained polysaccharide-protein conjugate is further purified to remove unreacted substances and impurities, and its polysaccharide-protein cross-linking degree and residual impurities are quantified through mass analysis to ensure the consistency and comparability of batches.
  • the purified polysaccharide is chemically activated to enable the sugar to react with the carrier protein.
  • different chemical activation methods can be selected.
  • Commonly used polysaccharide activation methods include cyanogen bromide method (US6375846B1), hydrolysis (US4761283), 1-cyano-4-dimethylaminopyridine tetrafluoroborate ( CDAP) (EP0720485), periodic acid oxidation method (US4711779).
  • the activated polysaccharides can be separated from other impurities by ultrafiltration. After activation, the polysaccharide has the activity to react with the carrier protein.
  • a periodate salt eg, sodium periodate
  • a protein carrier of choice for example, at room temperature, use about 1 ml of about 20 mM sodium periodate to dissolve The solution can suitably oxidize about 10 mg of polysaccharide for about 10-15 minutes; alternatively, add 0.05-2 equivalents of periodate to the polysaccharide for 12-24 hours.
  • the reaction time can be varied with other amounts of periodate to obtain equivalent oxidation.
  • the reduction and ring-opening of sugars activates the ortho-hydroxyl groups of reducing sugars into aldehyde groups. After the polysaccharide activation is completed, the small molecular substances in the reaction can be removed by ultrafiltration and concentration.
  • the conjugate is prepared by chemically coupling the polysaccharide fragment obtained by processing the purified and extracted pneumococcal outer membrane polysaccharide to the carrier protein.
  • Polysaccharide-protein conjugates can be obtained by direct chemical coupling or through a linker, such as adipic acid dihydrazide.
  • linker such as adipic acid dihydrazide.
  • Common methods include cyanogen bromide method, CDAP method or reductive amination method (US5952454, EP0720485, US4711779).
  • US Patent 4,644,059 incorporated herein by reference, describes conjugates prepared using adipic acid dihydrazide (ADH) as the homobifunctional linker.
  • ADH adipic acid dihydrazide
  • Patent 4,695,624 also incorporated by reference herein, describes methods for preparing polysaccharides and preparing conjugates using intergeneric spacers.
  • a general study of the various preparation methods and factors used to design conjugates is discussed in Dick, William E. and Michel Beurret, Contrib. Mrcrobil. Immunol. (1989), Vol. 10, pp. 48-114, which This document is also incorporated by reference.
  • the polysaccharide-protein conjugate obtained through coupling can induce relatively strong immunogenicity and can induce specific antibodies against polysaccharide and anti-protein at the same time.
  • a preferred method of conjugating the various pneumococcal capsular polysaccharide-protein conjugates of the invention is reductive amination.
  • the activated pneumococcal capsular polysaccharide is conjugated to the conjugated protein of choice by coupling the amino group of the carrier protein to the aldehyde group of the pneumococcal capsular polysaccharide in the presence of cyanoborohydride ions or another reducing agent. combine.
  • the pneumococcal capsular polysaccharide-protein conjugates resulting from the reductive amination process are preferably soluble in aqueous solutions. This makes the pneumococcal capsular polysaccharide-protein conjugates of the invention a preferred candidate for use as a vaccine.
  • the conjugation conditions can be adjusted based on the molecular weight of the polysaccharide and the specific protein.
  • the ratio of polysaccharide to carrier protein is controlled between 0.5-3.0.
  • 1.0-2.0 equivalents of sodium cyanoborohydride solution of the polysaccharide is added, and then 2.0 equivalents of sodium cyanoborohydride solution is added.
  • the incubation temperature may be room temperature, and the incubation time may be, for example, at least 12 hours.
  • use ultrafiltration to change the liquid or chromatography to remove impurities or unreacted substrates in the reaction.
  • sterile filter through a 0.22um filter, and store the polysaccharide-protein conjugate solution at 2-8°C.
  • capsular polysaccharide is conjugated to a carrier protein
  • the polysaccharide-protein conjugate is purified by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution, and column chromatography.
  • the immunogenic composition of the invention After purifying each complex sugar, they are mixed to formulate the immunogenic composition of the invention, which can be used as a vaccine.
  • Formulation of the immunogenic compositions of the present invention can be accomplished using methods well known in the art.
  • 20 or 24 individual pneumococcal conjugates may be formulated with pharmaceutically acceptable excipients to prepare a composition.
  • pharmaceutically acceptable excipients include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and glucose solutions.
  • immunogenic compositions can include one or more adjuvants.
  • an "adjuvant” is a substance used to enhance the immunogenicity of the immunogenic composition of the invention.
  • adjuvants are often administered to enhance immune responses and are well known to the skilled person.
  • Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, such as MF59, SAF, RibiTM Adjuvant System (RAS), (Corixa, Hamilton, MT); (3) Saponin adjuvants, such as QuilA or STIMULONTMQS-21; (4) Bacterial lipopolysaccharides (such as aminoalkyl glucosamine phosphate compounds ( AGP) or its derivatives or analogs), synthetic polynucleotides (such as oligonucleotides containing CpG motifs); (5) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL- 4.
  • aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.
  • IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. interferon (e.g., gamma interferon), granulocyte macrophage colony-stimulating factor (GM- CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; (6) Mucosal adjuvants, including protein toxins, recombinant protein subunits bases and nucleic acids, including detoxified mutants of bacterial ADP-ribosylating toxins, such as wild-type or mutant cholera toxin (CT), pertussis toxin (PT), or E.
  • CT cholera toxin
  • PT pertussis toxin
  • coli heat-labile toxin (LT) (see For example, WO93/13302 and WO92/19265); and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition.
  • the dosage of the adjuvant can be determined by those skilled in the art according to routine techniques.
  • the concentration of adjuvant (eg, aluminum phosphate) in the immunogenic compositions herein ranges from 0.125 mg/dose to 0.5 mg/dose.
  • the immunogenic compositions and vaccines of the present invention can be used to protect or treat mammals (eg, humans) susceptible to pneumococcal infection by administering the vaccine systemically or mucosally. and the use of the immunogenic compositions described herein for the preparation of a medicament for providing protection against a variety of pneumococcal infections.
  • the immunogenic compositions of the present invention are useful as a means of increasing antibodies for prophylactic and diagnostic purposes. Diagnostics are particularly useful for monitoring and detecting various infections and diseases caused by pneumococcal bacteria.
  • Another embodiment of the present invention uses an immunogenic composition as an immunogen for treating people at risk of pneumococcal infection or disease. Active and passive immunogenic protection of diseased individuals. Immunizing antibodies for passive protection are generated by immunizing a mammal with any of the immunogenic compositions of the invention and then recovering the bactericidal antibodies in the gamma-globulin fraction from the mammal, either as serum or as specific antibodies.
  • the vaccines of the invention used herein are capable of inducing antibodies that provide protection against pneumococcal infection.
  • pneumococcal capsular polysaccharide itself can be used as an immunizing agent, preferably in association with an adjuvant, such as an aluminum-based adjuvant, as an immunogenic composition.
  • an adjuvant such as an aluminum-based adjuvant
  • a further embodiment of the invention is the use of the immunogenic composition as immunogenic protection against pneumococcal infection.
  • Immunogenic compositions and vaccines of the present invention may typically be formed by dispersing the pneumococcal capsular polysaccharide or conjugate into suitable pharmaceutically acceptable excipients.
  • auxiliary materials include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants.
  • Excipients are preferably non-toxic to the recipient at the dosage and concentration employed, for example: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the compositions may contain ingredients to improve, maintain, or preserve, for example, the pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution, or release of the composition.
  • the rate, absorption or penetration of a substance are known from the prior art.
  • the optimal pharmaceutical composition will be determined based on the intended route of administration, mode of delivery and desired dosage.
  • Additives commonly used in vaccines may also be present, for example stabilizers such as lactose or sorbitol and adjuvants such as aluminum phosphate, aluminum hydroxide, aluminum sulfate, CpG, monophosphoryl lipid A, QS21, MF59 or stearoyltyrosine.
  • the protective or therapeutic effects of the immunogenic compositions and vaccines of the present invention can be accomplished by administering the vaccine via systemic or mucosal routes.
  • administration includes parenteral administration, such as injection by the intramuscular, intraperitoneal, intradermal or subcutaneous route, or mucosal administration to the oral/digestive, respiratory or genitourinary tracts.
  • intranasal administration is used to treat pneumonia or otitis media.
  • the dosage of the immunogenic composition should be such that an immunogenic effect is achieved.
  • the amount of conjugate per vaccine dose is selected as that amount that induces immune protection without significant adverse effects. This amount may vary depending on the pneumococcal serotype.
  • each dose will contain 0.01 ⁇ g to 100 ⁇ g of polysaccharide or conjugate, for example 0.1 ⁇ g. to 10 ⁇ g, or 1 ⁇ g to 5 ⁇ g. Doses generally range from about 0.01 ⁇ g to about 10 ⁇ g per kilogram of body weight. A range of optimal immunizing doses can be given.
  • a unit dosage form of the vaccine may contain an equivalent amount of about 0.01 ⁇ g to about 100 ⁇ g of the meningitis-causing bacterium capsular polysaccharide or conjugate, for example, 0.1 ⁇ g to 10 ⁇ g, or 1 ⁇ g to 5 ⁇ g.
  • the components of a specific vaccine are determined through standard studies that include observation of appropriate immune responses in subjects the optimal amount. After the initial vaccination, subjects may receive one or several booster immunizations spaced sufficiently apart.
  • the immunogenic compositions of the invention are suitable for use by infants, children, adolescents and adults.
  • the immunization regimen can be determined by experienced clinicians.
  • the 20 or 24 valent vaccine is serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, respectively, conjugated to CRM197 , 22F and 33F pneumococcal capsular polysaccharides and pneumococcal capsular polysaccharides of serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F each conjugated to TTD.
  • Each dose is formulated to contain: 2 ⁇ g of each sugar except 4 ⁇ g of polysaccharide 3, 6B, or 12F; approximately 20 to 80 ⁇ g of CRM197 carrier protein; approximately 10 to 40 ⁇ g of TTD carrier protein; 0.125 mg to 0.5 mg of aluminum phosphate adjuvant. Fill a single-dose syringe without preservatives. After shaking, the vaccine is homogeneous and the white suspension is ready for intramuscular administration.
  • the serotypes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 serotypes selected from the following: 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, and as described Serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.
  • the carrier is selected from one or more of saline, Ringer's solution and phosphate buffered saline.
  • the immunogenic composition according to item 1 or 2 characterized in that the immunogenic composition further contains an adjuvant,
  • the adjuvant includes one or more selected from aluminum-based adjuvants, monophosphoryl lipid A, QS21, CpG, MF59, stearoyltyrosine, Freund's adjuvant and other mucosal adjuvants. kind.
  • the immunogenic composition according to item 1 or 2 characterized in that the weight ratio of the capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is 10:1 to 1:10 , such as 5:1 to 1:5,
  • the composition is a preparation, and the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1 to 8ug/dose, and the concentrations of the remaining capsular polysaccharides are each independently 0.5 to 5ug/dose.
  • a multivalent immunogenic composition comprising a variety of polysaccharide-protein conjugates and pharmaceutically acceptable excipients, wherein each polysaccharide-protein conjugate contains pneumonia chains from different serotypes conjugated to a carrier protein Capsular polysaccharide of cocci,
  • the carrier protein contains at least two carrier proteins,
  • the serotypes include at least 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F; more preferably, the serotypes include the following 20 serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, or the following 24 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.
