WO2018080213A1 - Multivalent immunogenic composition having increased igg titer, and use thereof - Google Patents

Multivalent immunogenic composition having increased igg titer, and use thereof Download PDF

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WO2018080213A1
WO2018080213A1 PCT/KR2017/011959 KR2017011959W WO2018080213A1 WO 2018080213 A1 WO2018080213 A1 WO 2018080213A1 KR 2017011959 W KR2017011959 W KR 2017011959W WO 2018080213 A1 WO2018080213 A1 WO 2018080213A1
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serotype
composition
capsular
serotypes
derived
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PCT/KR2017/011959
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French (fr)
Korean (ko)
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김태현
제훈성
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주식회사 엘지화학
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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
    • 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
    • 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

Definitions

  • the present invention relates to a polyimmunogenic composition having improved IgG titer and its use, specifically to a vaccine composition for preventing pneumococcal disease comprising a capsular polysaccharide-carrying protein conjugate and immunogenicity against pneumococcus It relates to a composition.
  • Streptococcus pneumonia is a leading cause of meningitis, pneumonia and severely invasive diseases in infants and children around the world. More than 1.6 million people die each year from pneumococcal disease (2008 International Health Organization), and the incidence of invasive infectious diseases caused by pneumococcal in children under 5 years old and elderly people 65 years or older with low immunity high.
  • Pneumococci are classified into more than 90 serotypes according to the structural and immunological characteristics of the capsular polysaccharides, the main pathogenic factors surrounding them, of which 20 to 80% in humans It is known to be associated with pathogenicity.
  • the only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults).
  • the US Centers for Disease Control and Prevention (CDC) estimated that approximately 2.1 million children under 5 years of age died from pneumonia, and 1.2 million of them died from developing countries alone annually.
  • pneumococcal meningitis and sepsis are reported to be about 3,000 and 50,000 cases per year, respectively (Peters TR, Poehling KA et al . JAMA 2007; 297: 1825-6; Invasive pneumococcal disease). .
  • pneumoACTION which is a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in Korean children in 2000 and 47 of them died (www.pneumoadip.org).
  • pneumococci are the most common cause of invasive infections (43.7%) in infants aged 3 to 59 months. appear.
  • multidrug resistant bacteria that are resistant to not only penicillin but also to three or more drugs are increasing, further increasing the difficulty of treating pneumococcal infectious diseases. Therefore, the need for pneumococcal vaccination for children and the elderly, which is a high risk group of pneumococcal infectious diseases, has been continuously raised.
  • multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977, and these capsular polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients.
  • the immune system since the maturity of the immune system is lower than that of adults, when only the polysaccharide vaccine is received, the immune system does not recognize the polysaccharide antigen as an external invading factor, so it is difficult to expect a role as a vaccine.
  • a 7-valent pneumococcal conjugate vaccine a capsular polysaccharide-protein conjugated vaccine conjugated with a carrier protein that increases immunogenicity to polysaccharide antigens ( 7vPnC, Prevenar ® (Prevenar ®)) has been used in the development, has been reported to be effective for the prevention of invasive disease and otitis media in infants and children in many materials.
  • 7vPnC Prevenar ®
  • Prevenar ® Prevenar ®
  • the use of the 7-valent vaccine induced a decrease in invasive disease caused by the vaccine serotypes used in the vaccine, but together with the relative pneumococcus caused by some non-vaccine serotypes due to serotype replacement.
  • the capsular polysaccharide in order to provide a wider coverage - a protein conjugate vaccine in renal flow Rix ® (Synflorix ®), and the free the base serotypes of vena ®
  • a 13 add serotypes 6 kinds of pneumococcal conjugate vaccine Prevenar 13 ® (Prevenar13 ®) one of these has been developed, but presently on the market, resulting tried monitoring pneumococcal disease onset into after market, also 10 a and 13 a serotype other than the non-vaccine serotypes included in the vaccine Serotype replacement was observed (Weinberger DM et al . 2011; Lancet, 378: 9807,1962; Serotype replacement in disease following pneumococcal vaccination: A discussion of the evidence).
  • the pneumococcal conjugate vaccine of the present invention comprises 15 or more capsular polysaccharide antigens, specifically 17 capsular polysaccharide antigens, and may have broader serotype coverage than previously known pneumococcal conjugate vaccines. It can have an excellent effect in preventing.
  • One object of the present invention is to provide novel multivalent immunogenic compositions.
  • one object of the present invention includes capsular polysaccharide-carrying protein conjugates, wherein the conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B.
  • Vaccine composition for the prevention of pneumococcal disease wherein each of 15 capsular polysaccharides derived from 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, and It is to provide an immunogenic composition for pneumococcal.
  • one object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F,
  • a vaccine composition for preventing pneumococcal disease wherein each of 15 capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, wherein the composition is derived from serotype 1
  • Capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and capsular polysaccharides from serotype 6B exceeded 0.5 and a content ratio of 1.9 or less.
  • Eggplant is to provide a vaccine composition for the prevention of pneumococcal disease, and an immunogenic composition against pneumococcal.
  • Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C.
  • Vaccine composition for the prevention of pneumococcal disease and immunogenic composition for pneumococcal, wherein each of 17 capsular polysaccharides derived from 19A, 19F, 22F, 23F and 33F is covalently conjugated to a carrier protein.
  • another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14
  • a vaccine composition for preventing pneumococcal disease wherein each of 17 capsular polysaccharides from 15B, 18C, 19A, 19F, 22F, 23F, and 33F is covalently conjugated to a carrier protein, wherein
  • the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides derived from serotype 6B were greater than 0.5 and 1.9 or less, compared to the capsular polysaccharides derived from type 1, respectively.
  • Still another object of the present invention is to provide a method for preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.
  • Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 15 kinds of capsular polysaccharides from 18C, 19A, 19F and 23F are covalently conjugated to a carrier protein, to provide a use for the preparation of a vaccine composition for the prevention of pneumococcal disease.
  • the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 15 kinds of capsular polysaccharides from 18C, 19A, 19F and 23F are covalently conjugated to a carrier protein, to provide a use for the preparation of a vaccine composition for the prevention of pneumococcal disease.
  • another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F,
  • a vaccine composition for preventing pneumococcal disease wherein each of 15 capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, wherein the composition is derived from serotype 1
  • the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides from serotype 6B exceeded 0.5 and a content ratio of 1.9 or less.
  • Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 17 types of capsular polysaccharides derived from 18C, 19A, 19F, 22F, 23F and 33F are each covalently conjugated to a carrier protein, providing a use for the preparation of a vaccine composition for the prevention of pneumococcal disease It is.
  • another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F,
  • a vaccine composition for the prevention of pneumococcal disease wherein each of 17 capsular polysaccharides from 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F is covalently conjugated to a carrier protein, wherein
  • the capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F, respectively had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides derived from serotype 6B were greater than 0.5, compared to the capsular polysaccharides derived from serotype 1, respectively. It is to provide a use for use in the manufacture of a vaccine composition for the prevention of pneumococcal disease, having the following content ratio.
  • One aspect of the present invention for achieving the above object is a novel multivalent immunogenic composition.
  • one embodiment of the present invention is a vaccine composition for the prevention of pneumococcal disease comprising 15 species of capsular polysaccharide-carrying protein conjugates.
  • Another aspect of the present invention for achieving the above object is a vaccine composition for the prevention of pneumococcal disease comprising 17 species of capsular polysaccharide-carrying protein conjugate.
  • the 15 conjugates are 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F. It refers to each covalently conjugated to the carrier protein.
  • each of the capsular polysaccharides derived from Streptococcus pneumoniae serotypes 22F and 33F is covalently conjugated to each carrier protein. It may further comprise a conjugate of species.
  • the composition is a vaccine composition comprising 15 different polysaccharide-protein conjugates, each conjugate comprising a capsular polysaccharide from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein, wherein the capsular polysaccharide is Is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F.
  • composition is also a vaccine composition comprising 17 different polysaccharide-protein conjugates, in addition to the 15 conjugates described above, capsular polysaccharides derived from Streptococcus pneumoniae serotypes 22F and 33F covalently bound to their respective transport proteins. It is prepared by further comprising a conjugate to the conjugate.
  • the overall IgG titer was confirmed as the increase in the number of valent valences, 15 or 17 valences, and 15 or 17 valent immunogenic compositions were prepared.
  • the IgG titer can be actually increased by simultaneously controlling the conjugate content of serotypes 6A, 6B, 14, 19A and 19F.
  • Capsular polysaccharides derived from serotypes 6A, 14, 19A, and 19F as compositions of the present invention may each have a content ratio of greater than 0.25 and less than or equal to 0.95 relative to the capsular polysaccharides derived from serotype 1, specifically 0.3 to 0.95, 0.3 to It can represent a content ratio of 0.9, 0.4 to 0.95, 0.4 to 0.9, 0.45 to 0.95, 0.45 to 0.9, 0.5 to 0.95 or 0.5 to 0.9.
  • serotype 1-derived capsular polysaccharide serotype 6A, 14, 19A, or 19F-derived capsular polysaccharide was 1: 0.3 to 0.95, 1: 0.3 to 0.9, 1: 0.4 to 0.95, 1: 0.4 to 0.9, 1: 0.45 to It may represent a content ratio of 0.95, 1: 0.45 to 0.9, 1: 0.5 to 0.95 or 1: 0.5 to 0.9.
  • the capsular polysaccharide derived from serotype 6B may have a content ratio of more than 0.5 and 1.9 or less than the capsular polysaccharide derived from serotype 1.
  • the content ratio may be 0.6 to 1, 9, 0.6 to 1.8, 0.8 to 1.9, 0.8 to 1.8, 0.9 to 1.9, 0.9 to 1.8, 1.0 to 1.9, or 1.0 to 1.8. That is, the capsular polysaccharide derived from serotype 1: the capsular polysaccharide derived from serotype 6B is 1: 0.6 to 1.9, 1: 0.6 to 1.8, 1: 0.8 to 1.9, 1: 0.8 to 1.8, 1: 0.9 to 1.9, 1: 0.9 It may represent a content ratio of ⁇ 1.8, 1: 1 ⁇ 1.9, or 1: 1: 1.8.
  • polysaccharides other than the above-mentioned capsular polysaccharides are equivalent to serotype 1 derived capsular polysaccharides (1: 1 ratio).
  • Immunogenic compositions provided by the present invention comprising the capsular polysaccharides in the same amount as described above, by the specific content ratio of the five serotypes, serotypes 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F (additionally, 22F and 33F for 17 valent) may exhibit superior immunogenicity compared to immunogenic compositions in the same amount.
  • composition is specifically serotype 6A when serotype 1, 3, 4, 5, 7F, 9V, 12F, 15B, 18C and 23F derived capsular polysaccharides are respectively 4.4 ⁇ g / mL (eg, 2.2 ⁇ g / dose), respectively.
  • 14, 19A and 19F are greater than 1.1 ⁇ g / mL, respectively, 4.18 ⁇ g / mL, 1.32 ⁇ g / mL to 4.18 ⁇ g / mL, 1.32 ⁇ g / mL to 3.96 ⁇ g / mL, 1.76 ⁇ g / mL to 4.18 ⁇ g / mL, 1.76 ⁇ g, respectively.
  • concentrations of / mL to 3.96 ⁇ g / mL, 1.98 ⁇ g / mL to 4.18 ⁇ g / mL, 1.98 ⁇ g / mL to 3.96 ⁇ g / mL, 2.2 ⁇ g / mL to 4.18 ⁇ g / mL, or 2.2 ⁇ g / mL to 3.96 ⁇ g / mL Serotype 6B may be present in an amount greater than 2.2 ⁇ g / mL, 8.36 ⁇ g / mL, 2.64 ⁇ g / mL to 8.36 ⁇ g / mL, 2.64 ⁇ g / mL to 7.92 ⁇ g / mL, 3.52 ⁇ g / mL to 8.36 ⁇ g / mL, 3.52 ⁇ g / mL to 7.92 ⁇ g / mL, 3.96 ⁇ g / mL to 8.36 ⁇ g / mL, 3.
  • the composition may additionally include the capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F in the same amount as the capsular polysaccharide derived from serotype 1.
  • pneumococcal pneumonia accounts for about 50% of all pneumonia, severe chills, fever, cough and chest pain, sputum is often bloody, complications that can cause pleurisy, meningitis, endocarditis, peritonitis ( Stein GE et al. 2001; Diagn. Microbiol.Infect, Dis 39: 181; Comparative serum bactericidal activity of clarithromycin and azithromycin against macrolide-sensitive and resistant strains of Streptococcus pneumoniae ).
  • pneumococcus in the present invention refers to Streptococcus pneumoniae and is generally a commensal organism that colonizes the mucosal surface of the human nasopharynx. If the host's factor allows access to the lower respiratory tract of the organism, then a vigorous inflammatory response follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia May cause.
  • the pneumococci can synthesize more than 90 structurally unique capsular polysaccharides, and the serotypes of pneumococci are classified according to the structural and immunological characteristics of these capsular polysaccharides.
  • the immune response may be different depending on the type of capsular polysaccharide, that is, the serotype of pneumococcal from which capsular polysaccharide is derived.
  • the vaccine composition of the present invention specifically contains 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F. It can be prepared using.
  • 17-valent forms can be prepared, including capsular polysaccharides derived from serotypes 22F and 33F.
  • the capsular polysaccharide is recognized as an antigen when administered in the body, so that it can produce an antibody against it, thereby preparing a vaccine composition for preventing pneumococci.
  • the term “antigen” refers to a substance that can specifically induce an immune response when the substance invades the body.
  • 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F or
  • 17 capsular polysaccharides may act as antigens, respectively.
  • the capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and are not particularly limited in its method.
  • the capsular polysaccharide can be reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.
  • 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) Streptococcus pneumoniae with lysates were dissolved using sodium deoxycholate, respectively, and the polysaccharides bound to the cells were released.
