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WO2022031087A1 - Composition immunogène comprenant des conjugués polysaccharide pneumococcique-substance dérivée de paroi de cellulaire - Google Patents

Composition immunogène comprenant des conjugués polysaccharide pneumococcique-substance dérivée de paroi de cellulaire Download PDF

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WO2022031087A1
WO2022031087A1 PCT/KR2021/010359 KR2021010359W WO2022031087A1 WO 2022031087 A1 WO2022031087 A1 WO 2022031087A1 KR 2021010359 W KR2021010359 W KR 2021010359W WO 2022031087 A1 WO2022031087 A1 WO 2022031087A1
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capsular polysaccharide
immunogenic composition
cell wall
derived material
carrier protein
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Korean (ko)
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조경민
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Celltrion Inc
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Celltrion Inc
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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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • 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
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the present invention relates to an immunogenic composition
  • an immunogenic composition comprising a pneumococcal polysaccharide-cell wall-derived material conjugate, each conjugate comprising a cell wall-derived material bound to a pneumococcal-derived capsular polysaccharide of a specific serotype.
  • Streptococcus pneumoniae is a major causative agent of pneumonia. According to the National Statistical Office ⁇ Development of mortality by major cause of death in 2010 ⁇ , the death rate due to pneumonia in 2010 was 14.9 per 100,000 people, one of the top 10 causes of death, and it was found that it increased by 82.9% compared to 2000. In addition, according to the WHO in 2012, 476,000 HIV-negative children under the age of 5 worldwide died from infection with Streptococcus pneumoniae in 2008, and 5% of all deaths among children under the age of 5 died from diseases caused by this bacterium. did
  • Dr. A 14-valent polysaccharide vaccine was developed by Robert Austrian, which later evolved into a 23-valent polysaccharide vaccine.
  • Polyvalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in the elderly and high-risk patients. However, infants and children do not have a good immune response to most pneumococcal polysaccharides because of the T-cell-independent immune response.
  • Heptavalent pneumococcal conjugate vaccine (Prevnar®) contains capsular polysaccharides from the 7 most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.
  • the pneumococcal serotype used in the pneumococcal conjugate vaccine is usually in a state containing CPS (Capsular Polysaccharide), CWPS (Cell Wall Polysaccharide), LTA (Lipoteichoic acid), PG (Peptidoglycan), etc. and carrier protein (transfer protein).
  • CPS Capsular Polysaccharide
  • CWPS Cell Wall Polysaccharide
  • LTA Lipoteichoic acid
  • PG Poridoglycan
  • carrier protein transfer protein
  • apoptotic vaccines inactivated vaccines, etc.
  • Capsular polysaccharide a major antigen, is known to have low efficacy in inducing long-term immunity and low immune response in infants and children when used alone.
  • a specific serotype for example, serotype 3, is a serotype included in Prevena 13 (PCV13), but the serotype is known as a serotype with low protection after vaccination.
  • serotype 3 including other serotypes antibody titers and OPA titers appear after PCV13 inoculation, but in the case of serotype 3, the actual protection rate is low. The incidence rate of the type is still high.
  • the cell wall of pneumococcus is composed of LTA (Lipoteichoic acid), CWPS (cell wall polysaccharide), CPS (capsular polysaccharide, PG (Peptidoglycan), and cell membrane double layer, etc.
  • LTA Lipoteichoic acid
  • CWPS cell wall polysaccharide
  • CPS capsulear polysaccharide
  • PG Peptidoglycan
  • cell membrane double layer etc.
  • the present inventors conjugated a pneumococcal cell wall-derived material to pneumococcal capsular polysaccharide to induce structural stability of the immunogenic composition while increasing the functional antibody titer (OPA) and specific antibody titer (IgG) of a specific serotype.
  • OPA functional antibody titer
  • IgG specific antibody titer
  • an object of the present invention is to provide an immunogenic composition
  • an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall-derived material.
  • Another object of the present invention is to provide a pharmaceutical composition for inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • Another problem to be solved by the present invention is to provide a method for preparing the immunogenic composition.
  • the present invention provides an immunogenic composition comprising a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a cell wall derived material.
  • the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, It may contain a capsular polysaccharide of any one or more serotypes selected from the group consisting of 23F and 35B.
  • the capsular polysaccharide is at least one selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. serotype capsular polysaccharides.
  • the immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 22F, 23A, and 35B.
  • the immunogenic composition according to the present invention further comprises a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, It may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 7F, 9V, 14, 18C and 23F.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and cell wall derived material may further include a carrier protein.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material further comprises a linker.
  • the cell wall-derived material may be isolated from Streptococcus pneumoniae.
  • the cell wall-derived material may be covalently linked to the capsular polysaccharide.
  • the cell wall-derived material is LTA (Lipoteichoic acid), CWPS (Cell Wall Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP ( Muramyl tripeptide) and PC (Phosphatidyl Choline) may be any one or more selected from the group consisting of.