  • TTD is the C-terminal domain of TT
  • the TTD has the sequence shown in SEQ ID NO.2, and the TTD variant has a sequence with at least 90% sequence identity with SEQ ID NO.2.
  • the serotypes include the 20 serotypes from 8, 9, 10, 11, 12, 13, 14, 15 including serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F , 16, 17, 18, 19 or 20 serotypes of capsular polysaccharide, respectively, conjugated to the carrier protein TTD or a variant thereof, or
  • the serotypes include the 24 serotypes, including serotypes 3, 5, 6B, 12F, Capsular polysaccharides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 serotypes including 15B, 18C, 19F and 23F Conjugated to the carrier protein TTD or its variants, respectively,
  • the serotypes include the 20 serotypes in which capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are respectively conjugated to the carrier protein TTD or a variant thereof; from serotypes
  • the capsular polysaccharides of 1, 4, 6A, 7F, 8, 9V, 10A, 11A, 14, 19A, 22F, and 33F are respectively conjugated with the carrier protein CRM197, or
  • the serotypes include the 24 serotypes, of which the capsular polysaccharides from serotypes 3, 5, 6A, 6B, 9N, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 23F, and 33F are respectively conjugated to the carrier protein TTD or a variant thereof; capsular polysaccharides from serotypes 1, 2, 4, 7F, 8, 9V, 10A, 14, 22F, respectively, conjugated to the carrier protein CRM197, or
  • the serotypes include the 24 serotypes in which capsular polysaccharides from serotypes 3, 5, 6B, 12F, 15B, 18C, 19F and 23F are respectively conjugated to the carrier protein TTD or a variant thereof; from serotypes
  • the capsular polysaccharides of 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F, and 33F were conjugated to the carrier protein CRM197 respectively.
  • the weight ratio of the capsular polysaccharide from serotype 3, 6B or 12F to any other capsular polysaccharide is 10:1 to 1:10, such as 5:1 to 1:5,
  • composition is a preparation, and the concentrations of capsular polysaccharides from serotypes 3, 6B and 12F are each independently 1 to 8ug/dose, and the concentrations of the remaining capsular polysaccharides are each independently 0.5 to 5ug/dose,
  • composition further includes an adjuvant; preferably, the adjuvant includes an adjuvant selected from the group consisting of aluminum-based adjuvant, monophosphoryl lipid A, QS21, CpG, MF59, stearoyltyrosine, Freund's adjuvant and others one or more mucosal adjuvants,
  • the weight ratio of conjugate to adjuvant is 1:10 to 1:2
  • the composition also contains a surfactant; preferably, the concentration of the surfactant in the composition is 100 to 300 ⁇ g/dose,
  • the pH of the composition is 5.0-7.0.
  • the medicament is used to prevent or treat pneumococcal infection and/or tetanus toxin infection.
  • An immune composition that leads to passive immunity including a bactericidal antibody targeting pneumococcal bacteria, the antibody being obtained by immunizing a mammal with the immunogenic composition described in any one of items 1 to 8,
  • the bactericidal antibodies are present in serum, gamma globulin fractions or purified antibody preparations.
  • Pneumococcal bacterial culture ⁇ fermentation and polysaccharide preparation is a mature process (US4686102, US5847112).
  • Streptococcus pneumoniae strains of different serotypes (American Type Biological Resources Collection) were cultured to prepare a bacterial strain library.
  • a tube of Streptococcus pneumoniae strains in the bacterial strain library was inoculated into a shake flask containing soybean culture medium. Cultivation in an incubator at °C and 5-10% CO 2 overnight. After the culture is completed, microscopic examination is performed. If the result is normal, the culture solution is inoculated into a 10L fermentation tank and fermented and cultured at pH 7.0 and temperature 37°C. After the culture is completed, add 10% DOC solution for inactivation to ensure complete inactivation of pneumococcal bacteria.
  • the preparation and purification process mainly includes centrifuging the bacterial inactivated solution to separate the bacterial cells, harvesting the centrifugal supernatant, and then filtering through a 0.22 ⁇ m filter membrane and concentrating through 100KD membrane bag ultrafiltration to obtain the pneumonia polysaccharide fermentation concentrate.
  • Add CTAB solution to the pneumonia polysaccharide fermentation concentrate stir at room temperature, centrifuge at 8000 rpm, and collect the polysaccharide complex precipitate (for 7F, 14, and 33F polysaccharides, collect the supernatant).
  • the polysaccharide complex is then dissociated with 0.25-0.5M NaCl solution, and then purified through precipitation, column chromatography, ultrafiltration and other processes to obtain a purified polysaccharide solution.
  • the polysaccharide solution was filtered through a 0.22 ⁇ m membrane and stored at low temperature.
  • pneumococcal capsular polysaccharides prepared were used to identify serotypes and The chemical structure of pneumococcal polysaccharides can be analyzed by nuclear magnetic resonance (NMR), and the functional groups (such as rhamnose, uronic acid, O-acetyl and other components) contained in the polysaccharides can be analyzed by chemical color development methods. Content determination. It was determined that the physical and chemical indicators of the polysaccharide meet the standards set by the European Pharmacopoeia for pneumococcal polysaccharides, and the contents of major impurities such as protein and nucleic acid are less than 1%.
  • the DNA sequence expressing TTD (SEQ ID NO: 1) was optimized and cloned into the protein expression plasmid pET21 to construct E. coli BL21 (DE3) recombinantly expressing the TTD protein (SEQ ID NO: 2). Pick the recombinant BL21 (DE3) single clone colony in 10 mL of LB (Amp) liquid medium and culture it at 37°C and 250rpm until the OD reaches 600 to 0.8. Then add 0.1mM IPTG and continue culturing for 4 hours at 25°C and 250rpm. The culture medium was centrifuged at 8000 rpm and 4°C to collect the cells, then resuspended in PBS and disrupted.
  • the broken liquid was centrifuged at 8000 rpm and 4°C, and the supernatant was collected.
  • the TTD protein is purified through ammonium sulfate precipitation, clarification and filtration, and combined chromatography, with a purity of more than 95%.
  • the molecular weight determined by mass spectrometry was consistent with the theoretical molecular weight, and the homogeneity of the TTD protein was confirmed by molecular sieves.
  • the amino acid sequence of TTD is as follows:
  • the CRM197 carrier protein is a non-toxic variant of diphtheria toxin (SEQ ID NO: 3), which is non-toxic but retains the immunogenicity of diphtheria toxin. Its fermentation and purification methods have been reported in published articles and patents (US5614382). Usually, the Corynebacterium diphtheriae strain is inoculated into the culture medium, stirred at 37°C for 20-30 hours, and then removed by centrifugation, retaining the supernatant. After the fermentation supernatant is replaced by ultrafiltration, ammonium sulfate is added to precipitate and the precipitate is collected. Then sink The precipitate is dissolved, the liquid is changed, and the CRM197 protein is purified through column chromatography, with a purity of more than 95%, and is used for binding reactions.
  • SEQ ID NO: 3 diphtheria toxin
  • polysaccharide-protein conjugates by coupling bacterial polysaccharides with carrier proteins is an effective way to improve the immunogenicity of bacterial polysaccharides.
  • Polysaccharide-protein conjugates are widely used in the preparation of bacterial vaccines, such as Haemophilus influenzae type B polysaccharide PRP-TT conjugate vaccine, Neisseria meningitis bacteria A, C, Y, and W polysaccharide-carrier protein conjugate vaccines.
  • the preparation of polysaccharide-protein conjugates is mainly divided into two steps: hydrolysis or activation of polysaccharides, and polysaccharide-protein coupling.
  • Polysaccharide activation can be divided into different methods such as CNBr method (US 4619828), hydrolysis (US4761283), sodium periodate oxidation method (US5306492).
  • the activated polysaccharide can then be directly chemically coupled to the carrier protein (US4356170) or through small molecules
  • the connector is coupled to the carrier protein.
  • the preparation of pneumococcal polysaccharide-protein conjugates mainly includes the following steps: polysaccharide treatment, polysaccharide activation, and polysaccharide-protein coupling.
  • Pneumococcal polysaccharide pretreatment Some serotypes can be hydrolyzed by acid or alkali (US5847112A) before oxidation to reduce the molecular weight or improve the oxidative binding efficiency of polysaccharides, such as serotypes 1, 3, 4, 6A, 6B, 8, 10A, 11A, 12F, 15B, 18C, 22F polysaccharides. Some polysaccharides can reduce their molecular size through high-pressure homogenization, such as serotypes 2, 6B, 7F, 8, 10A, 11A, 12F, 14, 15B, 19A, 19F, and 33F.
  • Pneumococcal polysaccharide activation Polysaccharide activation is performed by adding sodium periodate solution (US4711779). The amount of periodate added is usually 0.05-2 equivalents of the polysaccharide. The oxidation time is 12-24 hours. After the polysaccharide is activated, the small molecular substances in the reaction are removed by ultrafiltration and concentration.
  • Preparation of pneumococcal polysaccharide-protein conjugates The chemical coupling of polysaccharide-protein is carried out by reductive amination method (US5952454, EP0720485, US4711779). Preparation of polysaccharide-protein conjugates of serotypes 1, 2, 4, 6A, 7F, 8, 9N, 9V, 10A, 11A, 14, 17F, 19A, 20, 22F and 33F polysaccharides by conjugation with CRM97; 3, 5 , 6B, 12F, 15B, 18C, 19F and 23F polysaccharides were prepared by coupling with TTD to prepare polysaccharide-protein conjugates.
  • the ratio of polysaccharide to carrier protein is controlled between 0.5-2.5.
  • 1.0-2.0 equivalents of sodium cyanoborohydride solution is added, and then 2.0 equivalents of sodium cyanoborohydride solution is added.
  • ultrafiltration to change the liquid or chromatography to remove impurities or unreacted substrates in the reaction.
  • sterile filter through a 0.22um filter, and store the polysaccharide-protein conjugate solution at 2-8°C.
  • All polysaccharide-protein conjugates pass mass analysis and free polysaccharide content is controlled within 20%, the free protein content is within 2%, the polysaccharide-protein ratio is between 0.5-2.0, and the endotoxin and other impurity contents are controlled within safe and acceptable ranges.
  • each polysaccharide-protein monovalent conjugate was prepared by diluting it with a pH 6.0 buffer solution and mixing, then adding aluminum phosphate adjuvant and stirring.
  • the polysaccharide content in various conjugates is 2 to 4 micrograms per milliliter, and the aluminum phosphate adjuvant content is 0.125 to 0.5 mg.
  • Mouse immunization experimental plan Add the polyvalent vaccine stock solution to the aluminum phosphate adjuvant to prepare an immune antigen.
  • Balb/c mice aged 6-8 weeks are selected for intraperitoneal immunization. There are 8 mice in each group, and the dose of each immunization is 0.5ml, immunized on days 0, 14, and 21 respectively.
  • Relevant vaccines that have been launched (Pfizer's Prevail 13, Watson Biotech's Wo'anxin) are used as positive controls, and aluminum phosphate adjuvant is used as negative controls.
  • Rabbit immunization experimental plan Add the polyvalent vaccine stock solution to aluminum phosphate adjuvant to prepare an immune antigen. Select New Zealand white rabbits of 2.0 to 2.5kg for thigh muscle immunization. There are 8 rabbits in each group. Each immunization dose is 0.5ml. At the same time, PCV13 (Pel 13) was used as the positive vaccine for two doses of immunization, and blood was drawn on 35 days to evaluate the immunogenicity of its polysaccharide.