  • serotypes 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F, and 23F are capable of ion bonding with CTAB (cetyltrimethylammonium bromide) to perform CTAB process 3 serotypes 7F, 14 and 33F that do not react with CTAB were purified using an aluminum phosphate gel (Algel) solution.
  • CTAB cetyltrimethylammonium bromide
  • the conjugate protein and the capsular polysaccharide are conjugated. It was prepared and used.
  • carrier protein refers to a protein that can be covalently conjugated with the capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen.
  • the carrier protein may be conjugated with the capsular polysaccharide through a standard conjugation method, and the capsular polysaccharide-carrying protein conjugate formed therefrom may be one or a plurality of capsular polysaccharides conjugated to one carrier protein.
  • the carrier protein may be, but is not limited to, a protein that is specifically nontoxic, nonreactive, and obtainable in sufficient amount and purity.
  • the carrier protein in the present invention is not limited thereto, but may be, for example, CRM197.
  • CRM197 in the present invention is a non-toxic variant of diphtheria toxin (ie, toxoid) isolated from the culture of Corynebacterium diphtheriae strain C7 ( ⁇ 197).
  • CRM197 can be purified via ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography.
  • the CRM197 may be recombinantly prepared according to US Pat. No. 5,614,382.
  • the carrier protein is also inactivated, such as tetanus toxoid, pertussis toxoid, cholera toxoid (WO2004 / 083251), E. coli LT, E. coli ST or Pseudomonas aeruginosa .
  • Bacterial toxins may be a variant of diphtheria toxins such as CRM173, CRM228, CRM45, but is not limited thereto.
  • OMPC outer complex c
  • porin porin
  • transferrin binding protein pneumolysin
  • pneumococcal surface protein A PspA
  • pneumococcal adhesin protein PsaA
  • group C5a peptidase from A or Group B streptococci or purified protein derivatives of Haemophilus influenzae protein D, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tuberculin (PPD)
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • PPD tuberculin
  • diphtheria toxins such as CRM173, CRM228, CRM45, but is not limited thereto.
  • the conjugate may have a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanylation method.
  • the vaccine compositions of the present invention are all compared to vaccine compositions conjugated by known reductive amination.
  • the serotype may show significantly better IgG titers.
  • the cyanation method may be appropriately performed by those skilled in the art through a known method, for example, may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.
  • purified capsular polysaccharides can be chemically activated and each chemically activated capsular polysaccharide can be conjugated to the carrier protein one by one to form a glycoconjugate.
  • Cyanation activity by treatment with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) converts the hydroxyl group of the capsular polysaccharide to a cyanate group, thereby covalently binding to the amino group of the carrier protein CRM197.
  • CDAP 1-cyano-4-dimethylaminopyridinium tetrafluoroborate
  • the cyanation reaction by the CDAP may be specifically terminated by adding 3 molar equivalents of glycine (glycine) solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto.
  • the reaction solution and reaction conditions can be adjusted accordingly.
  • the capsular polysaccharide-carrying protein conjugates obtained can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed and formulated into vaccine compositions of the invention and used respectively. Formulations of vaccine compositions of the invention can be carried out using methods known in the art. For example, compositions may be prepared by formulating individual capsular polysaccharide-carrying protein conjugates with a physiologically acceptable vehicle. Examples of such vehicles may include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.
  • a physiologically acceptable vehicle examples of such vehicles may include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.
  • the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by administering it to a human or an animal to prevent infection.
  • the vaccine composition may further include one or more selected from the group consisting of an adjuvant, a preservative, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and a free radical oxidation inhibitor.
  • adjuvant in the present invention refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention.
  • the adjuvant is often provided to enhance the immune response, which is well known to those skilled in the art.
  • adjuvants suitable for increasing the effectiveness of the vaccine composition of the present invention include, but are not limited to:
  • aluminum salts eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.
  • Oil-in-water emulsion formulations (with or without muramyl peptide (defined below) or other specific immune stimulants such as bacterial cell wall components), for example (a) MF59 (WO 90/14837): Contains 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally contains varying amounts of MTP-PE (although not required, see below)), Model 110Y Micro Formulated into submicron particles using a microfluidizer such as a microfluidizer (Microfluidics, Newton, Mass.), (B) SAF: 10% squalene, 0.4% Tween 80, 5% Pluronic -Block polymer L121 and thr-MDP (see below), microfluidized with a submicron emulsion, or vortexed to form a large particle size emulsion, and (c) Ribi TM adjuvant System (RAS) (Corixa, Hamilton, MT): 2% squalene, 0.
  • Tween 80 and from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL TM) (Corixa), trehalose dimicholate (TDM) and cell wall backbone (CWS) described in US Pat. No. 4,912,094. Contains at least one bacterial cell wall component, preferably MPL + CWS (Detox TM);
  • Saponin adjuvant such as Quill A or STIMULON TM QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540), may be used or produced from particles such as ISCOM ( Immunostimulatory complexes));
  • cytokines such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.) Interferon (eg gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and the like;
  • interleukins eg, interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.)
  • Interferon eg gamma interferon
  • GM-CSF granulocyte macrophage colony stimulating factor
  • MCSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • costimulatory molecules B7-1 and B7-2 costimulatory molecules
  • Wild type cholera toxin (CT) or mutant cholera toxin eg WO2002 / 098368
  • glutamic acid at amino acid position 29 according to WO2000 / 18434 is substituted with another amino acid, specifically histidine.
  • WO2002 / 098369 pertussis toxin (PT), or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PTK9 / G129 (WO93 / 13302 and WO92) / 19265), detoxified mutants of bacterial ADP-ribosylated toxins;
  • PT pertussis toxin
  • LT E. coli heat-labile toxin
  • LT-K63, LT-R72, CT-S109, PTK9 / G129 WO93 / 13302 and WO92
  • the muramyl peptides include N-acetyl-muramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1'-2 'dipalmityl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like, but is not limited thereto.
  • the aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine.
  • Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide, aluminum hydroxyphosphate adjuvant (APA), Amorphous alumina, and the like, but is not limited thereto.
  • APA refers to a suspension of aluminum hydroxyphosphate.
  • Aluminum chloride and sodium phosphate are mixed in a ratio of 1: 1, aluminum hydroxyphosphate sulfate is precipitated, and the precipitate is made to be 2 to 8 ⁇ m using a high shear mixer, and then prepared by dialysis and sterilization with physiological saline solution.
  • commercially available Al (OH) 3 eg Alhydrogel or Superfos
  • OH Alhydrogel or Superfos
  • 50 to 200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the pH of the protein and the pH of the solvent.
  • Low pI proteins bind more strongly than proteins with high pi.
  • Aluminum salts can form antigen reservoirs that slowly release antigens for two to three weeks to nonspecifically activate macrophages, complement, and innate immune mechanisms.
  • preservative means an anti-viral and / or antimicrobial agent that inhibits the growth of microorganisms in the vaccine composition, for example, chimerosal, phenoxyethanol, 2-phenoxyethanol. It may be, but is not limited to, formaldehyde, or mixtures thereof, all conventional preservatives used in the art may be used.
  • the vaccine composition may comprise one or more physiologically acceptable buffers.
  • the buffer may have buffering capacity at pH 4.0 to 10.0, specifically, pH 5.0 to 9.0, more specifically pH 6.0 to 8.0.
  • the buffer may be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanolamine buffer.
  • the buffer may be selected from buffers suitable for USP.
  • buffers include monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid; Dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid and phosphoric acid; It may be selected from the group consisting of ammonia, diethanolamine, glycine, triethanolamine, TRIS and the like.
  • the vaccine composition of the present invention may include a nonionic detergent.
  • a nonionic detergent for example, polysorbate 20 and polysorbate 80 in polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAX TM); Oxoxynols having a different repeating number of oxy-1,2-ethanediyl groups, especially ostoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethyleneglycol monolauryl ether (Brij 30); Sorbitan ethers, also known
  • Tween 80 may be included in the emulsion and a mixture of nonionic detergents such as Tween 80 / Span 85 may be used.
  • Combinations of polyoxyethylene sorbitan esters such as Tween 80 with octocinols such as Triton X-100 are also suitable, and combinations of Laureth 9 with Tween and or octosinol are also useful.
  • polyoxyethylene sorbitan esters such as Tween 80 may be used in an amount of 0.01% to 1% (w / v), in particular 0.1%; Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (such as Triton X-100) may range from 0.001% to 0.1%, in particular from 0.005% to 0.02%;
  • the polyoxyethylene ether eg laureth 9 may comprise 0.1% to 20%, preferably 0.1% to 10%, in particular 0.1% to 1% or about 0.5%.
  • composition of the present invention may be formulated in a single dose dose vial, multiple dose dose vial or prefilled syringe form, and may further comprise a physiologically acceptable carrier.
  • Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate.
  • Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions.
  • oils include vegetable or animal oils, peanut oils, soybean oils, olive oils, sunflower oils, synthetic oils such as liver oils, marine oils, and lipids derived from milk or eggs.
  • Vaccine compositions of the present invention may be isotonic, hypertonic or hypotonic, and pharmaceutical compositions administered by infusion or injection are preferably but is not limited to isotonicity. On the other hand, isotonicity or hypertension may be advantageous for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution can be ionic tonicity agents such as salts or nonionic tonicity agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.
  • the vaccine composition may further include aluminum element and sodium chloride, but is not limited thereto.
  • the vaccine composition may or may not contain a preservative depending on the purpose and use thereof.
  • the vaccine composition according to the present invention can be used to protect a subject susceptible to pneumococcal and to prevent pneumococcal disease by administering a pharmaceutically effective amount in a systemic or mucosal route.
  • prevention of the present invention refers to any action that inhibits or delays the infection caused by pneumococcal by administration of the vaccine composition of the present invention.
  • a “pharmaceutically effective amount” refers to a dosage required to elicit an antibody that is capable of significantly reducing the probability of infection or the severity of infection.
  • administration of the present invention refers to the introduction of certain substances into an individual in any suitable way.
  • the vaccine composition of the present invention may be administered by inhalation route through oral, nasal, rectal, transdermal or aerosol, but may be administered by bolus or infused slowly, but is not limited thereto.
  • the administration may be by injection via an intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract.
  • intranasal administration can be used for the treatment of pneumonia or otitis media, in which case more effective prevention of nasopharyngeal carriers of pneumococci can attenuate the infection at an early stage.
  • the term “individual” of the present invention means a living organism to which a pathogen can be infected, and in particular, may be a higher vertebrate, and more specifically, a mammal, but is not particularly limited thereto.
  • composition of the present invention may be administered in a single inoculation, or two, three, four or more times at appropriate intervals, but is not limited thereto.
  • routine inoculation plans for infants and newborns for invasive diseases caused by Streptococcus pneumoniae can be 2, 4, 6 and 12 to 15 months of age.
  • composition may further comprise one or more proteins from Streptococcus pneumoniae.
  • Streptococcus pneumoniae proteins suitable for inclusion include not only the proteins described in WO2002 / 053761, but also the proteins identified in WO2002 / 083855, all within the scope of the present invention.
  • each polysaccharide in a total of 0.5 mL provided that 6B is 8.8 ⁇ g / mL; About 29.3 ⁇ g CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 ⁇ g succinate buffer; About 3 mg of 2-phenoxyethanol and about 60 ⁇ g of formaldehyde were mixed (with preservatives) to prevent pneumococcal disease of the 13-valent (named 'LBVE013') and 17-valent (named 'LBVE017') Vaccine compositions were prepared. In addition, the content of the composition was 100%, and a vaccine composition was prepared separately by adjusting the content of some serotypes 6A, 6B, 14, 19A and 19F.
  • serum levels of rabbits inoculated with the LBVE013 vaccine composition confirmed higher serotype-specific IgG concentrations than prevena13 ® (Table 1), but other serotype-derived capsular polysaccharides were identified.
  • Table 1 serum levels of rabbits inoculated with the LBVE013 vaccine composition
  • the 17 transverse composition was prepared, it was confirmed that the IgG titer for each serotype was significantly decreased (FIG. 1).
  • serotypes 6A, 6B, 14, 19A and 19F conjugates at the same time and re-measuring the IgG titer for the entire serotype, it was confirmed that the IgG titer that decreased with the increase of the valence was restored again.
  • Another aspect of the invention is an immunogenic composition for pneumococci comprising capsular polysaccharide-carrying protein conjugates.
  • the conjugate and pneumococcus are as described above.
  • composition comprising the capsular polysaccharide-carrying protein conjugate of the present invention comprises Streptococcus pneumoniae-derived capsular polysaccharide having 15 or 17 different serotypes, and when administered to the body, it is recognized as an antigen It can be used as an immunogenic composition against pneumococcal to cause an immune response to produce an antibody.
  • Another aspect of the invention is a method of preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.
  • Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B for use in the preparation of vaccine compositions for the prevention of pneumococcal disease
  • 15 capsular polysaccharides derived from, 18C, 19A, 19F, and 23F each provide a use of 15 capsular polysaccharide-carrier protein conjugates covalently conjugated to a carrier protein.
  • Another aspect of the present invention provides a capsular polysaccharide derived from Streptococcus pneumoniae serotypes 22F and 33F, in addition to the aforementioned 15 conjugates, for use in the preparation of a vaccine composition for the prevention of pneumococcal disease. It is to provide the use of 17 kinds of capsular polysaccharide-carrier protein conjugate further comprising two conjugates covalently conjugated.
  • the capsular polysaccharide derived from serotypes 6A, 14, 19A, and 19F of the serotype 1-derived capsular polysaccharide in the composition has a content ratio of more than 0.25 and 0.95 or less, and the capsular polysaccharide derived from serotype 6B is 0.5. It may exceed and have a content ratio of 1.9 or less.
  • the capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F may be present in the composition in the same amount as the capsular polysaccharide derived from serotype 1.