  • the cell wall-derived material may be Streptococcus pneumoniae-derived CWPS (Cell Wall Polysaccharide).
  • the carrier protein is diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli-derived inactivated toxin, Pseudomonas aeruginosa-derived inactivated toxin and bacterial outer membrane protein (OMP) ) may be any one selected from the group consisting of.
  • the diphtheria toxoid may be any one selected from the group consisting of CRM 197 , CRM 173 , CRM 228 and CRM 45 .
  • the linker is composed of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond. It may be any one selected from the group.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein have a structure in which they are conjugated in one of the following splicing sequences. It may be an immunogenic composition comprising:
  • the cell wall-derived material may not directly bind to a carrier protein.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the structure conjugated in the conjugation sequence b) may be either linear or non-linear.
  • the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected.
  • the a) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • an adjuvant may be further included.
  • the adjuvant may be an aluminum salt.
  • the aluminum salt may be any one selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
  • the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 ⁇ g.
  • the present invention provides a pharmaceutical composition for inducing an immune response to the Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • the present invention provides a method for preparing the immunogenic composition.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the method of conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a thiol-malemide method. It may be any one or more selected from the group consisting of.
  • the a) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the b) conjugation sequence may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the immunogenic composition according to the present invention can induce a more increased immune response compared to the existing vaccine.
  • it is included in the existing pneumococcal conjugate vaccine, it has an excellent effect against a specific serotype that is prevalent around the world due to its low antibody titer or low OPA titer.
  • 1 is a diagram showing the manufacturing process and conjugation sequence of pneumococcal polysaccharide-cell wall-derived material conjugates A to C.
  • FIG. 2 is a diagram showing the results of ELISA of pneumococcal capsular polysaccharide 3 serotype-specific IgG concentration.
  • FIG. 3 is a diagram showing the IgG ELISA results for the 19-valent pneumococcal vaccine serotype and the 19-valent pneumococcal vaccine including the novel conjugate (3C).
  • FIGS. 4 and 5 are diagrams showing the results of serotype-specific IgG concentration ELISA and functional antibody titer (immunogenicity) OPA for a 19-valent pneumococcal vaccine serotype and a novel conjugate (3C).
  • the present invention provides an immunogenic composition comprising a conjugate of a cell wall-derived material and a capsular polysaccharide derived from Streptococcus pneumoniae .
  • the capsular polysaccharide according to the present invention can be prepared by standard techniques known to those skilled in the art. Capsular polysaccharide can be reduced in size to reduce viscosity and increase solubility of the activated capsular polysaccharide together. Pneumococcal conjugates can be prepared by separate procedures and formulated into a single dosage form. For example, each pneumococcal polysaccharide serotype can be grown in a soy-based medium, and then the individual polysaccharide can be purified by centrifugation, precipitation, ultrafiltration.
  • the capsular polysaccharide according to the present invention is a group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B It may contain capsular polysaccharides of any one or more serotypes selected from
  • the capsular polysaccharide according to the present invention is a capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. may include
  • the capsular polysaccharide according to the present invention may include capsular polysaccharide of any one or more serotypes selected from the group consisting of serotypes 3, 6A, 6B, 19A and 19F.
  • the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotypes 10A, 11A, 15B, 22F, 23A And it may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of 35B, but is not limited thereto.
  • the present invention may further include a conjugate of a capsular polysaccharide derived from Streptococcus pneumoniae and a carrier protein, wherein the capsular polysaccharide is serotype 1, 4, 5, 7F, 9V , 14, 18C and 23F may be a capsular polysaccharide of any one or more serotypes selected from the group consisting of, but is not limited thereto.
  • the immunogenic composition of the present invention may range from 13 different serotypes to at least 30 different serotypes in the number of conventional capsular polysaccharides, but is not limited thereto.
  • the immunogenic composition of the present invention induces a functional immune response in humans against diseases caused by pneumococcal infection.
  • the human subject is an elderly subject and the disease is pneumonia or invasive pneumococcal disease.
  • the elderly subject is at least 50 years of age. More specifically, in one embodiment, the elderly subject is at least 55 years of age. More specifically, in one embodiment, the elderly subject is at least 60 years of age.
  • the human subject is an infant and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).
  • IPD invasive pneumococcal disease
  • AOM acute otitis media
  • the infant is between 0 and 2 years of age or between 2 and 15 months of age.
  • the human subject is between 6 weeks of age and 17 years of age and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the human subject is between 6 weeks of age and 5 years of age. In another embodiment, the human subject is between 5 weeks of age and 17 years of age.
  • IPD invasive pneumococcal disease
  • AOM acute otitis media
  • the capsular polysaccharide of the present invention can be prepared by standard techniques known to those skilled in the art.
  • the capsular polysaccharide can be reduced in size to reduce viscosity or to increase solubility of the activated capsular polysaccharide.