  • Pneumonia polysaccharide was diluted with coating buffer, coated on a 96-well enzyme plate at 100 ⁇ l/well, and incubated at 37°C before washing the plate.
  • the serum is first diluted 1:100, then 2.5 times gradient diluted, diluted 8 gradients, and then added to the enzyme plate, 50 ⁇ l per well, and incubated overnight.
  • the secondary antibody to the enzyme plate at a dilution of 1:10,000, 100 ⁇ l per well, incubate for 2 hours, wash the plate, then add 1 mg/ml PNPP-Na chromogenic substrate, 100 ⁇ l per well, and incubate.
  • Example 1 Serotype 5 polysaccharide-CRM197 conjugate and pneumopolysaccharide-TTD conjugate in mice Comparison of immunogenicity
  • mice/group After adding aluminum phosphate adjuvant to the 5PS-CRM197 conjugate and 5PS-TTD conjugate, immunize BALB/c mice (5 mice/group) at 2 ⁇ g/dose on days 0, 14, and 28, respectively. Blood collection will be done on day 35 to detect antibody titers.
  • the immunogenicity differences of the 24-valent pneumonia polysaccharide conjugate vaccine with aluminum phosphate adjuvant dosages of 0.5 mg/dose, 0.25 mg/dose, and 0.125 mg/dose were evaluated in BALB/c mice.
  • BALB/c female mice aged 6 to 8 weeks were randomly divided into groups, 8 mice/group, and were immunized with 0.5ml/dose on days 0, 14, and 28 respectively. Blood was collected on day 35 to detect antibody titer levels.
  • the originality data are shown in Figures 2 and 3.
  • Example 3 Comparison of the immunogenicity of 24-valent pneumococcal polysaccharide conjugate vaccine in mice under different pH conditions
  • the immunogenicity differences of 24-valent pneumonia polysaccharide conjugate vaccines at different pHs were evaluated in BALB/c mice.
  • the 24-valent pneumonia polysaccharide conjugate vaccine was evaluated in 6- to 8-week-old BALB/c female mice. The mice were randomly divided into groups, with 8 mice/group. In the test, a total of two immunizations were conducted on D0 and D14, one dose per time. Serum was collected on D0 and D21, and the polysaccharide antibody titer level in the serum was detected by ELISA.
  • 24-valent pneumococcal polysaccharide conjugate vaccine with a pH range of 5.0 to 6.2. Comparative illustration of serum antibody titers one week after the second dose of immunization (D21) (see Figure 4). 24-valent pneumococcal polysaccharide conjugate vaccine with a pH range of 5.0 to 6.2 After immunization, there was no significant difference in the antibody titer for each serotype between groups (P>0.05), and good immunogenicity could be induced.
  • Example 4 Immunogens of 24-valent pneumococcal polysaccharide conjugate vaccine and positive vaccine in mice sexual comparison
  • Toxoid serves as a carrier protein.
  • the 24-valent pneumococcal conjugate vaccine can also induce better immunogenicity (Figure 6).
  • PCV24 pneumococcal polysaccharide conjugate vaccine was prepared to immunize rabbits (8/group), using PCV13 (Pel 13) as the positive control and physiological saline as the negative control. After the first immunization, the vaccine was administered three weeks apart, and two weeks after the second immunization. Blood was collected every week (D35) to detect the antibody titers of various types of polysaccharides in the serum ( Figure 7 and Figure 8).
  • the above two groups of vaccines were immunized against New Zealand white rabbits respectively, with 8 rabbits in each group. They were immunized twice in total, with an interval of three weeks. Serum was collected before immunization and two weeks after the second immunization to detect the titer levels of various types of polysaccharide antibodies. Two weeks after the first immunization, the vaccine titers of the two groups were converted into logarithms and statistically analyzed using Multiple t tests. The results are shown in Figure 9.
  • the dual-vector vaccine group can induce higher antibody titers than the single-vector vaccine group, and there are 7 serotypes (serotypes 4, 5, 9V, 12F, 19A , 19F, 23F), the induced antibody titers were significantly different (P ⁇ 0.05). It shows that the dual-carrier vaccine can avoid the immunosuppressive effect caused by a single vector.
  • Example 7 Comparison of the immunogenicity of dual-carrier and single-carrier 20-valent pneumococcal polysaccharide conjugate vaccines
  • the titer levels of 16 of the 20 serotypes in the dual-vector vaccine were higher than those in the single-vector vaccine group.
  • the titer levels of 16 of the 24 serotypes of the dual-vector vaccine were not lower than those of the single-vector vaccine group.
  • Example 9 BALB/c mouse protection test of 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 pneumococci
  • PCV13 Wood, Wo Anxin polysaccharide conjugate vaccine was used as the positive control substance and normal saline was used as the negative control substance.
  • 2 ⁇ 10 5 CFU of type 3 pneumonia was Streptococcal intranasal infection.
  • the survival rate within two weeks after challenge was observed, and the protective power of the 24-valent pneumococcal polysaccharide conjugate vaccine against type 3 pneumococcal challenge was analyzed.
  • Example 9 BALB/c mouse protection test of 24-valent pneumococcal polysaccharide conjugate vaccine against type 22F pneumococcus
  • the experiment used 23-valent pneumococcal polysaccharide vaccine (PPV23) as the positive control substance and physiological saline as the negative control substance.
  • PV23 23-valent pneumococcal polysaccharide vaccine