  • Vaccine compositions immunogenic compositions, and prevention of pneumococcal disease are as described above.
  • the present invention provides a composition comprising pneumococcal capsular polysaccharides having 15 or more different serotypes, specifically, capsular polysaccharides derived from pneumococcal having 17 different serotypes, and in particular, the composition comprises Optimal amounts of polysaccharides can be included to induce good serum IgG titers and functional antibody activity. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be usefully used to prevent diseases caused by pneumococci in infants, infants, children, and adults.
  • FIG. 1 shows that IgG titers are generally lowered in most serotypes as the valence increases from 13-valent (LBVE013) to 17-valent (LBVE017), and 6A, 6B, 14, 19A, and 19F have high IgG titers at 13-valent.
  • LBVE013 13-valent
  • LBVE017 17-valent
  • 6A, 6B, 14, 19A, and 19F have high IgG titers at 13-valent.
  • Increasing the conjugate's dose to 50% simultaneously suggests an overall increase in IgG titer (recovery).
  • 22F and 33F the standard used is different from the rest of the serotypes, so it is drawn separately from other serotypes.
  • Figure 2 shows the amount of serotypes 6A, 6B, 14, 19A, 19F conjugates in the 17-valent composition (LBVE017) simultaneously changed to 100%, 90%, 75%, 50%, 25%, 10%, 1% 17 shows serotypic patterns of change in each of the serotypes.
  • the standard is different from the rest of the serotypes.
  • FIG. 3 is a diagram of dose threshold setting of serotypes 6A, 6B, 14, 19A, and 19F conjugates in 17-valent composition (LBVE017), when all 17-valent (LBVE017) serotypes were all received without dose reduction (100 When each GMT value of the 6A, 6B, 14, 19A, and 19F serotypes of%) is taken as the (lower limit), the range of conjugate amounts of each of the five conjugates above 40 can show higher immunogenicity than the reference. To 95%.
  • Figure 4 shows that in the case of 13-valent (LBVE013), unlike 17-valent compositions, immunogenicity is rather reduced in most serotypes when simultaneously reducing the amount of conjugates of serotypes 6A, 6B, 14, 19A, and 19F. Shows.
  • Streptococcus pneumoniae with 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) Obtained from the Center for Disease Control and Prevention (USC), a trustee, and prepared a cell bank in the following manner.
  • USC Center for Disease Control and Prevention
  • Streptococcus pneumoniae strains were plated on blood agar medium to isolate single colonies. Among the 10 or more single colonies, a single good growing colony was selected, inoculated and cultured in a liquid medium containing no animal-derived components, and a research cell bank (RCB) containing synthetic glycerol was prepared.
  • RBC research cell bank
  • Master cell bank was prepared by taking out one vial from the cell bank for which the expression of polysaccharides with unique serotypes 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 to proliferate cells in a liquid medium containing no animal-derived components, and then synthetic glycerol was added to prepare a cell bank for production. The prepared cell bank was stored at -70 ° C or below for use in the next step.
  • One vial in the cell bank for preparation was thawed and seeded in a liquid medium containing no animal-derived component to start species fermentation.
  • Species were cultured at 37 ⁇ 2 ° C. in an unstirred state until reaching a certain cell concentration (Optical Density, OD 600 ) and reaching the end point of the mid-exponential growth phase.
  • the culture broth obtained from the species culture was inoculated into a fermentor containing a liquid medium containing no animal-derived components to start main fermentation.
  • This culture was then carried out while adjusting the pH of the medium with potassium hydroxide solution at 37 ⁇ 2 ° C. Every two hours, the optimal cell density and glucose concentration in the medium were measured. Fermentation was terminated when glucose in the medium was depleted.
  • Phosphoric acid was added to the sample treated with sodium deoxycholate, and the supernatant was recovered by centrifugation. The recovered supernatant was passed through a depth filter and then buffer exchanged with concentrated and phosphate buffer. After the buffer exchange, the sample was passed through an active carbon filter, and then impurities were removed by the following two methods.
  • CTAB cetyltrimethylammonium bromide
  • the sample was subjected to a depth filter and ultrafiltration (UF / DF), and then the amount of ethanol and sodium chloride was adjusted and stored in a raw form.
  • UF / DF depth filter and ultrafiltration
  • the equilibrated complex was dissolved by incubation (37 ⁇ 2 ° C) in sodium phosphate (Na 3 PO 4 ) buffer solution at a rate of 0.1 M per 20 g of saccharides, and then cyanoborohydride (100 mg / mL) was added. The conjugation reaction was started by doing so. After incubation at 37 ⁇ 2 ° C. for about 44-52 hours, the temperature was lowered to 23 ⁇ 2 ° C. and 1 mL of 0.9% NaCl solution was added to the reactor.
  • Sodium borohydride solution (100 mg / mL) is added to 1.8 to 2.2 molar equivalents of sodium borohydride per mole of saccharide, and the mixture is incubated with stirring at 23 ⁇ 2 ° C. to any unreacted saccharide present in the saccharide. Aldehydes were reduced. The mixture was diluted with 5 mL of 0.9% aqueous sodium chloride solution and the diluted conjugate mixture was diafiltered using a 100 kDa MWCO membrane.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to a 9V polysaccharide stock solution prepared without hydrolysis.
  • CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 0.5 w / w% to polysaccharide, added to a 9V polysaccharide solution, and stirred for 15 minutes to induce a polysaccharide activation reaction.
  • the sodium hydroxide solution was then raised until the pH was 9.5 ⁇ 0.1 ° C. and then stirred for 3 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP.
  • CRM197 was added at a ratio of CRM197 1.0 w / w% to polysaccharide in the polysaccharide solution that had undergone polysaccharide activation, and the conjugation reaction was performed at room temperature for 1 hour.
  • the conjugation reaction was terminated by adding 2 M glycine solution in 3 molar equivalents to 1 molar equivalent of CDAP and incubating overnight at room temperature with pH adjusted to 9.0.
  • the terminated conjugate was concentrated and diafiltered into an ultrafiltration filter through a buffer containing 0.9% sodium chloride.
  • the original capsular polysaccharide originated 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 6A, 12F and 19A were dissolved at 8-12 mg / ml, and 2-4 mg / ml for serotypes 7F, 14 and 23F.
  • serotypes 15B, 22F, and 33F the solution was dissolved in the range of 2-5 mg / ml and filtered.
  • Each serotype was run by incubating the solution in the pH and temperature ranges described below. Specifically, phosphoric acid at 70-80 ° C. overnight for serotypes 1, 3, 5, 6B, 7F, 14 and 23F, 70-80 ° C. for 1-4 hours for serotypes 6A and 19F, for serotypes 9V and 18C. The solution was incubated at pH 2.0, 65-80 ° C. for 1-3 hours. Serotypes 22F and 33F were incubated overnight at 75-85 °C and serotype 12F at pH 2.0, 75-85 °C for 1-3 hours using phosphate solution.
  • w% was dissolved at a weight ratio of 4 w / w% for serotypes 5, 9V, 18C, 19F, 22F and 23F, and 5 w / w% for serotype 12F and added to each polysaccharide solution.
  • sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP.
  • Conjugation reaction was performed for 1 hour to 4 hours by adding CRM197 0.5-1.0 w / w% to polysaccharide to each serotype polysaccharide solution. The reaction conversion was measured using HPLC-SEC, and CDAP was added as needed.
  • the reaction was terminated by adding 3-6 molar equivalents of glycine solution relative to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0.
  • the conjugation solution was stirred at 21-24 ° C. for 1 hour and then stored overnight at 2-8 ° C. low temperature.
  • the diluted conjugate mixture was concentrated and diafiltered into an ultrafiltration filter using at least 20 volumes of buffer.
  • the buffer was maintained in the range of pH 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used.
  • Fractional molecular weight of the ultrafiltration filter was carried out using 300 kDa in all serotypes, and the permeate was discarded.
  • Step 5 Sterile Filtration
  • the residue after diafiltration was diluted to less than 0.4 g / L based on polysaccharide 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 performed on the filtered residue to determine if further concentration, diafiltration and / or dilution were needed.
  • Aluminum salts (mainly aluminum phosphate) were added to the sterile filtrate so that the final concentration was 1 mg / mL based on aluminum ions, and extra salts were added to maintain a pH range of 5.5 to 6.5. After the adsorption, the stock solution was subjected to quality inspection to confirm quality suitability, and refrigerated at 2 to 8 ° C. until use.
  • the required amount of final bulk concentrate was calculated based on the batch volume and bulk polysaccharide concentration.
  • the required amount of 0.85% sodium chloride, succinate buffer, 2-phenoxyethanol and formaldehyde were added to a previously labeled formulation vessel, followed by the bulk concentrate. Then it was thoroughly mixed and filtered through a 0.22 ⁇ m filter.
  • the formulated bulk liquid was slowly mixed, then bulk aluminum phosphate was added and mixed well. pH was checked and adjusted if necessary.
  • the formulated bulk product was stored at 2-8 ° C.
  • the resulting vaccine composition contains 4.4 ⁇ g / mL of each polysaccharide (13, hereinafter, 'LBVE013') in 0.5 mL total, 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F.
  • For 17 is (hereinafter 'LBVE017') 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F), except that 6B is 8.8 ⁇ g / mL; About 29.3 ⁇ g CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 ⁇ g succinate buffer; It contains about 3 mg of 2-phenoxyethanol and about 60 ⁇ g of formaldehyde (applicable when preservatives are added). The content is set as the reference amount of 100%, and in the following examples, the content of some serotypes is adjusted as needed (eg, 90%, 75%, 50%, 25%, 10%, 1%) for further analysis. Was performed.
  • Example 3 A study was conducted to evaluate whether each multivalent pneumococcal vaccine composition prepared in Example 3 has the ability to induce an immune response in rabbits. This immunogenicity was confirmed by measuring serum IgG concentration via antigen-specific ELISA.
  • a solution in which 13 or 17 polysaccharide antigens were conjugated to magnetic beads was attached to a 96-well plate. Each individual serum was adsorbed by reacting with 1 mg / mL CWPS multi solution (CWPS multi ® , Statens Serum Institute) for 30 minutes at room temperature to minimize non-specific antigen-antibody reactions, and then buffered for antibody dilution containing Tween 20. Was diluted to a suitable dilution multiple using. Plates with 13 or 17 polysaccharide antigen-conjugated magnetic beads were washed twice with a wash buffer, and 50 ⁇ l of previously adsorbed and diluted serum was added to the plates and allowed to react at room temperature for 30 minutes.
  • CWPS multi ® CWPS multi ®
  • the reacted plate was washed three times in the same manner, and each well was added with R-PE Ghycoerythrin goat anti-Rabbit IgG (1: 500), followed by reaction at room temperature for 30 minutes.
  • the plate was washed three times in the same manner as above, and 80 ⁇ l of the buffer solution was added to each well, and fluorescence was measured using a multiplex reader.
  • Example 13 is an immunogenic composition (LBVE013) prepared in the present invention are all relative to Prevenar 13 ® IgG concentration levels higher than prevena 13 ® were identified in the serotype.
  • serotypes 1, 6B, 7F, 9V 14 and 19F showed two to six times better effects than prevena 13 ® (Table 1).
  • the immunogenicity of LBVE017 prepared by adding four or more serotypes to LBVE013 was compared with LBVE013.
  • the immunogenic composition was confirmed that the decrease in the overall IgG titer occurs when the 17-valent valence is increased from 13 (FIG. 1).
  • the inventors of the present invention have all administered the doses of each of the 6A, 6B, 14, 19A and 19F conjugates, which had relatively high IgG titers at 13 valent relative to the 17-valent immunogenic composition. It was confirmed that the reduction to the 50% level at the same time. As a result, it was confirmed that an overall increase in immunogenicity occurs (Fig. 1).
  • serotypes 5, 6A, 6B, 14, 18C, and 19A IgG titers at levels higher than 13 were found.
  • the immunogenic composition according to the present invention can be very useful for developing pneumococcal conjugate vaccines by providing a practical way to overcome possible immune interference which can negatively affect the overall immunogenicity of the vaccine.
  • the inventors have sought to see if recovery of the immune response as seen in the 17-valent composition can be similarly observed in the 13-valent composition by simultaneously reducing the dose of each of the conjugates. Accordingly, even at 13-valent, the amount of the conjugate conjugate of the five serotypes was reduced to 50% or 25%, respectively, to prepare a 13-valent immunogenic composition, and its immunogenicity was 100% of the 13-valent immunogenic composition of the present invention (LBVE013). ) And Prevena 13 ® . As a result, the total immunogenicity did not recover (rather, decreased) in the thirteen, even though the doses of the five serotypes were reduced, unlike the results of the seventeen. This result means that the result of reducing the dose of some serotypes in the 17-valent immunogenic composition of the present invention is beyond the level predictable by those skilled in the art.

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Abstract

The present invention relates to a multivalent immunogenic composition having an increased IgG titer, and a use thereof and, particularly, to: a vaccine composition for preventing pneumococcal diseases, containing a capsular polysaccharide-carrier protein conjugate; and an immunogenic composition for Streptococcus pneuoniae.

Description

향상된 IgG 역가를 갖는 다가면역원성 조성물 및 이의 용도Polyimmunogenic Compositions with Improved IgG Titers and Uses thereof
본 발명은 향상된 IgG 역가를 갖는 다가면역원성 조성물 및 이의 용도에 관한 것으로, 구체적으로 협막 다당류 (capsular polysaccharide)-운반 단백질 접합체를 포함하는 폐렴구균병의 예방을 위한 백신 조성물 및 폐렴구균에 대한 면역원성 조성물에 관한 것이다.The present invention relates to a polyimmunogenic composition having improved IgG titer and its use, specifically to a vaccine composition for preventing pneumococcal disease comprising a capsular polysaccharide-carrying protein conjugate and immunogenicity against pneumococcus It relates to a composition.