  • the present invention is an immunogenic composition
  • an immunogenic composition comprising at least 14 different polysaccharide-protein conjugates with a physiologically acceptable vehicle, each conjugate comprising a capsular polysaccharide from pneumococci of a different serotype conjugated to a carrier protein.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a carrier protein.
  • the carrier protein according to the present invention may be a protein which is preferably non-toxic and non-reactive, obtainable in sufficient quantity and purity.
  • the carrier protein may preferably be CRM 197 .
  • CRM 197 is a non-toxic variant (ie, toxoid) of diphtheria toxin isolated from a culture of Corynebacterium diphtheria strain C7 ( ⁇ 197) grown in casamino acids and yeast extract-based medium.
  • CRM 197 is purified via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography.
  • CRM 197 can also be prepared by genetic recombination according to US Pat. No. 5,614,382.
  • diphtheria toxoids may also be used as carrier proteins.
  • Other carrier proteins may include, in addition to diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, inactivated toxin from E. coli and inactivated toxin from Pseudomonas aeruginosa .
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group C5a peptidase from B streptococci, or Haemophilus influenzae protein D may also be used.
  • 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.
  • Variants of diphtheria toxin such as CRM 173 , CRM 228 , CRM 45 can also be used as carrier proteins.
  • a conventionally known conjugation method may be used.
  • a reductive amination or CDAP conjugation method may be used, but the present invention is not limited thereto.
  • Purified polysaccharides can be chemically activated to produce saccharides capable of reacting with carrier proteins. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment according to the present invention, each capsular polysaccharide may be conjugated to the same carrier protein. Chemical activation of polysaccharides and subsequent conjugation to carrier proteins may also be performed by known methods (US Pat. Nos. 4,673,574, 4,902,506, etc.).
  • the carrier protein conjugate obtained by the above method may be purified by various methods. Examples of these methods include concentration/diafiltration processes, column chromatography and multilayer filtration. Each of the purified conjugates is mixed to formulate the immunogenic composition of the present invention, which can be used as a vaccine. Formulation of the immunogenic composition of the present invention can be accomplished using art-recognized methods. For example, individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare a composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • a physiologically acceptable vehicle include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the conjugate of the Streptococcus pneumoniae-derived capsular polysaccharide and the cell wall-derived material may further include a linker, but is not limited thereto.
  • any one selected from the group consisting of adipic acid dihyrazied (ADH), Beta-propionamido, nitrophenyl-ethylamine, haloalkyl halide, hexane diamine, 6-aminocaproic acid and a glycosidic bond It may be one, but is not limited thereto.
  • the pneumococcal capsular polysaccharide may be conjugated to a carrier protein or a cell wall-derived material through a linker, for example, a functional linker.
  • the linker can be, for example, a heterobifunctional or homobifunctional linker having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups.
  • the linker may have, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms.
  • the cell wall-derived material may be one isolated from Streptococcus pneumoniae, and Streptococcus pneumoniae may be CWPS (Cell Wall Polysaccharide) derived from,
  • CWPS Cell Wall Polysaccharide
  • the cell wall-derived material may be covalently linked to the capsular polysaccharide, and all conjugates present in the immunogenic composition of the present invention may be prepared by any known conjugation technique. have.
  • the conjugation method may be to activate the capsular polysaccharide using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • the cell wall-derived material according to the present invention may not be directly bound to the protein in the polysaccharide-protein conjugate, and other components may be included between the corresponding components, or a linker for indirect connection may be included. may mean, but is not limited thereto.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences. It is not limited.
  • the cell wall-derived material may not be directly bound to a carrier protein.
  • a linker may be additionally included between each component of the conjugation order a), b) or c), and the structure conjugated in the conjugation order b) may be either linear or non-linear.
  • the present invention is not limited thereto.
  • the capsular polysaccharide or cell wall-derived material according to an embodiment of the present invention may be conjugated to an amino group on a carrier protein directly or through a linker (indirect method).
  • the cyanate ester is conjugated with hexane diamine or ADH
  • the amino-derived capsular polysaccharide can be conjugated to the carrier protein via a carbodiimide (e.g., EDAC or EDC) linkage via a carboxyl group on the carrier protein. .
  • a hydroxyl group on the capsular polysaccharide (optionally an activated hydroxyl group, for example, an activated hydroxyl group to produce a cyanate ester can be linked directly or indirectly (linker) to an amino or carboxyl group on the protein.
  • the hydroxyl group on the saccharide can be connected to the amino group on the linker using, for example, CDAP conjugation.
  • One additional amino group in the linker (eg ADH) can be connected to the carrier protein using the carbodiimide mode. It can be conjugated to the carboxylic acid group on the pneumococcal capsular polysaccharide is conjugated to the linker before the linker is conjugated to the carrier protein.