  • Three weeks after one dose of intraperitoneal immunization in BALB/c mice 1 ⁇ 10 8 CFU of 22F type Streptococcus pneumoniae abdominal infection. Based on the survival of mice after challenge, the protective power of the 24-valent pneumococcal polysaccharide conjugate vaccine against 22F pneumococci was analyzed.
  • Example 10 Protection test of 24-valent pneumococcal polysaccharide conjugate vaccine against tetanus toxin
  • the 24-valent pneumococcal polysaccharide conjugate vaccine contains TTD carrier protein, and the effectiveness of the vaccine was evaluated through a tetanus toxin challenge protection test in mice.
  • the TT antibody titer levels in groups B, C, and D were significantly increased after two doses of immunization compared with before immunization, P ⁇ 0.05.
  • the TT antibody titer in group B was higher than that in group C, possibly because the diphtheria-tetanus pertussis vaccine is composed of TT, and the 24-valent pneumococcal polysaccharide conjugate vaccine uses TTD.

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Abstract

本发明涉及多价肺炎球菌多糖蛋白质结合物及其免疫原性，具体提供一种免疫原性组合物，含有来自不同血清型的肺炎链球菌的荚膜多糖和载剂，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。本发明的免疫原性组合物，能够提高不同血清型多糖的免疫原性，可以预防多种不同血清型肺炎球菌引起的侵袭性感染。

Description

多价肺炎球菌多糖结合疫苗的成分及其应用 技术领域
本发明涉及生物医学领域，具体涉及通过免疫多价疫苗预防细菌病原体的感染。
背景技术
肺炎链球菌(S.pneumoniae，即肺炎球菌)是一种具有荚膜的革兰氏阳性双球菌。根据其荚膜多糖的组成差异，可区分为近100种血清型，其中荚膜多糖为重要致病因子。肺炎链球菌正常情况下寄生于健康人鼻咽部，当寄生的环境发生变化时，如机体抵抗力下降，麻疹、流感等呼吸道病毒感染，或营养不良、老年体弱等情况下，它可透过黏膜防御体系发生侵袭性感染，如进入下呼吸道引起肺炎，穿过血脑屏障引起细菌性脑膜炎，穿过肺泡上皮细胞、侵袭血管内皮细胞进入血液引起菌血症，还可从鼻咽部移行进入鼻窦，引起鼻窦炎，通过咽鼓管进入中耳，引起中耳炎非侵袭性地持续扩散至呼吸道的其他部位。
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年全球每年大约有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，各年龄组免疫功能低下者。
根据肺炎球菌感染部位不同，可将肺炎球菌性疾病分为侵袭性肺炎球菌性疾病(IPD)和非侵袭性肺炎球菌性疾病(NIPD)两大类。常见的治疗方法就是抗生物治疗，但在全球范围，肺炎球菌对常用抗菌药物产生耐药性已成为一个日益严重的问题。多年临床实践证明接种肺炎球菌疫苗是预防肺炎球菌疾病最经济有效的途径。
目前已上市的肺炎疫苗包括肺炎多糖疫苗(PPSV)和肺炎球菌多糖蛋白结合疫苗(PCV)两大类。23价肺炎多糖疫苗(PPSV23)由于其免疫原性低、没有免疫记忆和加强作用，所以不适用于婴幼儿和免疫力低下的人群。已上市的 可应用于婴幼儿的肺炎多糖结合疫苗包括PCV7、PCV10和PCV13，但其血清覆盖范围小、免疫保护覆盖率低，并且可能引起免疫抑制作用。例如，针对我国内地肺炎链球菌血清型分布的文献荟萃分析表明，2000～2016年期间PCV10血清型覆盖率为52.3％，PCV13血清型覆盖率为68.4％。另外，也发现疫苗使用后非疫苗血清型疾病的发生率在增加。另外，由于多糖-蛋白结合疫苗所用的载体蛋白也广泛应用于婴幼儿的免疫接种中，因为高剂量或重复使用载体蛋白而引起的免疫抑制作用也是多糖-蛋白结合疫苗在临床应用中潜在的风险。
所以，现有多糖-蛋白结合疫苗的血清型覆盖率尚不理想，开发多价肺炎结合疫苗，覆盖更广的致病性血清型的肺炎球菌，增加非疫苗血清型，提高新型多价疫苗的覆盖保护率和免疫原性，降低载体蛋白免疫抑制作用的风险，有其重要的临床价值。
发明内容
为了解决肺炎球菌多糖疫苗免疫原性差、多糖结合疫苗血清覆盖率低、免疫原性低、免疫抑制等问题，本发明提供适用更广、免疫原性更强且免疫抑制减弱的多价免疫原性组合物。
本发明第一方面提供一种免疫原性组合物，含有来自不同血清型的肺炎链球菌的荚膜多糖和载剂，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。
在一个或多个实施方案中，所述血清型包括选自以下的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24种血清型：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，并且所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。优选地，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F，或以下24种血清型：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。
在一个或多个实施方案中，所述载剂选自盐水、林格氏溶液和磷酸盐缓冲 盐水中的一种或多种。
在一个或多个实施方案中，所述免疫原性组合物还含有佐剂。
在一个或多个实施方案中，所述佐剂是基于铝的佐剂。
在一个或多个实施方案中，所述佐剂包括选自磷酸铝、硫酸铝、氢氧化铝、单磷酰基脂A、QS21、CpG、MF59、硬脂酰酪氨酸、弗氏佐剂和其他粘膜佐剂中的一种或多种。
在一个或多个实施方案中，所述组合物中，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，优选为2∶1。
在一个或多个实施方案中，所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂(优选4μg/剂)，其余荚膜多糖的浓度各自独立为0.5～5ug/剂(优选2μg/剂)。进一步地，磷酸铝佐剂的浓度为0.125mg/剂至0.5mg/剂。
在一个或多个实施方案中，所述组合物还包含表面活性剂，例如Tween 20或Tween 80。优选地，组合物中表面活性剂的浓度为100～300μg/剂，优选70～120μg/剂。
本发明还提供包含多种的多糖-蛋白质缀合物以及药学上可接受辅料的多价免疫原性组合物，其中每种多糖-蛋白质缀合物含有缀合到载体蛋白的来自不同血清型的肺炎链球菌的荚膜多糖。
在一个或多个实施方案中，所述载体蛋白至少包含两种载体蛋白。
在一个或多个实施方案中，所述载体蛋白包括(1)CRM197和(2)TTD或其变体。
在一个或多个实施方案中，TTD是TT的C端结构域。
在一个或多个实施方案中，TTD具有SEQ ID NO：2所示的序列，TTD变体与SEQ ID NO：2具有至少90％序列相同性的序列。
在一个或多个实施方案中，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。优选地，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、 19F、22F、23F、33F，或以下24种血清型：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。
在一个或多个实施方案中，在所述多糖蛋白缀合物中，至少来自血清型3、5、6B、12F、15B、18C、19F和23F的一种或多种或全部荚膜多糖分别与载体蛋白TTD或其变体缀合。
在一个或多个实施方案中，所述血清型包括所述20种血清型，其中来自包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19或20种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合。
在一个或多个实施方案中，所述血清型包括所述20种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、4、6A、7F、8、9V、10A、11A、14、19A、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合。
在一个或多个实施方案中，所述血清型包括所述24种血清型，其中来自包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合。
在一个或多个实施方案中，所述血清型包括所述24种血清型，其中来自血清型3、5、6A、6B、9N、11A、12F、15B、17F、18C、19A、19F、20、23F、33F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、7F、8、9V、10A、14、22F的荚膜多糖分别与载体蛋白CRM197缀合。
在一个或多个实施方案中，所述血清型包括所述24种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、6A，7F、8、9N、9V、10A、11A、14、17F、19A、20、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合。
在一个或多个实施方案中，所述组合物中，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，优选为2∶1。
在一个或多个实施方案中，所述组合物还包含佐剂，如基于铝的佐剂。
在一个或多个实施方案中，所述佐剂选自磷酸铝、硫酸铝和氢氧化铝，优选磷酸铝。
在一个或多个实施方案中，所述组合物中，缀合物与佐剂的重量比为1∶10至1∶2，优选为54∶500至54∶125。
在一个或多个实施方案中，所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂(优选4μg/剂)，其余荚膜多糖的浓度各自独立为0.5～5ug/剂(优选2μg/剂)，磷酸铝佐剂的浓度为0.125mg/剂至0.5mg/剂。
在一个或多个实施方案中，所述组合物还包含表面活性剂，例如Tween 20或Tween 80。优选地，组合物中表面活性剂的浓度为100～300μg/剂，优选70～120μg/剂。
在一个或多个实施方案中，所述组合物的pH为5.0-7.0，优选5.0～6.2。
本发明还提供本文第一方面所述免疫原性组合物在制备诱导对肺炎球菌荚膜多糖缀合物的免疫应答和/或对破伤风毒素的免疫应答的药物中的用途。
在一个或多个实施方案中，所述药物用于预防或治疗肺炎球菌感染和/或破伤风毒素感染。
本发明还提供诱导对肺炎球菌荚膜多糖缀合物的免疫应答和/或对破伤风毒素的免疫应答的方法，包括向对象施用免疫有效量的本发明第一方面所述的免疫原性组合物。
本发明还提供一种导致被动免疫的免疫组合物，包括靶向肺炎球菌的杀菌抗体，所述抗体是由本文任一实施方案所述的免疫原性组合物免疫哺乳动物而获得。在一个或多个实施方案中，所述杀菌抗体存在于血清、γ球蛋白部分或纯化的抗体制剂中。
本发明优点：
本发明的免疫原性组合物，可以有效预防24种不同血清型的肺炎球菌的侵袭性感染，对24种血清型都能诱导相对均衡的比较高的免疫原性，其免疫原性与各血清型的结合物含量配比有关。本发明的免疫原性组合物同时含有不同的载体蛋白，而且两种载体蛋白都没有毒性，所以不需要通过脱毒处理。该双载体设计降低了潜在安全及免疫抑制风险。该免疫原性组合物还对对破伤风毒素的感染具有保护力。
附图说明
图1，不同载体的5型肺炎球菌多糖结合物免疫原性比较。
图2，不同佐剂剂量下24价肺炎球菌多糖结合疫苗在小鼠体内的免疫原性比较(与阳性疫苗相同的13种血清型)。
图3，不同佐剂剂量下24价肺炎球菌多糖结合疫苗在小鼠体内的免疫原性比较(阳性疫苗的13种血清型以外的11种血清型)。