스트렙토코커스 뉴모니애 (Streptococcus pneumonia)는 전세계에서 유아 및 소아의 뇌수막염, 폐렴 및 심각한 침습성질환의 주요 원인균이다. 매년 160 만명 이상이 폐렴구균에 의한 병으로 사망하고 있으며 (2008, 국제보건기구자료), 특히 면역능력이 낮은 5 세 이하 소아와 65 세 이상의 노인 연령층에서 폐렴구균에 의한 침습성 감염질환의 발병 빈도가 높다. Streptococcus pneumonia is a leading cause of meningitis, pneumonia and severely invasive diseases in infants and children around the world. More than 1.6 million people die each year from pneumococcal disease (2008 International Health Organization), and the incidence of invasive infectious diseases caused by pneumococcal in children under 5 years old and elderly people 65 years or older with low immunity high.
폐렴구균은 그 외부를 둘러싸고 있는 주요 병원성인자 (virulence factor)인 협막 다당류의 구조적, 면역학적 특성에 따라 90 가지가 넘는 혈청형 (serotype)으로 분류되며 이중 20 여 가지가 인간에서 80 ~ 90 %의 병원성과 연관되어 있다고 알려져 있다. 폐렴구균의 유일한 숙주는 인간이며 이들은 보통 건강한 정상인 (유아의 20 ~ 40 %, 성인의 5 ~ 10 %)의 비인강 (nasopharynx)에 군락을 지어 존재한다. 2005 년 미국 질병통제예방센터 (Centers for Disease Control and Prevention; CDC)는 개발도상국에서만 연간 약 210 만 명의 5 세 이하의 소아가 폐렴에 의해 사망하고 이 중 120 만 명이 폐렴구균 감염에 의해 사망한 것으로 보고하였으며, 미국 내에서도 폐렴구균에 의한 뇌수막염과 패혈증이 각각 연간 3 천 건과 5 만 건 가량 발생하는 것으로 집계되었다 (Peters TR, Poehling KA et al. JAMA 2007; 297: 1825-6; Invasive pneumococcal disease). 또한, 폐렴구균병의 데이터 베이스라고 할 수 있는 pneumoACTION의 집계에 의하면 2000 년 우리나라 소아에서 폐렴구균 감염질환 발생은 연간 24,047 건이었으며 이 중 사망은 47 건이었다 (www.pneumoadip.org). 나아가 최근의 질병관리본부가 발표한 '국내 소아청소년에서의 폐렴구균 혈청형 분석에 관한 연구'에 따르면 폐렴구균이 3 개월 ~ 59 개월 사이의 영유아에서 침습성 감염의 가장 흔한 (43.7%) 원인균인 것으로 나타났다. 전세계적으로 침습성 감염질환을 일으키는 폐렴구균에 있어서, 페니실린뿐만 아니라 3 제 이상의 약제에 내성을 보이는 다제 내성균이 증가하고 있어, 폐렴구균 감염질환 치료의 어려움을 더욱 가중시키고 있다. 따라서 폐렴구균 감염질환의 고위험군에 해당하는 소아와 노인층에 대한 폐렴구균 백신 접종의 필요성이 지속적으로 제기되고 있는 상황이다. Pneumococci are classified into more than 90 serotypes according to the structural and immunological characteristics of the capsular polysaccharides, the main pathogenic factors surrounding them, of which 20 to 80% in humans It is known to be associated with pathogenicity. The only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults). In 2005, the US Centers for Disease Control and Prevention (CDC) estimated that approximately 2.1 million children under 5 years of age died from pneumonia, and 1.2 million of them died from developing countries alone annually. In the United States, pneumococcal meningitis and sepsis are reported to be about 3,000 and 50,000 cases per year, respectively (Peters TR, Poehling KA et al . JAMA 2007; 297: 1825-6; Invasive pneumococcal disease). . In addition, pneumoACTION, which is a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in Korean children in 2000 and 47 of them died (www.pneumoadip.org). Furthermore, according to a recent study on the analysis of pneumococcal serotypes in children and adolescents published by the Korea Centers for Disease Control and Prevention, pneumococci are the most common cause of invasive infections (43.7%) in infants aged 3 to 59 months. appear. In pneumococci causing invasive infectious diseases all over the world, multidrug resistant bacteria that are resistant to not only penicillin but also to three or more drugs are increasing, further increasing the difficulty of treating pneumococcal infectious diseases. Therefore, the need for pneumococcal vaccination for children and the elderly, which is a high risk group of pneumococcal infectious diseases, has been continuously raised.
폐렴구균병을 예방하기 위해, 1977 년 이후로 다가 폐렴구균 다당류 백신이 개발, 승인되어 왔고, 이러한 협막 다당류 백신은 노인 및 고위험 환자에서 폐렴구균 질환을 예방하는데 있어서 유용한 것으로 입증되었다. 하지만, 유아 및 소아의 경우에는 면역체계의 성숙도가 성인에 비해 떨어지므로 다당류 백신만을 맞았을 경우, 면역체계가 다당류 항원을 외부 침입인자로 인식하지 못하므로 백신으로서의 역할을 기대하기 어려웠다. 이와 같이 유아 및 소아에서의 다당류 백신의 면역원성 저하 문제를 해결하기 위해 다당류 항원에 면역원성을 증가시켜주는 운반 단백질 (carrier protein)을 접합한 협막다당-단백질 접합백신인 7 가 폐렴구균 접합체 백신 (7vPnC, 프리베나® (Prevenar®))이 개발되어 사용되었으며, 많은 자료들에서 유아 및 소아에서 침습성 질환 및 중이염의 예방에 대해 효과적인 것으로 보고되어 왔다. 다만, 상기 7 가 백신의 사용은, 백신에 사용된 백신 혈청형들에 의한 침습성 질병의 감소를 유도하였으나 이와 함께 혈청형 교체현상 (serotype replacement)에 의한 일부 비백신 혈청형들에 의한 상대적인 폐렴구균병 증가현상을 보여 주었다. 이들 비 백신 혈청형들은 접합백신 도입 이전에 우세적으로 군락을 형성하고 있던 백신 혈청형들을 밀어내며 비인강에서의 군락 우위를 확보해감으로써 폐렴구균병의 발병에 있어서 새로운 위협요소로 떠올랐다(Hanage W.P.2007; The Journal of Infectious Diseases, 196:9,1282; Serotype Replacement in Invasive Pneumococcal Disease: Where Do We Go from Here?). 이에, 더 넓은 커버리지를 제공하기 위해 10 가 협막다당-단백 접합 백신인 신플로릭스® (Synflorix®)와, 프리베나®의 기본 혈청형에 혈청형 6 종을 추가한 13 가 폐렴구균 접합 백신인 프리베나13® (Prevenar13®)이 개발되어 현재 시판되고 있으나, 시판 후의 폐렴구균병 발병 현황들을 모니터링 해본 결과, 역시 10가 및 13가 백신에 포함된 혈청형이외의 비 백신혈청형들에의한 혈청형 교체현상이 일어나고 있음이 관찰되었다(Weinberger D.M. et al. 2011; Lancet, 378:9807,1962; Serotype replacement in disease following pneumococcal vaccination: A discussion of the evidence). To prevent pneumococcal disease, multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977, and these capsular polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients. However, in the case of infants and children, since the maturity of the immune system is lower than that of adults, when only the polysaccharide vaccine is received, the immune system does not recognize the polysaccharide antigen as an external invading factor, so it is difficult to expect a role as a vaccine. In order to solve the problem of lowering immunogenicity of polysaccharide vaccines in infants and children, a 7-valent pneumococcal conjugate vaccine, a capsular polysaccharide-protein conjugated vaccine conjugated with a carrier protein that increases immunogenicity to polysaccharide antigens ( 7vPnC, Prevenar ® (Prevenar ®)) has been used in the development, has been reported to be effective for the prevention of invasive disease and otitis media in infants and children in many materials. However, the use of the 7-valent vaccine induced a decrease in invasive disease caused by the vaccine serotypes used in the vaccine, but together with the relative pneumococcus caused by some non-vaccine serotypes due to serotype replacement. The disease showed an increase. These non-vaccine serotypes emerged as a new threat to the development of pneumococcal disease by pushing away vaccine serotypes that predominantly formed before the introduction of the conjugate vaccine and gaining colony advantage in the nasopharynx (Hanage WP2007). ; The Journal of Infectious Diseases, 196: 9,1282; Serotype Replacement in Invasive Pneumococcal Disease: Where Do We Go from Here?). Thus, 10 the capsular polysaccharide in order to provide a wider coverage - a protein conjugate vaccine in renal flow Rix ® (Synflorix ®), and the free the base serotypes of vena ® A 13 add serotypes 6 kinds of pneumococcal conjugate vaccine Prevenar 13 ® (Prevenar13 ®) one of these has been developed, but presently on the market, resulting tried monitoring pneumococcal disease onset into after market, also 10 a and 13 a serotype other than the non-vaccine serotypes included in the vaccine Serotype replacement was observed (Weinberger DM et al . 2011; Lancet, 378: 9807,1962; Serotype replacement in disease following pneumococcal vaccination: A discussion of the evidence).
또한, 다가 폐렴구균 접합백신 개발시 직면할 수 있는 또다른 문제점은, 혈청형 가수를 추가함에 따라 증가될 수 있으며 일반적으로 백신내의 협막다당 혹은 캐리어 단백질들간의 상호작용에 의해 야기될 수 있는 면역간섭현상(immune interference)이다 (Dagan R et al. (2010) Vaccines, 28(5513); Glycoconjugate vaccines and immune interference: A review). 실제로 13가인 프리베나13®의 임상데이터를 보면 또한 대조군인 7가 백신인 프리베나® 와 공통되는 혈청형들의 면역원성이 프리베나®에 비해 전반적으로 낮아진 경향을 보임을 관찰할 수 있었으며(EMEA Assessment Report for Prevenar 13, 2009.EMA/798877/2009), 따라서, 이와 같은 현상은 혈청형 교체현상에 의해 요구될 수 있는 13가 이상의 차기 폐렴구균접합백신의 개발에 있어서 극복해야 할 중요한 과제가 될 수 있다.In addition, another problem that may be encountered when developing multivalent pneumococcal conjugate vaccines can be increased with the addition of serotype valences, and immune interference, which can generally be caused by interactions between the capsular polysaccharides or carrier proteins in the vaccine. Immune interference (Dagan R et al . (2010) Vaccines, 28 (5513); Glycoconjugate vaccines and immune interference: A review). Indeed, the clinical data of 13-valent prevena 13 ® also showed that the immunogenicity of serotypes common to the control 7-valent vaccine prevena ® was associated with prevena ® . It was observed that the overall trend was lower (EMEA Assessment Report for Prevenar 13, 2009.EMA / 798877/2009). Therefore, this phenomenon is more than 13 next pneumonia that may be required by serotype replacement. This can be an important challenge to overcome in the development of a conjugated vaccine.
이에, 본 발명자들은 보다 더 높고 안정적인 IgG 역가를 가지며, 종래 개발된 백신 제제에 비해 더욱 높은 가수를 갖는 폐렴구균병의 예방용 백신을 개발하기 위해 예의 노력한 결과, 15 종 이상의 혈청형을 포함하는 신규한 폐렴구균 접합백신, 구체적으로 17종의 혈청형을 포함하는 신규한 폐렴구균 접합백신을 개발하여 본 발명을 완성하였다. 본 발명의 폐렴구균 접합 백신은 15 종 이상의 협막다당 항원, 구체적으로 17종의 협막다당 항원을 포함하며, 기존에 알려진 폐렴구균 접합 백신에 비해 보다 넓은 혈청형 커버리지를 가질 수 있으므로, 침습성 폐렴구균병을 예방하는데 우수한 효과를 나타낼 수 있다.Accordingly, the present inventors have made intensive efforts to develop a vaccine for preventing pneumococcal disease having a higher and stable IgG titer and having a higher valence than a conventionally developed vaccine preparation. One pneumococcal conjugate vaccine, specifically a novel pneumococcal conjugate vaccine comprising 17 serotypes, has been developed to complete the present invention. The pneumococcal conjugate vaccine of the present invention comprises 15 or more capsular polysaccharide antigens, specifically 17 capsular polysaccharide antigens, and may have broader serotype coverage than previously known pneumococcal conjugate vaccines. It can have an excellent effect in preventing.
본 발명의 하나의 목적은 신규한 다가 면역원성 조성물을 제공하는 것이다.One object of the present invention is to provide novel multivalent immunogenic compositions.
구체적으로, 본 발명의 하나의 목적은 협막 다당류 (capsular polysaccharide)-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 (Streptococcus pneumoniae) 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물, 및 폐렴구균에 대한 면역원성 조성물을 제공하는 것이다.Specifically, one object of the present invention includes capsular polysaccharide-carrying protein conjugates, wherein the conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B. , Vaccine composition for the prevention of pneumococcal disease, wherein each of 15 capsular polysaccharides derived from 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, and It is to provide an immunogenic composition for pneumococcal.
보다 구체적으로, 본 발명의 하나의 목적은 협막 다당류-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물로서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고, 혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하인 함량비를 가지는, 폐렴구균병의 예방을 위한 백신 조성물, 및 폐렴구균에 대한 면역원성 조성물을 제공하는 것이다.More specifically, one object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, A vaccine composition for preventing pneumococcal disease, wherein each of 15 capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, wherein the composition is derived from serotype 1 Capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and capsular polysaccharides from serotype 6B exceeded 0.5 and a content ratio of 1.9 or less. Eggplant is to provide a vaccine composition for the prevention of pneumococcal disease, and an immunogenic composition against pneumococcal.
본 발명의 다른 목적은 협막 다당류 -운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물, 및 폐렴구균에 대한 면역원성 조성물을 제공하는 것이다.Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C. Vaccine composition for the prevention of pneumococcal disease, and immunogenic composition for pneumococcal, wherein each of 17 capsular polysaccharides derived from 19A, 19F, 22F, 23F and 33F is covalently conjugated to a carrier protein. To provide.