  • the present inventors also used a direct conjugation method for the production of a 13-valent or 20-valent or more multivalent vaccine, but in the present specification, an indirect conjugation method was used to compare and fabricate the effects of cell wall-derived substances.
  • the linker used in the indirect conjugation method was a commonly used ADH (adipic acid dihyrazied), and the same linker was used, and the conjugation sequence and the type of cell wall-derived material were varied and evaluated. As a result, even though the same linker and the same cell wall-derived material were used, it was confirmed that the immunogenicity (antibody and functional antibody titer) differed according to the conjugation sequence.
  • the capsular polysaccharide was activated using CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate), and then a linker was conjugated.
  • CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
  • CWPS cell wall polysaccharide, cell wall-derived polysaccharide
  • the primary material and the secondary material were finally bonded using a carbodiimide bonding method. (corresponding to embodiment A of the present invention, Fig. 1)
  • a carrier protein was conjugated.
  • Primary material and CWPS was activated using CDAP, followed by conjugation of a linker.
  • Secondary material the primary material and the secondary material were finally bonded using a carbodiimide bonding method.
  • CWPS was activated with CDAP
  • a linker was attached and it was conjugated with capsular polysaccharide using an EDC binding method.
  • the carrier protein was activated by CDAP to generate an amino group, and the CWPS-linker-CPS conjugate was conjugated with the carrier protein.
  • the cell wall-derived material is LTA (Lipoteichoic acid), PS (Polysaccharide) CPS (Capsular Polysaccharide), PG (Peptidoglycan), LPS (Lipopolysaccharide) MDP (Muramyl Dipeptide), MTP (Muramyl tripeptide) and Any one or more selected from the group consisting of PC (Phosphatidyl Choline) may be conjugated in the above manner, but is not limited thereto.
  • LTA Lipoteichoic acid
  • PS Polysaccharide
  • CPS Capsular Polysaccharide
  • PG Poridoglycan
  • LPS Lipopolysaccharide
  • MDP Muramyl Dipeptide
  • MTP Muramyl tripeptide
  • PC Phosphatidyl Choline
  • the method for conjugating the conjugate of the capsular polysaccharide with the cell wall-derived material and the carrier protein is selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected, but is not limited thereto.
  • the conjugation sequence a) may include the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the immunogenic composition according to the present invention may further comprise an adjuvant as a component.
  • An 'adjuvant' as defined herein is a substance used to increase the immunogenicity of the immunogenic composition of the present invention.
  • adjuvants are often given to boost the immune response and are well known to those skilled in the art.
  • Suitable adjuvants to increase the effectiveness of the composition include, but are not limited to:
  • aluminum salts may be used as adjuvants.
  • the aluminum salt may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine in its form.
  • Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), and the like.
  • Aluminum chloride and sodium phosphate are mixed in a ratio of 1:1, aluminum hydroxyphosphate sulfate is precipitated. It can be prepared by dialyzing with physiological saline and sterilizing the precipitate so that the size of the precipitate becomes 2-8 ⁇ m using a high shear mixer.
  • Al(OH)3 eg Alhydrogel or Superfos
  • 50-200 g of protein can be adsorbed per 1 mg of aluminum hydroxide, and this ratio is dependent on the pI of the protein and the pH of the solvent.
  • a protein with a low pI can bind strongly compared to a protein with a high pI.
  • Aluminum salt forms an antigen reservoir that releases antigens slowly for 2-3 weeks, non-specifically activating macrophages, complement, and innate immune mechanisms.
  • the immunogenic composition may contain a polysaccharide in an amount of 0.1 to 100 ⁇ g, but is not limited thereto.
  • the present invention also provides a pharmaceutical composition (eg, a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the immunogenic composition.
  • a pharmaceutical composition eg, a vaccine formulation
  • for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of the immunogenic composition.
  • the vaccine formulation according to the present invention can be used to protect or treat a person susceptible to pneumococcal infection by administration by a systemic or mucosal route.
  • an 'effective amount' refers to a dose required to elicit an antibody sufficient to significantly reduce the probability or severity of pneumococcal infection.
  • Administration includes injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or mucosal administration to the oral/digestive tract, airway tract, or genitourinary tract.
  • intranasal administration is used for the treatment of pneumonia or otitis media, since it can more effectively prevent nasopharyngeal carriers of pneumococci, thereby attenuating infection at an early stage.
  • each vaccine dose is selected such that it induces an immunoprotective response without significant side effects. This amount may vary depending on the serotype of the pneumococci. In general, each dose may contain 0.1 to 100 ⁇ g, preferably 0.1 to 10 ⁇ g, more preferably 1 to 5 ⁇ g of polysaccharide, but is not limited thereto. Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of an adequate immune response in subjects. For example, extrapolation of animal test results can be used to determine vaccination doses in humans. Dosage can also be determined empirically.