图4，在小鼠体内不同pH的24价肺炎球菌多糖结合疫苗的免疫原性比较。
图5，在小鼠体体内13价相同血清型免疫原性(D35)比较。
图6，在小鼠体内PCV13疫苗以外血清型多糖抗体滴度(D35)比较。
图7，在大兔体内13价相同血清型多糖抗体滴度比较(D35)。
图8，在兔体内13种以外血清型多糖抗体滴度(D35)比较。
图9，双载体与单载体24价肺炎多糖结合疫苗免疫二剂后血清抗体滴度水平。
图10，PCV20双载体与单载体疫苗免疫二剂后血清滴度比较。
图11，PCV24双载体与单载体疫苗免疫二剂后血清滴度比较。
图12，24价肺炎球菌多糖结合疫苗对3型肺炎球菌挑战的小鼠保护力生存曲线。
图13，24价肺炎球菌多糖结合疫苗对22F型肺炎球菌的保护力生存曲线。
图14，免疫二剂后与免疫前TT抗体滴度比较。A组为生理盐水；B组为百白破疫苗；C组为24价肺炎球菌多糖结合疫苗。
图15，破伤风毒素挑战后生存率曲线。
具体实施方式
本发明提供新型的20价或24价肺炎球菌荚膜多糖-蛋白缀合物以及相应免疫原性组合物和疫苗制剂。本发明的疫苗制剂与上市的13价多糖结合疫苗相比，在一些血清型上诱导的抗体滴度更高；对13种血清型以外的血清型也显示出较好的免疫原性；相比13价多糖结合疫苗和23价多糖疫苗能够在体内诱导更好的免疫保护作用。此外，发明人还发现，在不同缀合物上使用不同载体比单载体疫苗组能诱导更高的抗体滴度。
本发明首先提供一种诱导哺乳动物对肺炎球菌荚膜多糖缀合物的免疫应答、保护其不受肺炎球菌感染的免疫原性组合物。该免疫原性组合物含有来自不同血清型的肺炎链球菌的荚膜多糖，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。优选地，所述血清型包括：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。
存在于免疫原性组合物中的肺炎球菌荚膜多糖可以是游离多糖的形式，或作为缀合物的一个组分，在缀合物中多糖与蛋白质共价相连。除了多糖或多糖蛋白缀合物之外，免疫原性组合物还可以包含药学上可接受的载剂，例如盐水、林格氏溶液或磷酸盐缓冲盐水。
在本发明优选的实施方案中，荚膜多糖与蛋白质共价相连形成缀合物。因而，个体可接受的、并能诱导免疫细胞(例如T-细胞)依赖性应答的任何蛋白质或其片段都适宜与肺炎球菌荚膜多糖缀合。基本上任何蛋白质都能作为缀合蛋白。特别是所选择的蛋白质必须具有至少一个自由氨基，用于与多糖的缀合。优选蛋白质是任何天然或重组细菌蛋白，并且其本身是诱导年轻和成年哺乳动物中T-细胞依赖性应答的免疫原。这样的蛋白质的例子包括，但不限制于，破伤风类毒素、霍乱毒素、白喉类毒素和CRM197或它们的变体。其它缀合蛋白的候选物包括假单胞菌、葡萄球菌、链球菌、百日咳和包括大肠杆菌的产肠毒素细菌的毒素或类毒素。
在本发明优选的实施方案中，肺炎球菌荚膜多糖与蛋白质共价相连形成缀合物。因而，个体可接受的、并能诱导免疫细胞(例如T-细胞)依赖性应答的 任何蛋白质或其片段都适宜与肺炎球菌荚膜多糖缀合。基本上任何蛋白质都能作为缀合蛋白。特别是所选择的蛋白质必须具有至少一个自由氨基，用于与多糖的缀合。优选蛋白质是任何天然或重组细菌蛋白，并且其本身是诱导年轻和成年哺乳动物中T-细胞依赖性应答的免疫原。这样的蛋白质的例子包括，但不限制于，破伤风类毒素、霍乱毒素、白喉类毒素。其它缀合蛋白的候选物包括假单胞菌、葡萄球菌、链球菌、百日咳和包括大肠杆菌的产肠毒素细菌的毒素或类毒素。
与肺炎球菌荚膜多糖缀合的蛋白质(即载体蛋白)可以是个体可接受的、并能诱导免疫细胞(例如T-细胞)依赖性应答的任何蛋白质或其片段。基本上任何蛋白质都能作为缀合蛋白。特别是所选择的蛋白质必须具有至少一个自由氨基，用于与多糖的缀合。优选蛋白质是任何天然或重组细菌蛋白，并且其本身是诱导年轻和成年哺乳动物中T-细胞依赖性应答的免疫原。这样的蛋白质的例子包括，但不限制于，破伤风类毒素、霍乱毒素、白喉类毒素。其它缀合蛋白的候选物包括假单胞菌、葡萄球菌、链球菌、百日咳和包括大肠杆菌的产肠毒素细菌的毒素或类毒素。
另外，还可以使用蛋白毒素的非毒性变体，如CRM197。优选这样的突变保留了天然毒素的表位。这些突变后的毒素被称作“交叉反应物质”或CRM。CRM197载体蛋白(NCBI：AMV91693.1，SEQ ID NO：3)是白喉杆菌素的无毒变异体，该蛋白没有毒性，但保留了白喉类毒素(DT)的免疫原性。在公开的文章和专利中可获得其发酵和纯化方法(US5614382)。白喉类毒素无毒变异体蛋白(CRM197)是在临床已证明安全、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。其他白喉毒素变体也适于用作载体蛋白。还可以用这些蛋白质的片段肺炎球菌多糖缀合，其条件是这些片段应足够长，即优选至少10个氨基酸以确定T-细胞表位。
破伤风类毒素蛋白(TT)在公开文献中有很多报道。发明人发现了TT蛋白的变体TTD蛋白。该蛋白没有毒性，但保留了TT蛋白的免疫原性。TTD蛋白位于TT蛋白的重链C-末端，相对分子量50KDa，是毒素的受体结合区域，没有毒性，具有良好的免疫原性，其过敏性低于破伤风类毒素，是潜在的破伤风疫苗抗原成分及载体蛋白。TTD可以通过重组表达纯化得到(Immunobiology， Vol.216，Issue 4，2011，P 485-490)。示例性的TTD表达纯化方法包括：将表达TTD的DNA序列(SEQ ID NO：1)克隆到蛋白表达质粒pET21中，构建重组表达TTD蛋白(SEQ ID NO：2)的工程菌(例如大肠杆菌BL21(DE3))。挑取重组工程菌单克隆菌落在合适的培养基和条件中扩增(例如BL21(DE3)在10mL的LB(Amp)液体培养基中，37体，250rpm培养至OD600至0.8后)，加入诱导剂培养(例如加入0.1mM IPTG，在251，250rpm继续培养4h)，然后从培养物中分离TTD。从培养物中分离蛋白的过程本领域熟知，例如以下步骤：培养液8000rpm，4℃离心，然后用PBS重悬后破碎(例如超声波破碎)细胞，破碎液在8000rpm，4℃离心，收取包含TTD蛋白的上清。从上清中纯化TTD蛋白的方法可使用本领域常用从液体中纯化蛋白的方法，例如通过硫酸铵沉淀、澄清过滤、组合层析如离子交换层析、复合介质层析、和/或亲和层析等层析方法得到，纯度在95％以上。通过质谱测定的分子量与理论分子量一致。
其他合适的载体蛋白包括灭活的细菌毒素，如破伤风类毒素、百日咳类毒素、霍乱类毒素(参见例如WO2004/083251)、大肠杆菌LT、大肠杆菌ST、和来自铜绿假单胞菌(Pseudomonas aeruginosa)的外毒素。也可以使用细菌外膜蛋白，如外膜复合体c(OMPC)、孔蛋白、转铁蛋白结合蛋白、肺炎球菌溶血素、肺炎球菌表面蛋白A(PspA)、肺炎球菌黏附素蛋白(PsaA)、来自组A或者组B链球菌的C5a肽酶、或者流感嗜血杆菌蛋白D。其他蛋白质，如卵白蛋白、匙孔血蓝蛋白(KLH)、牛血清白蛋白(BSA)或者结核菌素的纯化的蛋白质衍生物(PPD)也可以用作载体蛋白。
在不实质性影响TTD活性(例如毒素结合活性)的前提下，本领域技术人员可以对本发明的TTD改变一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得TTD的变体。这些变体包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质的功能。可进行保守性取代的氨基酸残基为本领域所周知。这样的取代的氨 基酸残基可以是也可以不是由遗传密码编码的。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。它们都被视为包括在本发明保护的范围内。本文所述TTD变体包括与SEQ ID NO：2所示的TTD具有至少90％(例如至少95％，至少98％，至少99％)序列相同性并保留其毒素结合活性的TTD变体。
发明人发现，使用不同载体蛋白对多种血清型多糖进行缀合，混合后形成的免疫原性组合物具有更好的免疫原性和更低的免疫抑制风险。示例性地，免疫原性组合物中至少包含两种载体蛋白，例如CRM197和TTD或其变体。在优选的实施方案中，本发明的20或24种肺炎球菌多糖分别与CRM197和TTD载体蛋白通过化学偶联制备得到多糖蛋白缀合物。所得的不同血清型多糖-蛋白结合物具有良好的理化特性，如多糖蛋白比在0.5-3.0之间，游离糖含量在20％以下，其它残留杂质都控制在很低的范围内。
在一些实施方案中，在所述多糖蛋白缀合物中，至少来自血清型3、5、6B、12F、15B、18C、19F和23F的一种或多种或全部荚膜多糖分别与载体蛋白TTD或其变体缀合。进一步地，包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24种血清型分别与载体蛋白TTD或其变体缀合。优选地，来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、6A，7F、8、9N、9V、10A、11A、14、17F、19A、20、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合。
本文中，在多糖蛋白缀合物或免疫原性组合物中，来自不同血清型的荚膜多糖与载体蛋白的比例在0.2-3.0之间，例如0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0或上述任意两个数值之间的范围，优选0.5-2.5，0.5-2.0，0.5-1.5，更优选0.6-2.5，0.6-2.0，0.6-1.5。
在本文所述的多糖蛋白缀合物或免疫原性组合物中，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，优选为2∶1。例如，所述组合物可以是制剂，其中来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂(优选4优选u剂)，其余荚膜多糖的 浓度各自独立为0.5～5ug/剂(优选2优选5剂)，磷酸铝佐剂的浓度为0.125mg/剂至0.5mg/剂。
该免疫原性组合物可用作疫苗，进一步含有佐剂如基于铝的佐剂(氢氧化铝、磷酸铝或硫酸铝)。此外，本文所述的免疫原性组合物还可包含表面活性剂，例如Tween 20或Tween 80。本发明的免疫原性组合物能诱发对肺炎球菌感染的主动和被动保护。对于被动保护，免疫抗体是通过用本发明的免疫原性组合物制成的疫苗来免疫哺乳动物，然后从哺乳动物回收免疫抗体来生产的。本发明还提供了通过给予免疫有效量的本发明的组合物来免疫对象抵抗肺炎球菌感染的方法。
通过本领域技术人员已知的标准技术制备荚膜多糖。在本发明中，从肺炎链球菌的血清型1、3、4、5、6A、6B、7F、8、9V、9N、10A、11A、12F、14、15B、18C、19A、19F、20、22F、23F和33F制备荚膜多糖。通过单独的方法制备这些肺炎球菌缀合物并配制成单剂量制剂。
细菌荚膜多糖是细菌菌体的重要组成部分，也是致病菌重要的毒性因子。肺炎球菌细菌荚膜多糖会在细菌培养过程中释放到培养液中。本发明中的肺炎球菌细菌荚膜多糖是通过对不同血清型的肺炎球菌细菌的培养、发酵、纯化而得到。该类技术成熟，在行业内应用多年，有多个公开文章报道或专利报道(US4242501)。在一个实施方案中，在基于大豆的培养基中培养每种肺炎球菌多糖血清型。然后通过离心、沉淀、超滤和柱层析纯化各种多糖(US4686102)。
具体制备荚膜多糖的方法在美国专利US5714354有具体报道。肺炎球菌细菌培养\发酵及多糖制备是一个成熟的工艺(US4686102，US5847112)。简单来说将不同肺炎链球菌菌种接种到含有培养基(例如大豆培养基)的摇瓶中，在37℃二氧化碳培养箱培养过夜，将培养液接种到10升发酵罐，发酵培养6-12小时。培养结束后，加入DOC灭活剂杀菌灭活。离心收获澄清培养液，在澄清培养液液中加入CTAB沉淀剂，得到粗制沉淀复合糖，然后通过一系列沉淀、洗涤、层析纯化后得到精制多糖。
肺炎球菌荚膜多糖的纯化方法也是本领域常规技术，例如通过无有机溶剂的方法完成的(美国专利US 5714354)。通常精制多糖纯化工艺包括，但不限于，加入NaCl溶液对复合糖的溶解，用NaCl溶液溶解离心后取上清，加入酒 精去除杂质和沉糖，多次洗涤沉淀去杂，或可根据不同糖型，利用层析法如离子交换柱、复合型填料纯化得到精制多糖。在具体实施方案中，纯化过程主要包括，将细菌灭活液通过离心分离菌体，收获离心上清液，然后再通过0.22μm滤膜过滤及100KD膜包超滤浓缩，获得肺炎多糖发酵浓缩液。向肺炎多糖发酵浓缩液中加入CTAB溶液，室温搅拌，8000rpm离心，收取多糖复合物沉淀(对于7F、14、33F型多糖，收集上清液)。多糖复合物然后用0.25-0.5M NaCl溶液解离后，再通过沉淀、柱层析、超滤等工艺纯化得到纯化多糖溶液。多糖溶液通过0.22μm膜过滤后，低温保存。