보다 구체적으로, 본 발명의 다른 목적은 협막 다당류-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물로서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고, 혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하인 함량비를 가지는, 폐렴구균병의 예방을 위한 백신 조성물, 및 폐렴구균에 대한 면역원성 조성물을 제공하는 것이다.More specifically, another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14 A vaccine composition for preventing pneumococcal disease, wherein each of 17 capsular polysaccharides from 15B, 18C, 19A, 19F, 22F, 23F, and 33F is covalently conjugated to a carrier protein, wherein The capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides derived from serotype 6B were greater than 0.5 and 1.9 or less, compared to the capsular polysaccharides derived from type 1, respectively. To provide a vaccine composition for the prevention of pneumococcal disease, and immunogenic composition against pneumococcal having a content ratio.
본 발명의 또 다른 목적은 상기 백신 조성물 혹은 면역원성 조성물을 이를 필요로 하는 개체에 투여하여 폐렴구균병을 예방하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.
본 발명의 또 다른 목적은 협막 다당류-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한 용도를 제공하는 것이다.Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 15 kinds of capsular polysaccharides from 18C, 19A, 19F and 23F are covalently conjugated to a carrier protein, to provide a use for the preparation of a vaccine composition for the prevention of pneumococcal disease.
보다 구체적으로, 본 발명의 또 다른 목적은 협막 다당류-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물로서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고, 혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하의 함량비를 가지는, 조성물을 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한 용도를 제공하는 것이다.More specifically, another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, A vaccine composition for preventing pneumococcal disease, wherein each of 15 capsular polysaccharides derived from 14, 15B, 18C, 19A, 19F, and 23F is covalently conjugated to a carrier protein, wherein the composition is derived from serotype 1 The capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides from serotype 6B exceeded 0.5 and a content ratio of 1.9 or less. To provide a use for the use of the composition in the manufacture of a vaccine composition for the prevention of pneumococcal disease.
본 발명의 또 다른 목적은 협막 다당류 -운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한 용도를 제공하는 것이다.Another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 17 types of capsular polysaccharides derived from 18C, 19A, 19F, 22F, 23F and 33F are each covalently conjugated to a carrier protein, providing a use for the preparation of a vaccine composition for the prevention of pneumococcal disease It is.
보다 구체적으로, 본 발명의 또 다른 목적은 협막 다당류-운반 단백질 접합체를 포함하고, 여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 각각이 운반체 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물로서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고, 혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하의 함량비를 가지는, 조성물을 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한 용도를 제공하는 것이다.More specifically, another object of the present invention includes a capsular polysaccharide-carrying protein conjugate, wherein the conjugate is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, A vaccine composition for the prevention of pneumococcal disease, wherein each of 17 capsular polysaccharides from 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F is covalently conjugated to a carrier protein, wherein The capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F, respectively, had a content ratio of greater than 0.25 and 0.95 or less, and the capsular polysaccharides derived from serotype 6B were greater than 0.5, compared to the capsular polysaccharides derived from serotype 1, respectively. It is to provide a use for use in the manufacture of a vaccine composition for the prevention of pneumococcal disease, having the following content ratio.
본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는, 신규한 다가 면역원성 조성물이다. 구체적으로, 본 발명의 하나의 양태는 협막 다당류 -운반 단백질 접합체 15 종을 포함하는 폐렴구균병의 예방을 위한 백신 조성물이다. One aspect of the present invention for achieving the above object is a novel multivalent immunogenic composition. Specifically, one embodiment of the present invention is a vaccine composition for the prevention of pneumococcal disease comprising 15 species of capsular polysaccharide-carrying protein conjugates.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는, 협막 다당류-운반 단백질 접합체 17 종을 포함하는 폐렴구균병의 예방을 위한 백신 조성물이다. Another aspect of the present invention for achieving the above object is a vaccine composition for the prevention of pneumococcal disease comprising 17 species of capsular polysaccharide-carrying protein conjugate.
여기서 상기 15 종의 접합체는 스트렙토 코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반 단백질에 공유결합적으로 접합된 것을 말한다.Wherein the 15 conjugates are 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F. It refers to each covalently conjugated to the carrier protein.
또한 상기 17 종의 접합체를 포함하는 백신 조성물의 경우, 상술한 15 종의 접합체에 더하여 스트렙토 코커스 뉴모니애 혈청형 22F 및 33F 유래의 협막 다당류 각각이 각각의 운반 단백질에 공유결합적으로 접합된 2종의 접합체를 더 포함할 수 있다.In addition, in the case of the vaccine composition comprising the above 17 conjugates, in addition to the above 15 conjugates, each of the capsular polysaccharides derived from Streptococcus pneumoniae serotypes 22F and 33F is covalently conjugated to each carrier protein. It may further comprise a conjugate of species.
구체적으로, 상기 조성물은 15 종의 상이한 다당류-단백질 접합체를 포함하는 백신 조성물로서, 각각의 접합체는 운반 단백질에 접합된 상이한 혈청형의 스트렙토코커스 뉴모니애 유래의 협막 다당류를 포함하며, 상기 협막 다당류가 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F로부터 제조되는 것이다. Specifically, the composition is a vaccine composition comprising 15 different polysaccharide-protein conjugates, each conjugate comprising a capsular polysaccharide from Streptococcus pneumoniae of different serotypes conjugated to a carrier protein, wherein the capsular polysaccharide is Is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F.
또한, 상기 조성물은 17 종의 상이한 다당류-단백질 접합체를 포함하는 백신 조성물로서 상술한 15 종의 접합체에 추가로 스트렙토 코커스 뉴모니애 혈청형 22F 및 33F 유래의 협막 다당류가 각각의 운반 단백질에 공유결합적으로 접합된 접합체를 추가로 포함하여 제조되는 것이다.The composition is also a vaccine composition comprising 17 different polysaccharide-protein conjugates, in addition to the 15 conjugates described above, capsular polysaccharides derived from Streptococcus pneumoniae serotypes 22F and 33F covalently bound to their respective transport proteins. It is prepared by further comprising a conjugate to the conjugate.
본 발명에서는 다양한 혈청형의 협막 다당류를 포함하는 다가 면역원성 조성물에서 13 가 대비, 15 가 혹은 17 가로 가수가 증가함에 따라 전반적인 IgG 역가 저하 현상을 확인하였으며, 15 가 혹은 17 가 면역원성 조성물을 제조함에 있어서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 6B, 14, 19A 및 19F의 접합체 함량을 동시에 조절함으로써 IgG 역가를 실제로 증가시킬 수 있음을 확인하였다. 본 발명의 조성물로서 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 혈청형 1 유래의 협막 다당류 대비 0.25를 초과하고, 0.95 이하인 함량비를 가질 수 있고, 구체적으로 0.3 내지 0.95, 0.3 내지 0.9, 0.4 내지 0.95, 0.4 내지 0.9, 0.45 내지 0.95, 0.45 내지 0.9, 0.5 내지 0.95 또는 0.5 내지 0.9의 함량비를 나타낼 수 있다. 즉, 혈청형 1 유래 협막 다당류: 혈청형 6A, 14, 19A 또는 19F 유래 협막 다당류는 1 : 0.3 ~ 0.95, 1 : 0.3 ~ 0.9, 1 : 0.4 ~ 0.95, 1 : 0.4 ~ 0.9, 1 : 0.45 ~ 0.95, 1 : 0.45 ~ 0.9, 1 : 0.5 ~ 0.95 또는 1 : 0.5 ~ 0.9의 함량비를 나타낼 수 있다. 또한, 혈청형 6B 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류 대비 0.5를 초과하고, 1.9 이하인 함량비를 가질 수 있다. 구체적으로, 0.6 내지 1,9, 0.6 내지 1.8, 0.8 내지 1.9, 0.8 내지 1.8, 0.9 내지 1.9, 0.9 내지 1.8, 1.0 내지 1.9, 또는 1.0 내지 1.8의 함량비를 나타낼 수 있다. 즉, 혈청형 1 유래의 협막 다당류 : 혈청형 6B 유래 협막 다당류는 1 : 0.6 ~ 1.9, 1 : 0.6 ~ 1.8, 1 : 0.8 ~ 1.9, 1 : 0.8 ~ 1.8, 1 : 0.9 ~ 1.9, 1 : 0.9 ~ 1.8, 1 : 1 ~ 1.9, 또는 1 : 1 ~ 1.8의 함량비를 나타낼 수 있다. 상술한 종류의 협막 다당류 외 다른 다당류 (예, 혈청형 3, 4, 5, 7F, 9V, 12F, 15B, 18C, 22F, 23F, 및 33F 유래)는 혈청형 1 유래 협막 다당류와 동량 (1 : 1 비율)으로 존재할 수 있다. 상기와 같은 함량으로 협막 다당류를 포함하는 본 발명에서 제공하는 면역원성 조성물은 상기 5 종의 혈청형의 특정 함량비에 의해, 혈청형 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F (17가의 경우, 추가적으로, 22F 및 33F)가 동량으로 존재하는 면역원성 조성물에 비해 우수한 면역원성을 나타낼 수 있다.In the present invention, in the multivalent immunogenic composition containing various serotypes of the capsular polysaccharides, the overall IgG titer was confirmed as the increase in the number of valent valences, 15 or 17 valences, and 15 or 17 valent immunogenic compositions were prepared. In comparison with the capsular polysaccharide derived from serotype 1, it was confirmed that the IgG titer can be actually increased by simultaneously controlling the conjugate content of serotypes 6A, 6B, 14, 19A and 19F. Capsular polysaccharides derived from serotypes 6A, 14, 19A, and 19F as compositions of the present invention may each have a content ratio of greater than 0.25 and less than or equal to 0.95 relative to the capsular polysaccharides derived from serotype 1, specifically 0.3 to 0.95, 0.3 to It can represent a content ratio of 0.9, 0.4 to 0.95, 0.4 to 0.9, 0.45 to 0.95, 0.45 to 0.9, 0.5 to 0.95 or 0.5 to 0.9. Namely, serotype 1-derived capsular polysaccharide: serotype 6A, 14, 19A, or 19F-derived capsular polysaccharide was 1: 0.3 to 0.95, 1: 0.3 to 0.9, 1: 0.4 to 0.95, 1: 0.4 to 0.9, 1: 0.45 to It may represent a content ratio of 0.95, 1: 0.45 to 0.9, 1: 0.5 to 0.95 or 1: 0.5 to 0.9. In addition, the capsular polysaccharide derived from serotype 6B may have a content ratio of more than 0.5 and 1.9 or less than the capsular polysaccharide derived from serotype 1. Specifically, the content ratio may be 0.6 to 1, 9, 0.6 to 1.8, 0.8 to 1.9, 0.8 to 1.8, 0.9 to 1.9, 0.9 to 1.8, 1.0 to 1.9, or 1.0 to 1.8. That is, the capsular polysaccharide derived from serotype 1: the capsular polysaccharide derived from serotype 6B is 1: 0.6 to 1.9, 1: 0.6 to 1.8, 1: 0.8 to 1.9, 1: 0.8 to 1.8, 1: 0.9 to 1.9, 1: 0.9 It may represent a content ratio of ~ 1.8, 1: 1 ~ 1.9, or 1: 1: 1.8. Other polysaccharides other than the above-mentioned capsular polysaccharides (e.g., serotypes 3, 4, 5, 7F, 9V, 12F, 15B, 18C, 22F, 23F, and 33F derived) are equivalent to serotype 1 derived capsular polysaccharides (1: 1 ratio). Immunogenic compositions provided by the present invention comprising the capsular polysaccharides in the same amount as described above, by the specific content ratio of the five serotypes, serotypes 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F (additionally, 22F and 33F for 17 valent) may exhibit superior immunogenicity compared to immunogenic compositions in the same amount.
상기 조성물은 구체적으로 혈청형 1, 3, 4, 5, 7F, 9V, 12F, 15B, 18C 및 23F 유래 협막 다당류가 각각 4.4㎍/mL (한 예로, 2.2 ㎍/dose)인 경우, 혈청형 6A, 14, 19A 및 19F는 각각 1.1㎍/mL 초과 4.18㎍/mL, 1.32㎍/mL 내지 4.18㎍/mL, 1.32㎍/mL 내지 3.96㎍/mL, 1.76㎍/mL 내지 4.18㎍/mL, 1.76㎍/mL 내지 3.96㎍/mL, 1.98㎍/mL 내지 4.18㎍/mL, 1.98㎍/mL 내지 3.96㎍/mL, 2.2㎍/mL 내지 4.18㎍/mL, 또는 2.2㎍/mL 내지 3.96㎍/mL의 농도로 존재할 수 있으며, 혈청형 6B는 2.2㎍/mL 초과 8.36㎍/mL, 2.64㎍/mL 내지 8.36㎍/mL, 2.64㎍/mL 내지 7.92㎍/mL, 3.52㎍/mL 내지 8.36㎍/mL, 3.52㎍/mL 내지 7.92㎍/mL, 3.96㎍/mL 내지 8.36㎍/mL, 3.96㎍/mL 내지 7.92㎍/mL, 4.4㎍/mL 내지 8.36㎍/mL, 또는 4.4㎍/mL 내지 7.92㎍/mL의 농도로 존재할 수 있다. 그러나, 이에 제한되는 것은 아니며, 상술된 함량비의 범위 내라면 제조하고자 하는 목적에 따라 그 농도를 조절할 수 있다.The composition is specifically serotype 6A when serotype 1, 3, 4, 5, 7F, 9V, 12F, 15B, 18C and 23F derived capsular polysaccharides are respectively 4.4 μg / mL (eg, 2.2 μg / dose), respectively. , 14, 19A and 19F are greater than 1.1 μg / mL, respectively, 4.18 μg / mL, 1.32 μg / mL to 4.18 μg / mL, 1.32 μg / mL to 3.96 μg / mL, 1.76 μg / mL to 4.18 μg / mL, 1.76 μg, respectively. concentrations of / mL to 3.96 μg / mL, 1.98 μg / mL to 4.18 μg / mL, 1.98 μg / mL to 3.96 μg / mL, 2.2 μg / mL to 4.18 μg / mL, or 2.2 μg / mL to 3.96 μg / mL Serotype 6B may be present in an amount greater than 2.2 μg / mL, 8.36 μg / mL, 2.64 μg / mL to 8.36 μg / mL, 2.64 μg / mL to 7.92 μg / mL, 3.52 μg / mL to 8.36 μg / mL, 3.52 Μg / mL to 7.92 μg / mL, 3.96 μg / mL to 8.36 μg / mL, 3.96 μg / mL to 7.92 μg / mL, 4.4 μg / mL to 8.36 μg / mL, or 4.4 μg / mL to 7.92 μg / mL May be present in concentration. However, the present invention is not limited thereto, and the concentration may be adjusted according to the purpose to be prepared as long as it is within the above-described content ratio.