  • the pharmaceutical composition comprises 2 ⁇ g of each saccharide, 4 ⁇ g of capsular polysaccharide serotype; about 34 ⁇ g of CRM 197 transporter protein; 2 ⁇ g of extracellular material; 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.
  • the composition may be filled into single dose syringes without preservatives. After shaking, the vaccine is a homogeneous white suspension that can be administered immediately intramuscularly.
  • composition of the present invention may be formulated in the form of a single dose dose vial, a multiple dose dose vial or a prefilled syringe.
  • the formulation of the present invention may include a surfactant, and may include a mixture of surfactants such as Tween 80 or Span 85.
  • the present invention provides a method for preparing the immunogenic composition.
  • the conjugate of the Streptococcus pneumoniae -derived capsular polysaccharide and the cell wall-derived material and the carrier protein may be conjugated in any one of the following conjugation sequences.
  • a linker may be additionally included between each component of the conjugation sequence a), b), or c).
  • the method of conjugation between the capsular polysaccharide and the cell wall-derived material and the carrier protein is a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one or more selected from the group consisting of, but is not limited thereto.
  • the conjugation sequence a) may be conjugated by a method comprising the following steps, but is not limited thereto.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps, but is not limited thereto.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence b) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • the conjugation sequence c) may be conjugated by a method comprising the following steps.
  • CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate)
  • Pneumococcal culture and purification of capsular polysaccharide were performed by methods known to those skilled in the art.
  • Each of the pneumococcal serotypes can be obtained from the ATCC Ameircan Type Culture Collection, US.
  • Pneumococci were identified by capsular, nonmotile, Gram-positive, lancet-shaped dicocci, and alpha hemolysis in blood agar media. The serotype was confirmed based on the Banlung Test using a specific antisera.
  • Example 1-1 cell bank manufacturing
  • Pneumococci with 19 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B) was obtained from the US ATCC (Ameircan Type Culture Collection, US), which is a consignment institution.
  • a single colony was isolated by spreading the pneumococcal strain on a blood agar medium. After selecting a single colony with good growth among more than 10 single colonies, inoculating it in a liquid medium that does not contain animal-derived components and culturing, a Research Cell Bank (RCB) containing synthetic glycerol was prepared.
  • RBC Research Cell Bank
  • a master cell bank was prepared by adding synthetic glycerol after taking out one vial from the cell bank for research in which the expression of polysaccharides with a unique serotype was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol.
  • One vial was taken out of the bank, and the cells were grown in a liquid medium not containing animal-derived components, and then synthetic glycerol was added to prepare a manufacturing cell bank.
  • the prepared cell bank was stored at -70°C or lower for use in the next step.
  • Example 1-2 Fermentation and polysaccharide separation
  • One vial of the cell bank for production was thawed and inoculated into a liquid medium containing no animal-derived components to start the species fermentation.
  • Seed culture was performed at 37 ⁇ 2 °C without agitation until a certain cell concentration (Optical Density, OD600) was reached and the endpoint of the intermediate-exponential growth phase was reached.
  • the culture medium obtained from the seed culture was inoculated into a fermenter containing a liquid medium containing no animal-derived components to start the main fermentation (Main Fermentation).
  • the main culture was carried out while adjusting the pH of the medium with a potassium hydroxide solution at 37 ⁇ 2 °C.
  • the optimal cell density and glucose concentration in the medium were measured every 2 hours. Fermentation was terminated when the glucose in the medium was depleted.
  • the supernatant was recovered by centrifugation.
  • the recovered supernatant was passed through a depth filter, and then the buffer was exchanged with concentration and phosphate buffer solution. After the buffer exchange, the sample was passed through an active carbon filter, and impurities were removed by the following two methods.
  • CTAB Cetyltrimethylammonium Bromide
  • the two serotypes 7F and 14 that do not react with CTAB were reacted by adding an aluminum phosphate solution (Algel), and then the supernatant obtained by centrifugation was used.
  • Algel aluminum phosphate solution
  • the sample was subjected to depth filter and ultrafiltration (UF/DF) process, and then the amounts of ethanol and sodium chloride were adjusted and stored in raw form.
  • UF/DF depth filter and ultrafiltration
  • Capsular polysaccharide raw material derived from each serotype was dissolved in water for injection so that the final concentration range was within the range described below and filtered through a 0.45 ⁇ m filter.
  • serotypes 1, 3 and 4 were dissolved in the range of 0.8 - 2.0 mg/ml, serotypes 5, 6B, 9V, 18C and 19F were 4 - 8 mg/ml, serotypes 6A and 19A were 8 - In the case of 12 mg/ml, serotypes 7F, 10A, 11A and 23F, it was dissolved at 2 - 4 mg/ml and filtered. In addition, serotypes 15B, 22F and 35B were dissolved in the range of 2 - 5 mg/ml and filtration was performed.