对所得多糖进行质量分析的方法本领域已知，例如通过核磁共振。具体地，制备得到的不同类型肺炎球菌荚膜多糖通过特异性血清进行鉴别血清型、通过核磁共振波谱(Nuclear magnetic resonance，NMR)分析肺炎球菌多糖的化学结构、通过化学显色方法可对多糖中含有的官能团(如鼠李糖、糖醛酸、O-乙酰基等组分)进行含量测定。确定多糖各个理化指标符合欧州药典关于肺炎球菌多糖制定的标准，蛋白及核酸等主要杂质含量均低于1％。
可用许多缀合方法产生本发明的多糖-蛋白质缀合物。在多糖-蛋白结合反应过程中，通常包括多糖活化和蛋白缀合步骤。通过常规方法实现多糖的化学活化和随后缀合到载体蛋白。参见例如美国专利号4,673,574和4,902,506。通过控制多糖的分子量、多糖活化反应、多糖-蛋白的反应比例、反应温度、反应时间及反应液pH值等参数，得到最终多糖蛋白缀合物。所得的多糖蛋白缀合物通过进一步纯化，去除未反应物及杂质，通过质量分析，定量其多糖-蛋白交联度及残留杂质，确保批次的一致性和可比性。
将纯化的多糖化学活化以使得该糖能够与载体蛋白反应。根据多糖的特点，可以选择不同的化学活化方法，常用的多糖活化方法有溴化氰法(US6375846B1)、水解(US4761283)、1-氰基-4-二甲氨基砒啶四氟硼酸酯(CDAP)(EP0720485)、高碘酸氧化法(US4711779)。活化的多糖可经过超滤与其他杂质分离。多糖经过活化后具有与载体蛋白反应的活性。
例如，用高碘酸盐(例如高碘酸钠)或其等同物的选择性氧化反应来氧化先前糖部分的羟基而形成醛基。这形成了活化的肺炎球菌荚膜多糖，其现在能与选择的蛋白质载体共价相连。例如在室温，用约1ml约20mM的高碘酸钠溶 液适宜地氧化约10mg的多糖约10-15分钟；或者，加入多糖的0.05-2个当量的高碘酸盐氧化12-24小时。反应时间可随其它数量的高碘酸盐而变化以得到同等的氧化。糖的还原和开环使得还原糖的邻位羟基活化成醛基。多糖活化结束后，可通过超滤浓缩除去反应中的小分子物质。
缀合物的制备是通过对纯化提取的肺炎球菌外膜多糖经过处理得到的多糖片段通过化学偶联方式连接到载体蛋白上。多糖-蛋白结合物可以通过直接化学偶联或通过连接子实施得到，如利用连接子己二酸二酰肼进行偶联。将多糖与蛋白质缀合的各种化学方法是已知的并在文献中有所描述。通常方法有溴化氰法、CDAP法或还原胺化法(US5952454，EP0720485，US4711779)。例如，作为参考文献引入本发明的美国专利4,644,059描述了用己二酸二酰肼(ADH)作为同双功能连接物制备的缀合物。也作为参考文献引入本发明的美国专利4,695,624描述了制备多糖以及用属间间隔基制备缀合物的方法。在Dick、WilliamE.和MichelBeurret的Contrib.Mrcrobil.Immunol.(1989)，Vol.10，pp.48-114中讨论了对各种用于设计缀合物的制备方法和因素的概括性研究，其也作为参考文献引入本发明。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。
优选的缀合本发明的各种肺炎球菌荚膜多糖-蛋白质缀合物的方法是还原胺化法。在氰基硼氢化物离子或另一种还原剂的存在下，通过将载体蛋白的氨基与肺炎球菌荚膜多糖的醛基偶联使活化的肺炎球菌荚膜多糖与所选择的缀合蛋白缀合。得自还原性氨基化过程的肺炎球菌荚膜多糖-蛋白缀合物优选能溶于水溶液。这使本发明的肺炎球菌荚膜多糖-蛋白缀合物成为用作疫苗的优选候选物。缀合的条件可根据多糖的分子量和具体蛋白进行调整。在还原胺化法中，多糖与载体蛋白的比例控制在0.5-3.0之间，反应过程中加入多糖的1.0-2.0当量的氰基硼氢化钠溶液，然后加入2.0当量的硼氢化钠溶液。孵育温度可以是室温，孵育时间例如至少12小时。反应结束后通过超滤换液或层析，去除反应中的杂质或没有反应的底物，最后通过0.22um过滤器无菌过滤，将多糖-蛋白结合物溶液放在2-8℃下保存。
荚膜多糖缀合到载体蛋白后，通过多种技术纯化多糖-蛋白质缀合物。这些技术包括浓缩/渗滤操作、沉淀/洗脱和柱层析。
纯化各复合糖后，将它们混合以配制本发明的免疫原性组合物，其可以用作疫苗。使用本领域公知的方法可以完成本发明的免疫原性组合物的配制。例如，可以用药学上可接受的辅料配制20或24种各自的肺炎球菌缀合物以制备组合物。此类载体的实例包括，但不限于，水、缓冲盐水、多元醇(例如，甘油、丙二醇、液体聚乙二醇)和葡萄糖溶液。
在一些实施方案中，免疫原性组合物可包含一种或多种佐剂。如本文定义的，“佐剂”是用于增强本发明的免疫原性组合物的免疫原性的物质。从而，通常给予佐剂以加强免疫应答并且佐剂是技术人员公知的。用于增强组合物的有效性的合适的佐剂包括但不限于：(1)铝盐，如氢氧化铝、磷酸铝、硫酸铝，等等；(2)水包油乳液制剂，如MF59、SAF、RibiTM佐剂系统(RAS)，(Corixa，Hamilton，MT)；(3)皂苷佐剂，如QuilA或STIMULONTMQS-21；(4)细菌脂多糖(如氨基烷基葡糖胺磷酸酯化合物(AGP)或者其衍生物或类似物)，合成的多核苷酸(如含有CpG基序的寡核苷酸)；(5)细胞因子，如白介素(例如，IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12、IL-15、IL-18，等)，干扰素(例如，γ干扰素)，粒细胞巨噬细胞集落刺激因子(GM-CSF)，巨噬细胞集落刺激因子(M-CSF)，肿瘤坏死因子(TNF)，共刺激分子B7-1和B7-2，等等；(6)粘膜佐剂，包括蛋白质毒素、重组蛋白亚基和核酸等，包括细菌ADP-核糖基化毒素的脱毒突变体，如野生型或者突变型的霍乱毒素(CT)，百日咳毒素(PT)，或者大肠杆菌不耐热毒素(LT)(参见例如WO93/13302和WO92/19265)；和(7)作为免疫刺激剂以增强组合物的有效性的其他物质。佐剂的用量可由本领域技术人员根据常规技术确定。示例性地，本文的免疫原性组合物中佐剂(例如磷酸铝)浓度为0.125mg/剂至0.5mg/剂。
本发明的免疫原性组合物和疫苗可以用于保护或者治疗对肺炎球菌感染敏感的哺乳动物(例如人)，这可通过全身或者粘膜途径施用疫苗来完成。以及本文所述免疫原性组合物的用途，用于制备用于提供对抗多种肺炎球菌感染的保护的药物。
本发明的免疫原性组合物可用作增加用于预防和诊断目的的抗体的手段。诊断尤其适用于监视和检测各种由肺炎球菌引起的感染和疾病。本发明的另一个实施方案用免疫原性组合物作为免疫原，用于对有危险患肺炎球菌感染或疾 病的个体进行主动的和被动的免疫原性保护。用于被动保护的免疫抗体如下产生：用本发明的任意免疫原性组合物免疫哺乳动物，然后从哺乳动物回收γ-球蛋白部分中的杀菌抗体，或者作为血清或作为特异性抗体。此处所用的本发明的疫苗能诱导用于提供对肺炎球菌感染的保护的抗体。
另外，肺炎球菌荚膜多糖本身可以用作免疫剂，优选与佐剂如基于铝的佐剂缔合为免疫原性组合物。本发明进一步的实施方案是用该免疫原性组合物作为对抗肺炎球菌感染的免疫原性保护。
本发明的免疫原性组合物和疫苗可通过将肺炎球菌荚膜多糖或缀合物分散到适宜的药学上可接受的辅料而典型地形成。所述辅料包括但不限于稀释剂、载剂、增溶剂、乳化剂、防腐剂和/或佐剂。辅料优选地在所采用的剂量和浓度下对接受者无毒，例如：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。在某些实施方案中，组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的物质。这些物质为现有技术已知。可视预期的施用途径、递送方式和所需的剂量来确定最佳的药物组合物。也可以存在疫苗中常用的添加剂，例如稳定剂如乳糖或山梨糖醇和佐剂如磷酸铝、氢氧化铝、硫酸铝、CpG、单磷酰基脂A、QS21、MF59或硬脂酰酪氨酸。
本发明的免疫原性组合物和疫苗的保护或者治疗作用可通过全身或者粘膜途径施用疫苗来完成。这样的施用包括胃肠外给药，例如通过肌内、腹膜内、皮内或者皮下途径注射，或者通过粘膜施用于口/消化道、呼吸或者泌尿生殖道。在一个实施方案中，鼻内施用用于治疗肺炎或者中耳炎。
免疫原性组合物的剂量应是能达到致免疫作用效果的剂量。选择每个疫苗剂量中缀合物的量作为诱导免疫保护而无显著的不利作用的量。此类量可以取决于肺炎球菌血清型而变。通常，每剂将包含0.01μg到100μg多糖或缀合物，例如0.1μg.到10μg，或1μg到5μg。给药剂量通常在每千克体重约0.01μg至约10μg之间的范围。可以给出一系列最佳免疫剂量。单位剂型的疫苗可含有约0.01μg至约100μg相当量的脑膜炎致病菌荚膜多糖或缀合物，例如0.1μg到10μg，或1μg到5μg。
通过包括观察受试者中合适的免疫应答的标准研究确定具体疫苗的组分 的最佳量。最初接种后，受试者可以接受足够间隔的一次或几次加强免疫。本发明的免疫原性组合物适于婴儿、儿童、青少年和成人使用。其免疫方案可由有经验的临床医生确定。
在本发明的具体实施方案中，20或24价疫苗是各自缀合到CRM197的血清型1、2、4、6A，7F、8、9N、9V、10A、11A、14、17F、19A、20、22F和33F的肺炎球菌荚膜多糖和各自缀合到TTD的血清型3、5、6B、12F、15B、18C、19F和23F的肺炎球菌荚膜多糖的无菌液体制剂。每剂被配制成含有：除了4μg多糖的3，6B或12F之外，每种糖2μg；约20～80μg CRM197载体蛋白；约10～40μg TTD载体蛋白；0.125mg～0.5mg磷酸铝佐剂。将液体填充到没有防腐剂的单剂量注射器中。摇动后，疫苗是均匀的，白色混悬液可用于肌内施用。
本申请的示例性具体实施方案：
1、一种免疫原性组合物，含有来自不同血清型的肺炎链球菌的荚膜多糖和载剂，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F，
优选地，所述血清型包括选自以下的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24种血清型：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，并且所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。
2、如项目1所述的免疫原性组合物，其特征在于，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F，或以下24种血清型：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，
优选地，所述载剂选自盐水、林格氏溶液和磷酸盐缓冲盐水中的一种或多种。
3、如项目1或2所述的免疫原性组合物，其特征在于，所述免疫原性组合物还含有佐剂，
优选地，所述佐剂包括选自基于铝的佐剂、单磷酰基脂A、QS21、CpG、MF59、硬脂酰酪氨酸、弗氏佐剂和其他粘膜佐剂中的一种或多种。
4、如项目1或2所述的免疫原性组合物，其特征在于，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，
优选地，所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂，其余荚膜多糖的浓度各自独立为0.5～5ug/剂。
5、包含多种的多糖-蛋白质缀合物以及药学上可接受辅料的多价免疫原性组合物，其中每种多糖-蛋白质缀合物含有缀合到载体蛋白的来自不同血清型的肺炎链球菌的荚膜多糖，
优选地，所述载体蛋白至少包含两种载体蛋白，
优选地，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F；更优选地，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F，或以下24种血清型：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。
6、如项目5所述的多价免疫原性组合物，其特征在于，所述载体蛋白包括(1)CRM197和(2)TTD或其变体，
优选地，TTD是TT的C端结构域，
更优选地，TTD具有SEQ ID NO.2所示的序列，TTD变体具有与SEQ ID NO：2有至少90％序列相同性的序列。
7、如项目5或6所述的多价免疫原性组合物，其特征在于，在所述多糖蛋白缀合物中，至少来自血清型3、5、6B、12F、15B、18C、19F和23F的一种或多种或全部荚膜多糖分别与载体蛋白TTD或其变体缀合，
优选地，
所述血清型包括所述20种血清型，其中来自包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19或20种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合，或
所述血清型包括所述24种血清型，其中来自包括血清型3、5、6B、12F、 15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合，
更优选地，