상기 조성물은 추가적으로 혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류가 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 포함되는 것일 수 있다.The composition may additionally include the capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F in the same amount as the capsular polysaccharide derived from serotype 1.
본 발명에서 용어 “폐렴”은 폐실질의 급성 염증질환의 일종으로 감염원은 주로 스트렙토코커스 뉴모니아 와 크렙실라 뉴모니아 (Klebsiella pneumoniae)이다. 특히 폐렴구균성 폐렴은 모든 폐렴의 약 50 %를 차지하며, 심한 오한, 발열, 기침 및 흉통이 나타나고 객담은 혈담인 경우가 많으며 합병증으로 흉막염, 뇌막염, 심내막염, 복막염 등을 일으킬 수 있는 질환이다 (Stein GE et al.2001; Diagn. Microbiol. Infect, Dis 39:181; Comparative serum bactericidal activity of clarithromycin and azithromycin against macrolide-sensitive and resistant strains of Streptococcus pneumoniae).In the present invention, the term "pneumonia" is a kind of acute inflammatory disease of the lung parenchyma, and the infectious agents are mainly Streptococcus pneumoniae and Klebsiella pneumoniae . In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, severe chills, fever, cough and chest pain, sputum is often bloody, complications that can cause pleurisy, meningitis, endocarditis, peritonitis ( Stein GE et al. 2001; Diagn. Microbiol.Infect, Dis 39: 181; Comparative serum bactericidal activity of clarithromycin and azithromycin against macrolide-sensitive and resistant strains of Streptococcus pneumoniae ).
본 발명에서 용어 “폐렴구균 (pneumococcus)”은 스트렙토코커스 뉴모니애 를 지칭하며 일반적으로 인간 비인강 (nasopharynx)의 점막 표면을 콜로니화하는 편리공생 (commensal) 생물체이다. 숙주의 인자 (factor)가 생물체의 하기도 (lower respiratory tract)로 접근을 허용하는 경우, 왕성한 염증 반응이 뒤이어 일어나고, 이로써 폐포 공간이 삼출물 (exudate)를 채울 때 밀집한 경화 (consolidation)를 야기시켜, 폐렴을 유발할 수 있다. 상기 폐렴구균은 90 개 이상의 구조적으로 독특한 협막 다당류 를 합성할 수 있고, 이러한 협막 다당류의 구조적, 면역학적 특성에 따라 폐렴구균의 혈청형 (serotype)이 분류된다. 이에, 폐렴구균의 협막 다당류를 이용하여 백신용 제제를 제조할 경우 협막 다당류의 종류, 즉 협막 다당류가 유래된 폐렴구균의 혈청형에 따라 면역 반응이 다르게 나타날 수 있다. 본 발명의 백신 조성물은 특히 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류를 이용하여 제조될 수 있다. 또한, 추가적으로 혈청형 22F 및 33F 유래의 협막 다당류를 포함하여 17 가 형태로 제조될 수 있다.The term “pneumococcus” in the present invention refers to Streptococcus pneumoniae and is generally a commensal organism that colonizes the mucosal surface of the human nasopharynx. If the host's factor allows access to the lower respiratory tract of the organism, then a vigorous inflammatory response follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia May cause. The pneumococci can synthesize more than 90 structurally unique capsular polysaccharides, and the serotypes of pneumococci are classified according to the structural and immunological characteristics of these capsular polysaccharides. Thus, when a vaccine preparation is prepared using pneumococcal capsular polysaccharide, the immune response may be different depending on the type of capsular polysaccharide, that is, the serotype of pneumococcal from which capsular polysaccharide is derived. The vaccine composition of the present invention specifically contains 15 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F and 23F. It can be prepared using. In addition, 17-valent forms can be prepared, including capsular polysaccharides derived from serotypes 22F and 33F.
상기 협막 다당류는 체내에 투여되었을 때 항원으로 인식되어 이에 대한 항체를 생산할 수 있도록 하므로, 이를 이용하여 폐렴구균의 예방용 백신 조성물을 제조할 수 있다. 본 발명에서 용어 “항원”이란 물질이 체내에 침입한 경우 면역응답을 특이적으로 유발할 수 있는 물질을 말한다. 본 발명에서는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류, 혹은 여기에 추가적으로 22F 및 33F 유래 협막 다당류가 포함될 경우 17 종의 협막 다당류가 각각 항원으로 작용할 수 있다.The capsular polysaccharide is recognized as an antigen when administered in the body, so that it can produce an antibody against it, thereby preparing a vaccine composition for preventing pneumococci. As used herein, the term “antigen” refers to a substance that can specifically induce an immune response when the substance invades the body. In the present invention, 15 kinds of capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, and 23F, or In addition, when the 22F- and 33F-derived capsular polysaccharides are included, 17 capsular polysaccharides may act as antigens, respectively.
상기 협막 다당류는 당업자에게 공지된 표준 기술에 의해서 제조될 수 있으며, 그 방법에 특별히 제한되지 않는다. 상기 협막 다당류는 점도를 감소시키고 효과적인 면역원성을 유도하기 위해서 가수분해를 통해 그 크기를 줄일 수 있다. The capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and are not particularly limited in its method. The capsular polysaccharide can be reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.
본 발명의 구체적인 일 실시예에서는 17 종의 각기 다른 혈청형 (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F)을 가지는 스트렙토코커스 뉴모니애를 각각 소듐 데옥시콜레이트 (sodium deoxycholate)를 이용하여 용해시키고, 세포에 결합된 다당류를 유리시켰다. 그 다음 14 개의 혈청형 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F 및 23F의 경우 CTAB (cetyltrimethylammonium bromide)와 이온 결합이 가능하므로 CTAB 공정을 수행하여 정제하였고, CTAB와 반응하지 않는 3 개의 혈청형 7F,14 및 33F는 인산알루미늄겔 (Algel) 용액을 이용하여 정제하였다.In one specific embodiment of the present invention, 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) Streptococcus pneumoniae with lysates were dissolved using sodium deoxycholate, respectively, and the polysaccharides bound to the cells were released. Fourteen serotypes 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F, and 23F are capable of ion bonding with CTAB (cetyltrimethylammonium bromide) to perform CTAB process 3 serotypes 7F, 14 and 33F that do not react with CTAB were purified using an aluminum phosphate gel (Algel) solution.
상기 협막 다당류만을 이용하여 백신 조성물로 사용하는 경우 면역체계가 성인 보다 떨어지는 유아 및 소아는 이를 항원으로 인식하지 못하여 면역반응이 일어나지 않을 수 있으므로, 본 발명에서는 운반 단백질과 협막 다당류를 결합시킨 접합체 형태로 제조하여 사용하였다.In the case of using the capsular polysaccharide as a vaccine composition, infants and children whose immune system is lower than adults may not recognize this as an antigen, and thus an immune response may not occur. Thus, in the present invention, the conjugate protein and the capsular polysaccharide are conjugated. It was prepared and used.
본 발명에서 용어 “운반 단백질 (carrier protein)”은 협막 다당류와 공유결합적으로 접합되어 다당류 항원의 면역원성을 증가시켜줄 수 있는 단백질을 의미한다. 상기 운반 단백질은 표준 접합 방법을 통해 협막 다당류와 접합될 수 있으며, 이를 통해 형성된 협막 다당류-운반 단백질 접합체는 하나 또는 다수의 협막 다당류가 하나의 운반 단백질에 접합된 것일 수 있다. 상기 운반 단백질은 구체적으로 무독성이고 비반응원성이며, 충분한 양 및 순도로 수득할 수 있는 단백질일 수 있으나, 이에 제한되는 것은 아니다. 본 발명에서 상기 운반 단백질은 이에 제한되는 것은 아니나, 예를 들어 CRM197일 수 있다.As used herein, the term "carrier protein" refers to a protein that can be covalently conjugated with the capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen. The carrier protein may be conjugated with the capsular polysaccharide through a standard conjugation method, and the capsular polysaccharide-carrying protein conjugate formed therefrom may be one or a plurality of capsular polysaccharides conjugated to one carrier protein. The carrier protein may be, but is not limited to, a protein that is specifically nontoxic, nonreactive, and obtainable in sufficient amount and purity. The carrier protein in the present invention is not limited thereto, but may be, for example, CRM197.
본 발명에서 용어 “CRM197”은 코리네박테리움 디프테리아 (Corynebacterium diphtheriae) 균주 C7 (β197)의 배양물로부터 분리된 디프테리아 독소의 무독성 변이체 (즉, 톡소이드)이다. CRM197은 한외여과, 암모늄 설페이트 침전 및 이온 교환 크로마토그래피를 통해서 정제될 수 있다. 또한, 이에 제한되는 것은 아니나 상기 CRM197은 미국특허 제5,614,382호에 따라 재조합적으로 제조될 수 있다.The term “CRM197” in the present invention is a non-toxic variant of diphtheria toxin (ie, toxoid) isolated from the culture of Corynebacterium diphtheriae strain C7 (β197). CRM197 can be purified via ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography. In addition, the CRM197 may be recombinantly prepared according to US Pat. No. 5,614,382.
상기 운반 단백질은 또한, 파상풍톡소이드, 백일해 톡소이드, 콜레라 톡소이드 (제WO2004/083251호), 대장균 (E. coli) LT, 대장균 ST 또는 슈도모나스 애루지노사 (Pseudomonas aeruginosa) 유래의 외독소 A와 같은 불활성화된 세균 독소, 세균 외막 단백질, 예를 들면, 외막복합체 c (OMPC), 포린, 트랜스페린 결합 단백질, 뉴모리신, 폐렴구균 표면 단백질 A (PspA), 폐렴구균 어드헤신 (adhesin) 단백질 (PsaA), 그룹 A 또는 그룹 B 연쇄구균 유래의 C5a 펩티다제, 또는 헤모필러스 인플루엔자(Haemophilus influenzae) 단백질 D, 오브알부민, 키홀 림펫 헤모시아닌 (KLH), 소 혈청 알부민 (BSA), 투베르쿨린의 정제된 단백질 유도체 (PPD), CRM173, CRM228, CRM45와 같은 디프테리아 독소의 변이체일 수 있으나, 이에 제한되는 것은 아니다.The carrier protein is also inactivated, such as tetanus toxoid, pertussis toxoid, cholera toxoid (WO2004 / 083251), E. coli LT, E. coli ST or Pseudomonas aeruginosa . Bacterial toxins, bacterial outer membrane proteins such as outer complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group C5a peptidase from A or Group B streptococci, or purified protein derivatives of Haemophilus influenzae protein D, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tuberculin (PPD), may be a variant of diphtheria toxins such as CRM173, CRM228, CRM45, but is not limited thereto.
협막 다당류 및 운반 단백질의 접합체를 제조하기 위한 공지의 방법은 모두 본 발명의 범위에 포함될 수 있다. 구체적인 일 예로서, 본 발명자들은 폐렴구균 백신의 면역원성을 증진시키기 위한 방법을 찾기 위해 문헌조사와 함께 초기연구로 대표적인 두 가지 접합방식인 환원적 아민화 (reductive amination) 반응과 시아닐화 (cyanylation) 반응을 비교하였으며 이 결과 시아닐화 방법이 접합수율 및 소요 시간 측면에서 환원적 아민화 반응에 비해 월등히 우수하다는 사실을 확인하였다. 이를 바탕으로, 현재 가장 커버리지가 높은 프리베나 13® 와 동일한 13 개의 혈청형을 사용하여 프리베나 13®와는 다른 시아닐화 반응을 통한 혈청형별 최적화된 접합을 시도해본 결과, 13 개의 혈청형에서 표준품인 프리베나 13® 대비 모든 혈청형의 IgG 역가가 매우 우수하다는 사실을 확인하였다. 이에, 본 발명의 면역원성 조성물 제조 시 시아닐화 반응을 이용한 결합방식을 사용할 수 있다. 다가 면역원성 조성물에서 면역성의 방해현상이 있을 수 있기 때문에, 사용된 모든 혈청형에서 동시에 면역원성을 증가시키는 것은 당업자에게 많은 노력을 필요로 하는 것이다.All known methods for preparing conjugates of capsular polysaccharides and transport proteins can be included within the scope of the present invention. As a specific example, the present inventors conducted an initial study along with a literature search to find a method for enhancing the immunogenicity of pneumococcal vaccines. Reductive amination and cyanylation are two representative methods of conjugation. The reactions were compared and the results showed that the cyanation method was much better than the reductive amination reaction in terms of bonding yield and time required. Based on this, 13 serotypes that are identical to those of Pribena 13 ® , which is currently the highest coverage, have been tested for serotype-optimized conjugation using cyanation reactions different from Pribena 13 ® , and the standard of 13 serotypes It was confirmed that IgG titers of all serotypes were very good compared to Prevena 13 ® . Thus, in the preparation of the immunogenic composition of the present invention can be used a binding method using a cyanation reaction. Since there may be immunological disturbances in multivalent immunogenic compositions, increasing immunogenicity simultaneously in all serotypes used requires a lot of effort for the skilled person.