  • the solution was incubated at the pH and temperature ranges described below for each serotype. Specifically for serotypes 1, 3, 5, 6B, 7F, 10A, 11A, 14 and 23F 70 - 80 °C overnight, for serotypes 6A and 19F 70 - 80 °C for 1-4 hours, serotypes 9V and 18C In the case of , the incubation process was performed at pH 2.0, 65 - 80 °C for 1 - 3 hours using a phosphoric acid solution. For serotypes 22F, 23A and 35B, 75-85° C. overnight, and for serotypes 4, 15B and 19A, no hydrolysis was performed. Hydrolysis was then stopped by cooling to 21-24° C. and adding sodium hydroxide to a target pH of 6.0 ⁇ 1.0.
  • CRM 197 protein transporter was prepared through a conventional production process. After inoculation into a tube containing a freeze-dried and withered seed culture medium of Corynebacterium diphtheria (ATCC 39255), it was incubated at 36° C. for 48 hours. After culturing, diphtheria cells grown on the surface were gram salted to check for contamination, and the main culture was performed. After filtration of the culture medium, primary purification was performed using the TFF system, and the process of precipitation using ammonium sulfate was repeated. Thereafter, impurities were purified through Capto Q (Anion exchange chromatography), and final TFF was performed using a buffer including the formulation composition to prepare a CRM 197 protein carrier.
  • ATCC 39255 Corynebacterium diphtheria
  • a 2 M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • the reaction was terminated by adding 3 to 6 molar equivalents of a glycine solution to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0.
  • the conjugation solution was stirred at 21 to 24° C. for 1 hour and then stored at a low temperature of 2 to 8° C. overnight.
  • the diluted conjugation mixture was concentrated and diafiltered through an ultrafiltration filter using at least 20 volumes of buffer.
  • the buffer was maintained in a pH range of 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used.
  • the molecular weight cutoff of the ultrafiltration filter was performed using 300 kDa for all serotypes, and the permeate was discarded.
  • Example 2-5 sterilization filtration
  • the residual solution after diafiltration was diluted to less than 0.4 g/L based on the polysaccharide content concentration using a buffer, and filtered through a 0.22 ⁇ m filter.
  • the filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were applied to the filtered retentate to determine whether further concentration, diafiltration and/or dilution was required.
  • Example 3-1 cell wall-derived substances
  • Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes.
  • appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • Example 3-4 junction termination and filtration
  • Example 4-1 cell wall-derived substances
  • Cell wall polysaccharide (CWPS, cat No. 3549) and lipo-teichoic acid (LTA, F-antigen, Cat No. 88840) were purchased from SSI Diagnostica.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • ADH adipic acid dihydrazide, linker
  • the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • CRM 197 was added at a weight ratio of 1 w / w % compared to CWPS after 7 minutes. .
  • 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CWPS-CRM 197 conjugate.
  • EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added in a ratio of 1 w/w% compared to the Pn 3-ADH conjugate. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later
  • ADH was added at a weight ratio of 10 w/w % of the polysaccharide.
  • the CWPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
  • an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3-CRM 197 conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to CWPS (cell wall polysaccharide, a cell wall-derived material).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • the CPS 3-ADH conjugate was obtained by purification in PBS buffer (pH 7.4) using a 6-8 kDa dialysis sack.
  • EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)
  • CWPS -ADH conjugate was added in a ratio of 1 w/w%.
  • 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain a CWPS-ADH-CPS 3 conjugate.
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. .
  • a 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, and 7 minutes later
  • ADH was added at a weight ratio of 10 w/w % of the polysaccharide. After stirring at room temperature for 4 hours, it was purified using a 6-8 kDa dialysis sack in PBS buffer (pH 7.4) to obtain an LTA-ADH conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to pneumococcal capsular polysaccharide serotype 3 (CPS 3).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. . After stirring at room temperature for 4 hours, 2M glycine in the same amount as the added CDAP was added, stirred for 1 hour, and filtered to obtain a CPS 3-CRM 197 conjugate.
  • an EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5) was added to the CWPS-ADH conjugate 1 w /w% was added. After adding 0.1M HCl solution to lower the pH to 5, the mixture was stirred at room temperature for 2 hours. Thereafter, 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3-CRM 197 conjugate.
  • a 2M NaCl solution was prepared by adding sodium chloride powder to LTA (Lipo-teichoic acid, Cat No. 88840).
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution.
  • v/v acetonitrile/water for injection
  • EDC.HCl solution (20 mg/mL in 0.1M Tris HCl pH 7.5)
  • LTA -ADH conjugate was added in a ratio of 1 w/w%.
  • 1M Tris HCl solution was added to raise the pH to 7, stirred at room temperature for 30 minutes, and purified by a tangential flow filtration system to obtain an LTA-ADH-CPS 3 conjugate.