所述血清型包括所述20种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、4、6A、7F、8、9V、10A、11A、14、19A、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合，或
所述血清型包括所述24种血清型，其中来自血清型3、5、6A、6B、9N、11A、12F、15B、17F、18C、19A、19F、20、23F、33F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、7F、8、9V、10A、14、22F的荚膜多糖分别与载体蛋白CRM197缀合，或
所述血清型包括所述24种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、6A，7F、8、9N、9V、10A、11A、14、17F、19A、20、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合。
8、如项目5或6所述的多价免疫原性组合物，其特征在于，所述多价免疫原性组合物还具有选自以下的一项或多项特征：
所述组合物中，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，
所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂，其余荚膜多糖的浓度各自独立为0.5～5ug/剂，
所述组合物还包含佐剂；优选地，所述佐剂包括选自基于铝的佐剂、单磷酰基脂A、QS21、CpG、MF59、硬脂酰酪氨酸、弗氏佐剂和其他粘膜佐剂中的一种或多种，
所述组合物中，缀合物与佐剂的重量比为1∶10至1∶2，
所述组合物还包含表面活性剂；优选地，组合物中表面活性剂的浓度为100～300μg/剂，
所述组合物的pH为5.0-7.0。
9、项目1-8中任一项所述的免疫原性组合物在制备诱导对肺炎球菌荚膜多糖缀合物的免疫应答和/或对破伤风毒素的免疫应答的药物中的用途，
优选地，所述药物用于预防或治疗肺炎球菌感染和/或破伤风毒素感染。
10、一种导致被动免疫的免疫组合物，包括靶向肺炎球菌的杀菌抗体，所述抗体是由项目1-8中任一项所述的免疫原性组合物免疫哺乳动物而获得，
优选地，所述杀菌抗体存在于血清、γ球蛋白部分或纯化的抗体制剂中。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的材料和方法。
实施例
实验方法
1、肺炎球菌荚膜多糖的制备
肺炎球菌细菌培养\发酵及多糖制备是一个成熟的工艺(US4686102，US5847112)。不同血清型的肺炎球菌菌种(美国典型生物资源保藏中心)通过培养制备得到菌种库，将一管菌种库中的肺炎链球菌菌种接种到含有大豆培养基的摇瓶中，在37℃、5～10％CO2培养箱培养过夜，培养完成后，镜检，结果正常则将培养液接种到10L发酵罐，在pH 7.0，温度37℃下发酵培养。培养结束后，加入10％的DOC溶液，灭活，确保肺炎球菌细菌完全灭活。
肺炎球菌荚膜多糖的纯化是通过无有机溶剂的方法完成的(美国专利US 5714354)。其制备纯化过程主要包括，将细菌灭活液通过离心分离菌体，收获离心上清液，然后再通过0.22μm滤膜过滤及100KD膜包超滤浓缩，获得肺炎多糖发酵浓缩液。向肺炎多糖发酵浓缩液中加入CTAB溶液，室温搅拌，8000rpm离心，收取多糖复合物沉淀(对于7F、14、33F型多糖，收集上清液)。多糖复合物然后用0.25-0.5M NaCl溶液解离后，再通过沉淀、柱层析、超滤等工艺纯化得到纯化多糖溶液。多糖溶液通过0.22μm膜过滤后，低温保存。
制备得到的不同类型肺炎球菌荚膜多糖通过特异性血清进行鉴别血清型、 通过核磁共振波谱(Nuclear magnetic resonance，NMR)分析肺炎球菌多糖的化学结构、通过化学显色方法可对多糖中含有的官能团(如鼠李糖、糖醛酸、O-乙酰基等组分)进行含量测定。确定多糖各个理化指标符合欧州药典关于肺炎球菌多糖制定的标准，蛋白及核酸等主要杂质含量均低于1％。
2、载体蛋白TTD的制备
将表达TTD的DNA序列优化后(SEQ ID NO：1)克隆到蛋白表达质粒pET21中，构建重组表达TTD蛋白(SEQ ID NO：2)的大肠杆菌BL21(DE3)。挑取重组BL21(DE3)单克隆菌落在10mL的LB(Amp)液体培养基中，37℃，250rpm培养至OD600至0.8后，加入0.1mM IPTG，在25℃，250rpm继续培养4h。培养液在8000rpm，4℃离心，收集菌体，然后用PBS重悬后破碎。破碎液在8000rpm，4℃离心，收取上清。TTD蛋白的纯化是通过硫酸铵沉淀、澄清过滤、组合层析方法得到，纯度在95％以上。通过质谱测定的分子量与理论分子量一致，通过分子筛确证TTD蛋白的均一性。TTD的氨基酸序列如下所示：
3、白喉杆菌素无毒变异体CRM197蛋白的制备
CRM197载体蛋白是白喉杆菌素无毒变异体(SEQ ID NO：3)，该蛋白没有毒性，但保留了白喉毒素的免疫原性。在公开的文章和专利中都有报道其发酵和纯化方法(US5614382)。通常通过将Corynebacterium diphtheriae菌种接种到培养基中，在37℃下，搅拌20-30小时，然后通过离心去除菌体，保留上清。将发酵上清通过超滤换液后，加入硫酸铵沉淀，收取沉淀物。然后将沉 淀物溶解，换液，再通过柱层析，纯化得CRM197蛋白，其纯度在95％以上，用于结合反应。
4、多糖-蛋白结合物的制备
通过细菌多糖与载体蛋白偶联制备多糖-蛋白结合物是提高细菌多糖免疫原性的有效途径。多糖-蛋白结合物广泛应用于细菌性疫苗的制备，如B型流感嗜血杆菌多糖PRP-TT结合疫苗，奈氏脑膜炎细菌A、C、Y、W多糖-载体蛋白结合疫苗。多糖-蛋白结合物的制备主要分为两个步骤：多糖的水解或活化、多糖-蛋白的偶联。多糖活化又可分为不同方法如CNBr法(US 4619828)，水解(US4761283)、高碘酸钠氧化法(US5306492)，活化的多糖然后可以直接与载体蛋白化学偶联(US4356170)或通过小分子连接器与载体蛋白偶联。
肺炎球菌多糖-蛋白结合物的制备主要包括如下步骤：多糖处理、多糖活化、多糖-蛋白的偶联。
肺炎球菌多糖前处理：部分血清型在氧化前，可以通过酸或碱水解方式(US5847112A)以降低分子量或提高多糖氧化结合效率，如血清型1、3、4、6A、6B、8、10A、11A、12F、15B、18C、22F多糖。部分多糖则可以通过高压均质方式降低其分子大小，如血清型2、6B、7F、8、10A、11A、12F、14、15B、19A、19F、33F。
肺炎球菌多糖活化：多糖活化是通过加入高碘酸钠溶液进行的(US4711779)。高碘酸盐加入量通常在多糖的0.05-2个当量氧化时间在12-24小时，多糖活化结束后，通过超滤浓缩除去反应中的小分子物质。
肺炎球菌多糖-蛋白结合物制备：多糖-蛋白的化学偶联是通过还原胺化法进行(US5952454、EP0720485、US4711779)。血清型1、2、4、6A、7F、8、9N、9V、10A、11A、14、17F、19A、20、22F和33F多糖通过与CRM97偶联制备多糖-蛋白缀合物；3、5、6B、12F、15B、18C、19F和23F多糖通过与TTD偶联制备多糖-蛋白缀合物。在偶联反应中，多糖与载体蛋白的比例控制在0.5-2.5之间，反应过程中加入1.0-2.0当量的氰基硼氢化钠溶液，然后加入2.0当量的硼氢化钠溶液。反应结束后通过超滤换液或层析，去除反应中的杂质或没有反应的底物，最后通过0.22um过滤器无菌过滤，将多糖-蛋白结合物溶液放在2-8°下保存。所有多糖-蛋白结合物通过质量分析，游离多糖含量控制 在20％以内，游离蛋白含量在2％以内，多糖-蛋白比在0.5-2.0之间，内毒素及其它杂质含量都控制在安全、可接受范围内。
5、多价肺炎球菌多糖-蛋白结合疫苗的配制
肺炎球菌主要致病性的血清型有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。将每种多糖-蛋白单价结合物原液通过使用pH 6.0的缓冲溶液稀释后混合，然后加入磷酸铝佐剂搅拌后制备得到。其中各种结合物中的多糖含量为每毫升2～4微克，磷酸铝佐剂含量为0.125～0.5毫克。
6、多糖-蛋白缀合物在动物体内的免疫原性评价
小鼠免疫实验方案：将多价疫苗原液加入磷酸铝佐剂，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8个小鼠，每次免疫剂量为0.5ml，分别在0天、14天、21天进行免疫，已上市相关疫苗(辉瑞的沛儿13，沃森生物的沃安欣作为阳性对照，磷酸铝佐剂为阴性对照。在35天抽血评价其多糖的免疫原性。
大兔免疫实验方案：将多价疫苗原液加入磷酸铝佐剂，配制成免疫抗原，选择2.0～2.5kg的新西兰大白兔进行大腿肌肉免疫，每组8只，每次免疫剂量为0.5ml，同时以PCV13(沛儿13)为阳性疫苗免疫二剂，并在35天抽血评价其多糖的免疫原性。
7、ELISA法评价多糖免疫原性
将肺炎多糖用包被缓冲液稀释，包被到96孔酶标板，100μl/孔，并在37℃孵育后洗板。将小鼠、大兔抗血清通过吸附剂处理后，血清先以1∶100稀释，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∶1万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OD405数值。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。
实施例1：血清型5多糖-CRM197结合物和肺炎多糖-TTD结合物在小鼠 的免疫原性比较
将5PS-CRM197结合物、5PS-TTD结合物，加入磷酸铝佐剂后，在0天、14天、28天分别2μg/剂免疫BALB/c小鼠(5只/组)，在21天和35天采血检测抗体滴度。
结果显示(图1)，CRM197载体的结合物D35的滴度水平为866.43，TTD载体的结合物D35的滴度水平为16260.67，以TTD为载体的多糖结合物的免疫原性高于以CRM197为载体的多糖结合物免疫原性，经Mann Whitney test分析差异显著(P＝0.0079)。
实施例2：不同佐剂剂量下24价肺炎球菌多糖结合疫苗在小鼠体内的免疫原性比较
在BALB/c小鼠体内评价磷酸铝佐剂加入量分别为0.5mg/剂、0.25mg/剂、0.125mg/剂的24价肺炎多糖结合疫苗的免疫原性差异。在6～8周龄的BALB/c雌性小鼠随机分组，8只/组，在0天、14天、28天分别0.5ml/剂免疫一剂，在35天采血检测抗体滴度水平，免疫原性数据如图2和图3所示。
结果表明，不同佐剂剂量下24价肺炎球菌多糖结合疫苗对13种血清型(阳性疫苗包含的血清型)的免疫原性与上市的13价结合疫苗相当，而且部分血清型比13价结合疫苗有更高的抗体滴度水平，如6B，18C，23F。同时在13种血清型以外的11种血清型也达到了良好的免疫原性。
实施例3：不同pH条件下24价肺炎球菌多糖结合疫苗在小鼠体内的免疫原性比较
在BALB/c小鼠体内评价不同pH的24价肺炎多糖结合疫苗的免疫原性差异。在6～8周龄的BALB/c雌性小鼠体内评价24价肺炎多糖结合疫苗，小鼠随机分组，8只/组。试验在D0、D14共免疫二次，1剂/次，在D0、D21、采血清，以ELISA检测血清中的多糖抗体滴度水平。
pH范围在5.0～6.2范围内的24价肺炎球菌多糖结合疫苗，免疫二剂后一周(D21)血清抗体滴度比较说明(见图4)，5.0～6.2范围内pH的24价肺炎多糖结合疫苗免疫后，对每种血清型的抗体滴度组间无显著差异(P＞0.05)，都能诱导良好的免疫原性。
实施例4：24价肺炎球菌多糖结合疫苗与阳性疫苗在小鼠体内的免疫原 性比较
目前上市的13价肺炎结合疫苗有两种，一种是沛儿13，由美国辉瑞公司生产，使用CRM197作为载体蛋白，另一种是沃安欣，由沃森生物股份公司生产，使用破伤风类毒素(TT)作为载体蛋白。
将24价肺炎结合疫苗与沛儿13、沃安欣及佐剂对照组分别在0，14，28天免疫BALB/c小鼠后，评价在35天的免疫原性，如图5、图6所示。
结果表明：
(1)24价肺炎球菌结合疫苗与基于CRM197载体蛋白的沛儿13比较，血清型18C的免疫原性明显高于沛儿13，另外5，6A、7F，23F的抗体滴度也高于对照组。
(2)24价肺炎球菌结合疫苗与基于TT载体蛋白的沃安欣比较，D35血清抗体滴度，经非参数Kruskal-Wallis test检验，4型、6A型、23F型有显著性差异，免疫原性高于沃安欣。另外，5，7F，9V的抗体滴度也高于对照组。
(3)对于非PCV13疫苗血清型，24价肺炎球菌结合疫苗也能诱导较好的免疫原性(图6)。将24价肺炎球菌结合疫苗(PCV24)与佐剂对照组在35天的抗体滴度用Mann-Whitney test分析，血清型2、8、9N、11A、15B、17F、20、33F有显著性差异(P＜0.05)，表明PCV24疫苗对PCV13以外的血清型有较好的免疫原性。10A型疫苗组与佐剂对照组比较，没有显著差异(P＝0.3068)，可能是10A型在实验动物小鼠中的本底抗体滴度水平较高所致。