구체적으로, 이에 제한되는 것은 아니나 본 발명에서 상기 접합체는 시아닐화 방법을 사용하여 협막 다당류 및 운반 단백질이 -O-C(NH)-NH- 기로 연결된 구조를 가지는 것일 수 있다. 다른 방법으로 제조된 PCV (Pneumococcal conjugate vaccine) 제제와의 구조적 차이점 및 세부적인 접합 실시의 최적화를 바탕으로, 본 발명의 백신 조성물은 기존에 공지된 환원적 아미노화에 의해 접합된 백신 조성물에 비하여 모든 혈청형에 있어서 현저히 우수한 IgG 역가를 보일 수 있다.Specifically, the present invention is not limited thereto, but in the present invention, the conjugate may have a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanylation method. Based on the structural differences from the Pneumococcal conjugate vaccine (PCV) formulations prepared by other methods and optimization of detailed conjugation practices, the vaccine compositions of the present invention are all compared to vaccine compositions conjugated by known reductive amination. The serotype may show significantly better IgG titers.
상기 시아닐화 방법은 공지된 방법을 통해 당업자가 적절히 수행할 수 있으며, 예를 들어 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) 또는 CNBr을 사용하여 수행될 수 있으나, 이에 제한되는 것은 아니다.The cyanation method may be appropriately performed by those skilled in the art through a known method, for example, may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.
협막 다당류 및 운반 단백질의 접합체를 제조하기 위한 일례로, 정제된 협막 다당류를 화학적으로 활성화시키고, 화학적으로 활성화된 각 협막 다당류를 운반 단백질에 하나씩 접합시켜서 당접합체 (glycoconjugate)를 형성할 수 있다. CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) 처리에 의한 시아닐레이션에 의한 활성은 협막 다당류의 히드록실 그룹을 사이아네이트 그룹으로 변화시키고, 이를 이용하여 운반체 단백질인 CRM197의 아미노 구룹과 공유결합을 형성하게 할 수 있다. 상기 CDAP에 의한 시아닐레이션 반응은 구체적으로 CDAP 1 몰 당량 대비 3 몰 당량의 글리신 (glycine) 용액을 첨가하고 pH를 9.0으로 조정하여 종결하는 것일 수 있으나, 이에 제한되는 것은 아니며 당업자는 그 목적에 따라 반응 용액 및 반응 조건을 적절히 조절할 수 있다.As an example for preparing conjugates of capsular polysaccharides and carrier proteins, purified capsular polysaccharides can be chemically activated and each chemically activated capsular polysaccharide can be conjugated to the carrier protein one by one to form a glycoconjugate. Cyanation activity by treatment with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) converts the hydroxyl group of the capsular polysaccharide to a cyanate group, thereby covalently binding to the amino group of the carrier protein CRM197. Can form. The cyanation reaction by the CDAP may be specifically terminated by adding 3 molar equivalents of glycine (glycine) solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto. The reaction solution and reaction conditions can be adjusted accordingly.
수득한 협막 다당류-운반 단백질 접합체는 다양한 방법에 의해 정제할 수 있다. 이들 방법의 예는 농축/투석 여과 공정, 칼럼 크로마토그래피 및 다층 여과를 포함한다. 정제된 다당류-단백질 접합체들은 각각을 혼합하여 본 발명의 백신 조성물로 제제화하고, 이를 사용할 수 있다. 당업계에서 인정된 방법을 사용하여 본 발명의 백신 조성물의 제제화를 수행할 수 있다. 예를 들면, 개개의 협막 다당류-운반 단백질 접합체를 생리학적으로 허용되는 비히클과 함께 제형화하여 조성물을 제조할 수 있다. 이러한 비히클의 예에는, 물, 완충 식염수, 폴리올 (예: 글리세롤, 프로필렌 글리콜, 액체 폴리에틸렌 글리콜) 또는 덱스트로스 용액일 수 있으나, 이에 제한되는 것은 아니다.The capsular polysaccharide-carrying protein conjugates obtained can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed and formulated into vaccine compositions of the invention and used respectively. Formulations of vaccine compositions of the invention can be carried out using methods known in the art. For example, compositions may be prepared by formulating individual capsular polysaccharide-carrying protein conjugates with a physiologically acceptable vehicle. Examples of such vehicles may include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.
본 발명의 구체적인 일 실시예에서는, 1) 각각의 협막 다당류의 용해 및 가수분해, 2) CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 이용한 각각의 협막 다당류와 CRM197의 접합 반응 공정, 3) 접합 반응 종결, 4) 한외 여과, 5) 제균 여과 및 6) 흡착 단계를 거쳐 각각의 협막 다당류-운반 단백질 접합체를 제조하였다.In one specific embodiment of the present invention, 1) dissolution and hydrolysis of each capsular polysaccharide, 2) conjugation reaction process of each capsular polysaccharide and CRM197 using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate), 3) conjugation Each capsular polysaccharide-carrying protein conjugate was prepared through reaction termination, 4) ultrafiltration, 5) bactericidal filtration, and 6) adsorption steps.
본 발명에서 용어 “백신”은 생체에 면역을 주는 항원을 함유한 생물학적인 제제로서, 감염증의 예방을 위하여 사람이나 동물에 투여함으로써 생체에 면역이 생기게 하는 면역원 또는 항원성 물질을 말한다.As used herein, the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by administering it to a human or an animal to prevent infection.
상기 백신 조성물은, 애쥬번트, 보존제, 완충제, 냉동보호제, 염, 2 가 양이온, 비이온성 세제 및 자유 라디칼 산화 억제제로 이루어진 군에서 선택된 하나 이상을 더 포함하는 것일 수 있다.The vaccine composition may further include one or more selected from the group consisting of an adjuvant, a preservative, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and a free radical oxidation inhibitor.
본 발명에서 용어 "애쥬번트"는 본 발명의 면역원성 조성물의 면역원성을 증가시키는데 사용되는 물질을 말한다. 상기 애쥬번트는 종종 면역 반응을 증진시키기 위하여 제공되며, 이는 당업자에게 잘 알려져 있다. 본 발명의 백신 조성물의 유효성을 증가시키기에 적당한 애쥬번트는 다음을 포함하나, 이에 제한되는 것은 아니다: The term "adjuvant" in the present invention refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. The adjuvant is often provided to enhance the immune response, which is well known to those skilled in the art. Adjuvants suitable for increasing the effectiveness of the vaccine composition of the present invention include, but are not limited to:
(1) 알루미늄 염 (명반) (예: 알루미늄 하이드록사이드, 알루미늄 포스페이트, 알루미늄 설페이트 등);(1) aluminum salts (alum) (eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);
(2) 수중유형 에멀젼 제형 (무라밀 펩티드 (아래에서 정의됨) 또는 세균 세포벽 성분과 같은 다른 특정한 면역 자극제를 함유하거나 함유하지 않음), 예를 들면 (a) MF59 (제WO90/14837호): 5 % 스쿠알렌 (Squalene), 0.5 % 트윈 (Tween) 80 및 0.5 % 스판 (Span) 85를 함유하며 (임의로 다양한 양의 MTP-PE (필요하지는 않지만, 아래를 참조)를 함유함), Model 110Y 마이크로플루이다이저 (microfluidizer, Microfluidics, Newton, MA)와 같은 마이크로플루이다이저를 사용하여 서브마이크론 입자로 제형화됨, (b) SAF: 10 % 스쿠알렌, 0.4 % 트윈 80, 5 % 플루로닉 (pluronic)-블럭 중합체 L121 및 thr-MDP (아래를 참조)를 함유하며, 서브마이크론 에멀젼으로 미세유동화 (microfluidization)되거나, 와동시켜 큰 입자 크기의 에멀젼을 형성시킴, 및 (c) 리비 (Ribi)™ 애쥬번트 시스템 (RAS) (Corixa, Hamilton, MT): 2 % 스쿠알렌, 0.2 % 트윈 80 및, 미국특허 제4,912,094호에 기술된 3-O-탈아실화된 모노포스포릴 리피드 A (MPL™)(Corixa), 트레할로스 디미콜레이트 (TDM) 및 세포벽 골격 (CWS)으로 이루어진 그룹으로부터의 하나 이상의 세균 세포벽 성분, 바람직하게는 MPL + CWS (디톡스 (Detox)™)를 함유함;(2) Oil-in-water emulsion formulations (with or without muramyl peptide (defined below) or other specific immune stimulants such as bacterial cell wall components), for example (a) MF59 (WO 90/14837): Contains 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally contains varying amounts of MTP-PE (although not required, see below)), Model 110Y Micro Formulated into submicron particles using a microfluidizer such as a microfluidizer (Microfluidics, Newton, Mass.), (B) SAF: 10% squalene, 0.4% Tween 80, 5% Pluronic -Block polymer L121 and thr-MDP (see below), microfluidized with a submicron emulsion, or vortexed to form a large particle size emulsion, and (c) Ribi ™ adjuvant System (RAS) (Corixa, Hamilton, MT): 2% squalene, 0. 2% Tween 80 and from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL ™) (Corixa), trehalose dimicholate (TDM) and cell wall backbone (CWS) described in US Pat. No. 4,912,094. Contains at least one bacterial cell wall component, preferably MPL + CWS (Detox ™);
(3) 퀼 에이 (Quil A) 또는 스티뮬론 (STIMULON)™ QS-21 (Antigenics, Framingham, MA, 미국특허 제5,057,540호)과 같은 사포닌 애쥬번트가 사용되거나 이로부터 생성된 입자 (예: ISCOM (면역자극 복합체));(3) Saponin adjuvant, such as Quill A or STIMULON ™ QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540), may be used or produced from particles such as ISCOM ( Immunostimulatory complexes));
(4) 세균 지질다당류, 합성 리피드 A 동족체 (예: 아미노알킬 글루코스아민 포스페이트 화합물 (AGP)), 또는 이의 유도체 또는 동족체 (이는 Corixa로부터 구입할 수 있고, 미국특허 제6,113,918호에 기술되어 있음; 상기 AGP의 일 예는 2-[(R)-3-테트라데카노일옥시테트라데카노일아미노]에틸 2-데옥시-4-O-포스포노-3-O-[(R)-3-테트라데카노일옥시테트라데카노일]-2-[(R)-3-테트라데카노일옥시테트라데카노일아미노]-b-D-글루코피라노시드이고, 이는 또한 529로도 알려져 있으며 (이전에는 RC529로도 알려짐), 이는 수성형 또는 안정한 에멀젼으로서 제형화됨),(4) bacterial lipopolysaccharides, synthetic lipid A homologues (eg, aminoalkyl glucoseamine phosphate compounds (AGP)), or derivatives or homologues thereof (commercially available from Corixa and described in US Pat. No. 6,113,918; An example of is 2-[(R) -3-tetradecanoyloxytetradecanoylamino] ethyl 2-deoxy-4-O-phosphono-3-O-[(R) -3-tetradecanoyloxy Tetradecanoyl] -2-[(R) -3-tetradecanoyloxytetradecanoylamino] -bD-glucopyranoside, which is also known as 529 (formerly also known as RC529), which is aqueous or Formulated as a stable emulsion),
(5) 합성 폴리뉴클레오타이드 (예: CpG 모티프를 함유하는 올리고뉴클레오타이드 (미국특허 제6,207,646호));(5) synthetic polynucleotides (eg oligonucleotides containing CpG motifs (US Pat. No. 6,207,646));
(6) 사이토카인, 예를 들어, 인터루킨 (예: IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18 등), 인터페론 (예: 감마 인터페론), 과립구 대식세포 콜로니 자극 인자 (GM-CSF), 대식세포 콜로니 자극 인자 (MCSF), 종양 괴사 인자 (TNF), 공동자극 분자 B7-1 및 B7-2 등;(6) cytokines, such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.) Interferon (eg gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and the like;
(7) 야생형 콜레라 독소 (CT) 또는, 예를 들면, 제WO2000/18434호에 따라 아미노산 29 번 위치에 있는 글루탐산이 다른 아미노산, 구체적으로는 히스티딘으로 치환된, 돌연변이형 콜레라 독소 (제WO2002/098368호 및 제WO2002/098369호), 백일해 독소 (PT), 또는 대장균 열-불안정성 독소 (LT), 특히 LT-K63, LT-R72, CT-S109, PTK9/G129 (제WO93/13302호 및 제WO92/19265호)와 같은 세균 ADP-리보실화 독소의 무독화된 돌연변이체; 및 (7) Wild type cholera toxin (CT) or mutant cholera toxin (eg WO2002 / 098368) wherein glutamic acid at amino acid position 29 according to WO2000 / 18434 is substituted with another amino acid, specifically histidine. And WO2002 / 098369), pertussis toxin (PT), or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PTK9 / G129 (WO93 / 13302 and WO92) / 19265), detoxified mutants of bacterial ADP-ribosylated toxins; And
(8) Complement component C3d의 trimer와 같은 보체.(8) Complement component C3d, the same complement as the trimer.
상기 무라밀 펩티드에는 N-아세틸-무라밀-L-트레오닐-D-이소글루타민 (thr-MDP), N-아세틸-노르무라밀-L-알라닌-2-(1'-2' 디팔미토일-sn-글리세로-3-하이드록시포스포릴옥시)-에틸아민 (MTP-PE) 등이 포함될 수 있으나, 이에 제한되는 것은 아니다.The muramyl peptides include N-acetyl-muramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1'-2 'dipalmityl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like, but is not limited thereto.