  • CDAP was dissolved in a ratio of 1 g of CDAP per 100 ml of a 50/50 acetonitrile/water for injection (v/v) solution. It was dissolved in a weight ratio of 10 w/w % of CDAP to polysaccharide, and after 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7. After 7 minutes, CRM 197 was added in a weight ratio of 1 w/w % to polysaccharide. .
  • conjugates prepared above are novel, and it was confirmed as shown in Table 1 below that the finally produced conjugates A B, C, E, F had no problems in sugar content, protein content, sugar/protein ratio and concentration as a vaccine.
  • CPS 3 pneumococcal capsular polysaccharide serotype 3
  • serotype 3 for pneumococcal capsular polysaccharide.
  • Example 5-1 Serotype 3 specific IgG concentration ELISA measurement
  • Capsular polysaccharides (PnPs) for each serotype were coated at 0.5 ⁇ g/well to 1 ⁇ g/well on 96-well plates. Equal amounts of serum were sampled from each subject and pooled into groups. The serum pool was serially diluted up to 2.5 times with an antibody dilution buffer containing Tween 20 and 5 ⁇ g/mL of CWPS, and then reacted at room temperature for 30 minutes. Plates were washed 5 times with wash buffer, then 50 ⁇ l of pre-adsorbed and diluted serum was added to the coated well plates, followed by incubation at room temperature for 2 to 18 hours.
  • the well plate was washed in the same manner, and then goat anti-mouse IgG-alkaline phosphatase conjugate was added to each well and incubated for 2 hours at room temperature.
  • the plate was washed as described above, and 1 mg/mL p-nitrophenylamine buffer as a substrate was added to each well, followed by reaction at room temperature for 2 hours.
  • the reaction was quenched by addition of 50 ⁇ l of 3 M NaOH and the absorbance was measured at 405 nm and 690 nm.
  • a commercially available 13-valent vaccine (PREVNAR13) was applied in the same procedure.
  • the Pn3C (serotype 3, Example C) group had a slightly higher antibody titer specific to serotype 3 than PCV13, and in the case of Pn3A, it showed a slightly higher tendency.
  • the antibody titer was low. This means that even in the case of the same cell wall-derived material, there is a difference in immunogenicity depending on the splicing sequence and method (FIG. 2).
  • Example 5-2 Measurement of serotype 3 functional antibody titer (OPA)
  • Functional antibody titers were assessed by testing sera in an OPA assay. Streptococcus MOPA strains stored at -70° C. or lower were diluted to the corresponding final dilution so that the concentration of each strain was about 50,000 CFU/mL. Equal amounts of serum were sampled from each subject, pooled into groups, and serially diluted 2-fold so that 20 ⁇ l of serum remained in U-bottom plates. After diluting the sample, 10 ⁇ l of the strain prepared for each serotype was mixed with the diluted sample, and the mixture was reacted at room temperature for 30 minutes to mix well with Streptococcus and the antibody.
  • a mixture of pre-differentiated HL-60 cells and complement was added and incubated in a CO 2 incubator (37° C.) for 45 minutes.
  • the phagocytosis was stopped by lowering the temperature and 10 ⁇ l of the reaction solution was spotted on a pre-dried agar plate for 30 to 60 minutes and then allowed to absorb on the plate for 20 minutes until dry.
  • a 25 mg/mL TTC stock solution was added to the prepared overlay agar, and an antibody suitable for the corresponding strain was added thereto.
  • the mixture was thoroughly mixed, then about 25 mL of the mixture was added to the plate and allowed to cure for about 30 minutes.
  • the fully cured plates were incubated in a CO 2 incubator (37° C.) for 12 to 18 hours and then colonies were counted.
  • MOPA titers were expressed as the dilution at which 50% killing was observed.
  • PREVNAR13 a commercially available, 13-valent vaccine
  • serotype 3 for pneumococcal capsular polysaccharide.
  • Example 6-1 Serotype 3 specific IgG concentration ELISA measurement
  • IgG ELISA analysis for serotype 3 was performed in the same manner as in Example 5 above. As a result, as shown in FIG. 3 , a similar level of IgG titer was confirmed in the group of Pn3C (serotype 3, Example C) compared to PCV13. However, in the case of Pn3F using the same conjugation method and conjugated with another cell wall-derived material, LTA, a lower antibody titer at the full dose than in the Pn3c group was confirmed. In Example 5, using the same conjugation method as that of Pn3B, which showed a low antibody titer, Pn3F conjugated with LTA showed a relatively low antibody titer. This shows that the antibody titers differ according to the conjugation of different cell wall-derived materials despite being prepared with the same conjugation sequence and method (Fig. 3).
  • Example 7-1 Serotype-specific IgG concentration ELISA measurement
  • Example 4-1 Except for the experimental group, this experiment was performed in the same manner as in Example 4-1, and the results are shown in Table 6 below.
  • 'Prevnar13' and 'PCV19-CRM 197 (Naive)' described in the table below are specifically in the form of existing vaccines containing only the capsular polysaccharide-CRM 197 structure, and refer to 13-valent and 19-valent vaccines, respectively.