实施例5：24价肺炎球菌多糖结合疫苗与阳性疫苗在大兔体内的免疫原性比较
配制PCV24肺炎球菌多糖结合疫苗免疫大兔(8只/组)，以PCV13(沛儿13)为阳性对照、生理盐水为阴性对照，首次免疫后，间隔三周免疫，在第二次免疫后二周(D35)采血检测血清中各型多糖的抗体滴度(图7和图8)。
与PCV13(沛儿13)相比(图7)，PCV24免疫二剂经T检验分析13种血清型，免疫原性可以达到已上市疫苗效果。13种血清型以外的11种血清型(图8)也显示出较好的免疫原性。
实施例6：双载体与单载体的24价肺炎球菌多糖结合疫苗免疫原性比较
为了对比24价肺炎球菌多糖结合疫苗中，使用两种载体蛋白与使用一种 载体蛋白免疫原性的差异，分别配制两种不同24价肺炎球菌多糖结合疫苗。A组疫苗中血清型3、5、6B、12F、15B、18C、19F、23F共8个型以TTD为载体蛋白，其它16个血清型为CRM197载体蛋白；B组疫苗中24个血清型均使用CRM197做为载体蛋白。
将以上二组疫苗分别免疫新西兰大白兔，每组8只，共免疫二次，间隔三周，在免疫前和第二次免疫后二周采血清检测各型多糖抗体滴度水平，将第二次免疫二周后二组疫苗滴度转换成对数后以Multiple t tests统计分析，结果见图9。
从图9可知，在所有24种血清型中，双载体疫苗组比单载体疫苗组能诱导更高的抗体滴度，而且有7种血清型，(血清型4、5、9V、12F、19A、19F、23F)，诱导的抗体滴度有显著性差别(P＜0.05)。说明双载体疫苗可以避免单一载体引起的免疫抑制作用。
实施例7：双载体与单载体的20价肺炎球菌多糖结合疫苗免疫原性比较
分别配制两种不同20价(1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F)肺炎球菌多糖结合疫苗。A组疫苗中血清型3、5、6B、12F、15B、18C、19F、23F共8个型以TTD为载体蛋白，其它12个血清型为CRM197载体蛋白；B组疫苗中20个血清型均使用CRM197做为载体蛋白。
将以上二组疫苗分别免疫新西兰大白兔，每组8只，共免疫二次，间隔三周，在免疫前和第二次免疫后二周采血清检测各型多糖抗体滴度水平，将第二次免疫后二周血清滴度值转换成对数后以Multiple t tests统计分析。结果如图10所示。
结果显示：双载体疫苗组共有8个血清型采用TTD载体(3、5、6B、12F、15B、18C、19F、23F)，此8个血清型的抗体滴度水平均高于单载体疫苗组，并且5型、23F型有显著性差异，P＜0.05。
双载体疫苗20个血清型中有16个血清型(除6A、7F、22F、33F)滴度水平均高于单载体疫苗组。
实施例8，双载体与单载体的24价肺炎球菌多糖结合疫苗免疫原性比较
分别配制两种不同24价(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)肺炎球菌多糖结合疫苗。A组疫苗中血清型3、5、6A、6B、9N、11A、12F、15B、17F、18C、19A、19F、20、23F、33F共15个型以TTD为载体蛋白，其它9个血清型为CRM197载体蛋白；B组疫苗中24个血清型均使用CRM197做为载体蛋白。
将以上二组疫苗分别免疫新西兰大白兔，每组8只，共免疫二次，间隔三周，在免疫前和第二次免疫后二周采血清检测各型多糖抗体滴度水平，将第二次免疫后二周血清滴度值转换成对数后以Multiple t tests统计分析。结果如图11所示。
结果显示：双载体疫苗组共有15个血清型采用TTD载体(3、5、6A、6B、9N、11A、12F、15B、17F、18C、19A、19F、20、23F、33F)，其中12个血清型的抗体滴度水平不低于单载体疫苗组，并且23F有显著性差异，P＜0.05。
双载体疫苗24个血清型中有16个血清型(除1、2、3、4、8、10A、14、19F)滴度水平不低于单载体疫苗组。
实施例9：24价肺炎球菌多糖结合疫苗对3型肺炎球菌的BALB/c小鼠保护力试验
在BALB/c小鼠体内以PCV13(沃森，沃安欣)多糖结合疫苗为阳性对照品、生理盐水为阴性对照品，腹腔免疫三剂后二周，以2×105CFU的3型肺炎链球菌滴鼻感染。观察攻毒后二周内的存活率，分析24价肺炎多糖结合疫苗对3型肺炎球菌攻毒的保护力。
结果(图12)表明，24价肺炎球菌多糖结合疫苗诱导的抗体具有100％的免疫保护作用，其效果优于保护率为89.9％的上市13价肺炎球菌多糖结合疫苗。
实施例9：24价肺炎球菌多糖结合疫苗对22F型肺炎球菌的BALB/c小鼠保护力试验
试验以23价肺炎球菌多糖疫苗(PPV23)为阳性对照品、生理盐水为阴性对照品，在BALB/c小鼠体内腹腔免疫一剂后三周，以1×108CFU的22F型 肺炎链球菌腹腔感染。通过攻毒后小鼠的生存情况，分析24价肺炎多糖结合疫苗对22F型肺炎球菌的保护力。
结果(图13)显示，24价疫苗具有100％的存活率，对照品PPV23多糖疫苗30％的存活率，生理盐水20％存活率。经Log-rank(Mantel-Cox)test检验，24价疫苗与对照品相比，具有显著性差异，P＜0.05(P值为0.0027)。
综上，说明24价肺炎球菌多糖结合疫苗对22F型肺炎球菌，与PPV23多糖疫苗比，具有显著的保护效果。
实施例10：24价肺炎球菌多糖结合疫苗对破伤风毒素的保护力试验
24价肺炎球菌多糖结合疫苗包含TTD载体蛋白，通过在小鼠体内破伤风毒素攻毒保护试验评价疫苗的有效性。
在BALB/c小鼠体内，0天、14天腹腔注射免疫24价肺炎球菌多糖结合疫苗，同时以百白破疫苗为阳性对照、生理盐水为阴性对照。第28天采血检测血清中对破伤风类毒素(TT)的抗体滴度水平(图14，A组为生理盐水；B组为百白破疫苗；C组为24价肺炎球菌多糖结合疫苗)，并腹腔注射破伤风毒素(20LD50)，观察二周内的小鼠存活情况(图15)。
由图14可知，B组、C组、D组免疫二剂后与免疫前相比，TT抗体滴度水平均有显著性提高，P＜0.05。B组对于TT抗体滴度高于C组，可能是由于百白破疫苗是由TT组成，24价肺炎球菌多糖结合疫苗使用的是TTD。
由图15可知，生理盐水组破伤风毒素腹腔感染后第1天全部死亡，24价肺炎球菌多糖结合疫苗保护率80％，对照组百白破疫苗保护率100％。说明24价肺炎球菌多糖结合疫苗对破伤风毒素的感染具有保护作用，其效果明显优于阴性对照。

Claims (10)

  1. 一种免疫原性组合物，含有来自不同血清型的肺炎链球菌的荚膜多糖和载剂，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F，
    优选地，所述血清型包括选自以下的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24种血清型：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，并且所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F。
  2. 如权利要求1所述的免疫原性组合物，其特征在于，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F，或以下24种血清型：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，
    优选地，所述载剂选自盐水、林格氏溶液和磷酸盐缓冲盐水中的一种或多种。
  3. 如权利要求1或2所述的免疫原性组合物，其特征在于，所述免疫原性组合物还含有佐剂，
    优选地，所述佐剂包括选自基于铝的佐剂、单磷酰基脂A、QS21、CpG、MF59、硬脂酰酪氨酸、弗氏佐剂和其他粘膜佐剂中的一种或多种。
  4. 如权利要求1或2所述的免疫原性组合物，其特征在于，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，
    优选地，所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂，其余荚膜多糖的浓度各自独立为0.5～5ug/剂。
  5. 包含多种的多糖-蛋白质缀合物以及药学上可接受辅料的多价免疫原性组合物，其中每种多糖-蛋白质缀合物含有缀合到载体蛋白的来自不同血清型的肺炎链球菌的荚膜多糖，
    优选地，所述载体蛋白至少包含两种载体蛋白，
    优选地，所述血清型至少包括2、8、9N、10A、11A、12F、15B、17F、20、22F和33F；更优选地，所述血清型包括以下20种血清型：1、3、4、5、6A、6B、7F、8、9V、10A、11A、12F、14、15B、18C、19A、19F、22F、23F、33F，或以下24种血清型：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。
  6. 如权利要求5所述的多价免疫原性组合物，其特征在于，所述载体蛋白包括(1)CRM197和(2)TTD或其变体，
    优选地，TTD是TT的C端结构域，
    更优选地，TTD具有SEQ ID NO.2所示的序列，TTD变体具有与SEQ ID NO：2有至少90％序列相同性的序列。
  7. 如权利要求5或6所述的多价免疫原性组合物，其特征在于，在所述多糖蛋白缀合物中，至少来自血清型3、5、6B、12F、15B、18C、19F和23F的一种或多种或全部荚膜多糖分别与载体蛋白TTD或其变体缀合，
    优选地，
    所述血清型包括所述20种血清型，其中来自包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19或20种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合，或
    所述血清型包括所述24种血清型，其中来自包括血清型3、5、6B、12F、15B、18C、19F和23F在内的8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24种血清型的荚膜多糖分别与载体蛋白TTD或其变体缀合，
    更优选地，
    所述血清型包括所述20种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、4、6A、7F、8、9V、10A、11A、14、19A、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合，或
    所述血清型包括所述24种血清型，其中来自血清型3、5、6A、6B、9N、11A、12F、15B、17F、18C、19A、19F、20、23F、33F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、7F、8、9V、10A、14、22F的荚膜多糖分别与载体蛋白CRM197缀合，或
    所述血清型包括所述24种血清型，其中来自血清型3、5、6B、12F、15B、18C、19F和23F的荚膜多糖分别与载体蛋白TTD或其变体缀合；来自血清型1、2、4、6A，7F、8、9N、9V、10A、11A、14、17F、19A、20、22F、33F的荚膜多糖分别与载体蛋白CRM197缀合。
  8. 如权利要求5或6所述的多价免疫原性组合物，其特征在于，所述多价免疫原性组合物还具有选自以下的一项或多项特征：
    所述组合物中，来自血清型3，6B或12F的荚膜多糖与其他任一荚膜多糖的重量比为10∶1至1∶10，例如5∶1至1∶5，
    所述组合物是制剂，来自血清型3，6B和12F的荚膜多糖的浓度各自独立为1～8ug/剂，其余荚膜多糖的浓度各自独立为0.5～5ug/剂，
    所述组合物还包含佐剂；优选地，所述佐剂包括选自基于铝的佐剂、单磷酰基脂A、QS21、CpG、MF59、硬脂酰酪氨酸、弗氏佐剂和其他粘膜佐剂中的一种或多种，
    所述组合物中，缀合物与佐剂的重量比为1∶10至1∶2，
    所述组合物还包含表面活性剂；优选地，组合物中表面活性剂的浓度为100～300μg/剂，
    所述组合物的pH为5.0-7.0。
  9. 权利要求1-8中任一项所述的免疫原性组合物在制备诱导对肺炎球菌荚膜多糖缀合物的免疫应答和/或对破伤风毒素的免疫应答的药物中的用途，
    优选地，所述药物用于预防或治疗肺炎球菌感染和/或破伤风毒素感染。
  10. 一种导致被动免疫的免疫组合物，包括靶向肺炎球菌的杀菌抗体，所述抗体是由权利要求1-8中任一项所述的免疫原性组合物免疫哺乳动物而获得，
    优选地，所述杀菌抗体存在于血清、γ球蛋白部分或纯化的抗体制剂中。
PCT/CN2023/089161 2022-04-19 2023-04-19 多价肺炎球菌多糖结合疫苗的成分及其应用 WO2023202607A1 (zh)

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