  • 'PCV19-CRM 197 (CWPS Conjugate)' has a structural difference from the existing vaccine form as capsular polysaccharide serotype 3 is linked to CWPS and carrier protein.
  • 23A and 35B are serotypes not included in PPV23, a pneumococcal polysaccharide vaccine (PPV), and serotypes not used in pneumococcal vaccines so far are one of the serotypes that have arrived in Asia and Europe after serotype replacement.
  • PCV19-CRM 197 including 23A and 35B had an excellent level of serotype-specific IgG concentration for all serotypes.
  • CWPS was conjugated to serotype 3
  • the antibody was formed at a similar level compared to PCV13 and PCV19 (naive)-CRM.
  • the serotype common to PREVNAR13 is similar to PREVNAR13, and the added serotypes 10A, 11A, 15B, 22F, and 23A, 35B each also showed superior serotype-specific IgG concentrations. (Table 6, Fig. 4)
  • Example 7-2 functional antibody titer (OPA)
  • Example 3 An experiment was performed to evaluate whether each of the pneumococcal vaccine compositions prepared in Example 3 had the ability to induce an immune response in mice. This immunogenicity was measured via functional antibody titer (OPA). Measurements were made in the same manner as in Example 4-1 except for the experimental group, and the results are shown in Table 7 below.
  • OPA functional antibody titer
  • antibiotic-resistant strains do not exist. Accordingly, antibiotic-resistant strains were prepared and used for MOPA analysis, and the production of resistant strains was carried out based on the UAB antibiotic-resistant strain production method (MOPA protocol). was confirmed.
  • PCV19 bonded with CWPS In the (3C Conjugated) group, serotype 3 was PCV13 and PCV19 (naive) showed a similar level of functional antibody titer to the group, and the interference effect by new conjugation and increase in serotype was not confirmed in the PCV19 (naive) and PCV19 (3C conjugated) groups.
  • Table 7, FIG. 5 In the monovalent experiment in which CWPS was conjugated, a significantly higher antibody titer and functional antibody titer were confirmed compared to the naive conjugated form, but a similar level of functional antibody titer was confirmed in the multivalent (PCV19) test.

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Abstract

La présente invention concerne une composition immunogène comprenant des conjugués polysaccharide pneumococcique-substance dérivée de paroi cellulaire comprenant chacun une substance dérivée de paroi cellulaire lié à un polysaccharide capsulaire dérivé d'un sérotype spécifique de pneumocoque. La composition immunogène selon la présente invention peut induire une réponse immunitaire plus accrue par comparativement aux vaccins existants. En particulier, la composition immunogène est incluse dans un vaccin pneumococcique conjugué existant mais possède un faible titre d'anticorps ou un faible titre d'OPA, et présente ainsi un excellent effet contre un sérotype spécifique qui est prévalent dans le monde.
PCT/KR2021/010359 2020-08-07 2021-08-05 Composition immunogène comprenant des conjugués polysaccharide pneumococcique-substance dérivée de paroi de cellulaire Ceased WO2022031087A1 (fr)

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Citations (5)

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WO2014124228A1 (fr) * 2013-02-07 2014-08-14 Children's Medical Center Corporation Antigènes de protéine qui confèrent une protection contre une colonisation et/ou une maladie pneumococcique
US9125863B2 (en) * 2008-05-22 2015-09-08 Children's Medical Center Corporation Synergistic immunogenic fusion protein-polysaccharide conjugate
KR20180105590A (ko) * 2017-03-15 2018-09-28 주식회사 엘지화학 다가 폐렴구균 백신 조성물
WO2019036313A1 (fr) * 2017-08-16 2019-02-21 Merck Sharp & Dohme Corp. Formulations de vaccin pneumococcique conjugué
KR20190055063A (ko) * 2016-08-05 2019-05-22 사노피 파스퇴르 인코포레이티드 다가 폐렴구균 다당류-단백질 접합체 조성물

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9125863B2 (en) * 2008-05-22 2015-09-08 Children's Medical Center Corporation Synergistic immunogenic fusion protein-polysaccharide conjugate
WO2014124228A1 (fr) * 2013-02-07 2014-08-14 Children's Medical Center Corporation Antigènes de protéine qui confèrent une protection contre une colonisation et/ou une maladie pneumococcique
KR20190055063A (ko) * 2016-08-05 2019-05-22 사노피 파스퇴르 인코포레이티드 다가 폐렴구균 다당류-단백질 접합체 조성물
KR20180105590A (ko) * 2017-03-15 2018-09-28 주식회사 엘지화학 다가 폐렴구균 백신 조성물
WO2019036313A1 (fr) * 2017-08-16 2019-02-21 Merck Sharp & Dohme Corp. Formulations de vaccin pneumococcique conjugué

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