상기 알루미늄 염 애쥬번트는 알루미늄-침전 백신 (alumprecipitated vaccine)이거나 알루미늄-흡착 백신 (alum-adsorbed vaccine)일 수 있다. 알루미늄 염에는 수화된 알루미나, 알루미나 수화물, 알루미나 3수화물 (ATH), 알루미늄 수화물, 알루미늄 3수화물, 알하이드로겔, Superfos, 암포젤, 수산화알루미늄, 알루미늄 히드록시포스페이트 설페이트 (Aluminum Phosphate Adjuvant (APA)), 무정형 알루미나 등이 포함될 수 있으나, 이에 제한되는 것은 아니다. APA는 알루미늄 히드록시포스 페이트의 현탁액을 말한다. 염화알루미늄과 인산나트륨을 1 : 1의 비율로 혼합하면 알루미늄 히드록시포스페이트 설페이트가 침전되며, High shear mixer를 이용하여 침전물의 크기가 2 ~ 8 ㎛이 되도록 한 다음 생리식염수로 투석하고 멸균하여 제조할 수 있다. 일 구현예로서, 상업적으로 이용가능한 Al(OH)3 (예를 들어 알하이드로겔 또는 Superfos)를 사용하여 단백질을 흡착한다. 수산화알루미늄 1 mg당 50 ~ 200 g의 단백질이 흡착될 수 있으며 이 비율은 단백질의 pI와 용매의 pH에 의존적이다. 낮은 pI의 단백질은 높은 pI를 가진 단백질에 비해 강하게 결합한다. 알루미늄 염은 2 ~ 3 주간 서서히 항원을 방출하는 항원 저장소를 형성하여 비특이적으로 대식세포, 보체, 선천성 면역 메커니즘을 활성화시킬 수 있다.The aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine. Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide, aluminum hydroxyphosphate adjuvant (APA), Amorphous alumina, and the like, but is not limited thereto. APA refers to a suspension of aluminum hydroxyphosphate. When aluminum chloride and sodium phosphate are mixed in a ratio of 1: 1, aluminum hydroxyphosphate sulfate is precipitated, and the precipitate is made to be 2 to 8 μm using a high shear mixer, and then prepared by dialysis and sterilization with physiological saline solution. Can be. In one embodiment, commercially available Al (OH) 3 (eg Alhydrogel or Superfos) is used to adsorb the protein. 50 to 200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the pH of the protein and the pH of the solvent. Low pI proteins bind more strongly than proteins with high pi. Aluminum salts can form antigen reservoirs that slowly release antigens for two to three weeks to nonspecifically activate macrophages, complement, and innate immune mechanisms.
본 발명에서 용어 “보존제”는 상기 백신 조성물 내에서 미생물의 증식을 억제하는 항-바이러스 및/또는 항균 작용을 하는 물질을 의미하며, 예를 들어 치메로살 (thimerosal), 페녹시에탄올 (2-phenoxyethanol), 포름알데히드 (formaldehyde), 또는 이들의 혼합물일 수 있으나 이에 제한되지 않고 당업계에서 사용되는 모든 통상적인 보존제가 사용될 수 있다.As used herein, the term “preservative” means an anti-viral and / or antimicrobial agent that inhibits the growth of microorganisms in the vaccine composition, for example, chimerosal, phenoxyethanol, 2-phenoxyethanol. It may be, but is not limited to, formaldehyde, or mixtures thereof, all conventional preservatives used in the art may be used.
또한, 상기 백신 조성물은 한 가지 이상의, 생리학적으로 허용되는 완충제를 포함할 수 있다. 예를 들면, 상기 백신 조성물이 주입제 (infusion)나 주사제일 경우 상기 완충제는 pH 4.0 내지 10.0, 구체적으로, pH 5.0 내지 9.0, 더욱 구체적으로 pH 6.0 내지 8.0에서 완충능을 가지는 것일 수 있다. 상기 완충제는 TRIS, 아세테이트, 글루타메이트, 락테이트, 말리에이트, 타트레이트, 포스페이트, 시트레이트, 카보네이트, 글리시네이트, 히스티딘, 글리신, 석시네이트, 트리에탄올아민 완충제로 구성된 군에서 선택되는 것일 수 있다.In addition, the vaccine composition may comprise one or more physiologically acceptable buffers. For example, when the vaccine composition is an infusion or injectable, the buffer may have buffering capacity at pH 4.0 to 10.0, specifically, pH 5.0 to 9.0, more specifically pH 6.0 to 8.0. The buffer may be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanolamine buffer.
특히 본 발명의 백신 조성물이 비경구 투여를 목적으로 할 경우, 완충제는 USP에 적합한 완충제 중에서 선택할 수 있다. 예를 들면, 완충제는 아세트산, 벤조산, 글루콘산, 글리세르산, 젖산과 같은 일염기산; 아코니트산, 아디프산 (adipic), 아스코르빈산, 탄산 (carbonic), 글루타민산, 말산, 석신산, 주석산과 같은 이염기산; 시트르산, 인산과 같은 다염기산; 암모니아, 다이에탄올아민, 글리신, 트리에탄올아민, TRIS와 같은 염기로 구성된 군에서 선택되는 것일 수 있다.In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffer may be selected from buffers suitable for USP. For example, buffers include monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid; Dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid and phosphoric acid; It may be selected from the group consisting of ammonia, diethanolamine, glycine, triethanolamine, TRIS and the like.
또한, 본 발명의 백신 조성물은 비이온성 세제를 포함할 수 있다. 예를 들어, 폴리옥시에틸렌 소르비탄 에스테르 (보통 Tweens이라고 불리는) 중 특히 폴리소르베이트 20과 폴리소르베이트 80; 에틸렌 옥시드 (EO), 프로필렌 옥시드 (PO), 부틸렌 옥시드 (BO)의 공중합체 (예: DOWFAXTM); 에톡시 (oxy-1,2-ethanediyl) 그룹의 반복 수가 서로 다른 옥스톡시놀류, 특히 오스톡시놀-9 (Triton-100); 에틸페녹시폴리에톡시에탄올 (IGEPAL CA-630/NP-40); 레시틴과 같은 인지질; NP 시리즈와 같은 노닐페놀 에톡시레이트; 라우릴, 세틸, 스테아릴, 올레일 알코올에서 유도된 폴리옥시에틸렌 지방산 에테르 (Brij 계면활성제), 특히 트리에틸렌글리콜 모노라우릴 에테르 (Brij 30); SPANs으로 알려진 소르비탄 에테르, 특히 소르비탄 트리올레이트(Span 85)와 소르비탄 모노라우레이트와 같은 계면활성제를 포함할 수 있으나, 이에 제한되는 것은 아니다. Tween 80은 에멀젼에 포함될 수 있으며, Tween 80/Span 85와 같은 비이온성 세제의 혼합물도 사용할 수 있다. Tween 80과 같은 폴리옥시에틸렌 소르비탄 에스테르와 Triton X-100과 같은 옥토시놀의 조합도 적합하며, Laureth 9과 Tween, 그리고 혹은 옥토시놀의 조합도 유용하다. 구체적으로는 폴리옥시에틸렌 소르비탄 에스테르 (예: Tween 80)를 0.01 % 내지 1 % (w/v), 특히 0.1 %; 옥틸페녹시 폴리옥시에탄올 또는 노닐페녹시 폴리옥시에탄올 (예: Triton X-100)은 0.001 % 내지 0.1 %, 특히 0.005 % 내지 0.02 %; 폴리옥시에틸렌 에테르 (예: laureth 9)는 0.1 % 내지 20 %, 가급적 0.1 % 내지 10 %, 특히 0.1 % 내지 1 % 또는 약 0.5 %를 포함할 수 있다. In addition, the vaccine composition of the present invention may include a nonionic detergent. For example, polysorbate 20 and polysorbate 80 in polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAX ™); Oxoxynols having a different repeating number of oxy-1,2-ethanediyl groups, especially ostoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethyleneglycol monolauryl ether (Brij 30); Sorbitan ethers, also known as SPANs, may include, but are not limited to, surfactants such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Tween 80 may be included in the emulsion and a mixture of nonionic detergents such as Tween 80 / Span 85 may be used. Combinations of polyoxyethylene sorbitan esters such as Tween 80 with octocinols such as Triton X-100 are also suitable, and combinations of Laureth 9 with Tween and or octosinol are also useful. Specifically, polyoxyethylene sorbitan esters such as Tween 80 may be used in an amount of 0.01% to 1% (w / v), in particular 0.1%; Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (such as Triton X-100) may range from 0.001% to 0.1%, in particular from 0.005% to 0.02%; The polyoxyethylene ether (eg laureth 9) may comprise 0.1% to 20%, preferably 0.1% to 10%, in particular 0.1% to 1% or about 0.5%.
본 발명의 조성물은 단회 투여 용량 바이얼, 다회 투여 용량 바이얼 또는 프리필드시린지 형태로 제제화할 수 있고, 생리학적으로 허용되는 담체를 더 포함하는 것일 수 있다. 액상 제제에 사용되는 생리학적으로 허용되는 담체에는 수성 또는 비수성 용매, 현탁액, 에멀젼, 오일이 있다. 비수성 용매의 예로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 에틸 올레이트가 있다. 수성 담체는 물, 알코올/수성 용매, 에멀젼 또는 현탁액, 생리식염수, 버퍼 용액을 포함한다. 오일의 예로는 식물성 또는 동물성오일, 피넛 오일, 대두유, 올리브 오일, 해바라기 오일, 간유, 수산유지 (marine oil)와 같은 합성 오일, 우유나 달걀에서 얻은 지질이 있다. 본 발명의 백신 조성물은 등장성, 고장성 또는 저장성일 수 있고, infusion이나 주사로 투여되는 약제학적 조성물은 기본적으로 등장성이 바람직하나 이에 제한되는 것은 아니다. 한편, 등장성이나 고장성이 조성물의 저장에 유리할 수 있다. 상기 백신 조성물이 고장성일 경우, 투여 전에 등장성이 되도록 희석할 수 있다. 희석을 위한 등장화제는 염과 같은 이온성 등장화제이거나 탄수화물과 같은 비이온성 등장화제일 수 있다. 이온성 등장화제에는 염화나트륨, 염화칼슘, 염화칼륨, 염화마그네슘 등이 포함되나, 이에 제한되는 것은 아니다. 비이온성 등장화제에는 솔비톨, 글리세롤 등이 포함되나, 이에 제한되는 것은 아니다. The composition of the present invention may be formulated in a single dose dose vial, multiple dose dose vial or prefilled syringe form, and may further comprise a physiologically acceptable carrier. Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions. Examples of oils include vegetable or animal oils, peanut oils, soybean oils, olive oils, sunflower oils, synthetic oils such as liver oils, marine oils, and lipids derived from milk or eggs. Vaccine compositions of the present invention may be isotonic, hypertonic or hypotonic, and pharmaceutical compositions administered by infusion or injection are preferably but is not limited to isotonicity. On the other hand, isotonicity or hypertension may be advantageous for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution can be ionic tonicity agents such as salts or nonionic tonicity agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.
상기 백신 조성물은 알루미늄 원소 및 염화나트륨을 더 포함하는 것일 수 있으나 이에 제한되지 않는다. 또한, 상기 백신 조성물은 그 목적 및 용도에 따라 보존제를 포함하거나, 또는 포함하지 않을 수 있다. The vaccine composition may further include aluminum element and sodium chloride, but is not limited thereto. In addition, the vaccine composition may or may not contain a preservative depending on the purpose and use thereof.
본 발명에 따른 백신 조성물은, 전신 또는 점막 경로로 약학적으로 유효한 양을 투여함으로써, 폐렴구균에 감염되기 쉬운 개체를 보호하고 폐렴구균병을 예방하는데 사용될 수 있다. 본 발명의 용어 “예방”은 본 발명의 백신 조성물의 투여로 상기 폐렴구균에 의한 감염을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명에서 정의되는 "약학적으로 유효한 양"이란 폐렴구균에 감염될 확률 또는 감염의 심각성을 상당히 감소시킬 수 있을 정도의 항체를 유발하는데 필요한 투여량을 말한다. 본 발명의 용어 “투여”는 어떠한 적절한 방법으로 개체에 소정의 물질을 도입하는 것을 말한다. 본 발명의 백신 조성물은 경구, 비강, 직장, 경피 또는 에어로졸을 통한 흡입 경로로 투여될 수 있으며, 볼루스로 투여하거나 또는 서서히 주입할 수도 있으나, 이에 제한되는 것은 아니다. 상기 투여는 근육내, 복강내, 피내 또는 피하 경로를 통한 주사; 또는 구강/소화관, 기도관 또는 비뇨생식관으로의 점막 투여가 포함될 수 있다. 일 구현예로서, 폐렴 또는 중이염의 치료를 위하여 비내 투여가 사용될 수 있으며, 이 경우 폐렴구균의 비인강 보균을 보다 효과적으로 예방하여, 초기 단계에서 감염을 약화시킬 수 있다. The vaccine composition according to the present invention can be used to protect a subject susceptible to pneumococcal and to prevent pneumococcal disease by administering a pharmaceutically effective amount in a systemic or mucosal route. The term "prevention" of the present invention refers to any action that inhibits or delays the infection caused by pneumococcal by administration of the vaccine composition of the present invention. As defined herein, a "pharmaceutically effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the probability of infection or the severity of infection. The term "administration" of the present invention refers to the introduction of certain substances into an individual in any suitable way. The vaccine composition of the present invention may be administered by inhalation route through oral, nasal, rectal, transdermal or aerosol, but may be administered by bolus or infused slowly, but is not limited thereto. The administration may be by injection via an intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract. In one embodiment, intranasal administration can be used for the treatment of pneumonia or otitis media, in which case more effective prevention of nasopharyngeal carriers of pneumococci can attenuate the infection at an early stage.
본 발명의 용어 “개체”는 병원균이 감염될 수 있는 살아있는 유기체를 의미하며, 구체적으로는 고등 척추동물이 될 수 있고, 더욱 구체적으로는 포유동물이 될 수 있으나, 특별히 이에 제한되지는 않는다.The term “individual” of the present invention means a living organism to which a pathogen can be infected, and in particular, may be a higher vertebrate, and more specifically, a mammal, but is not particularly limited thereto.
본 발명의 다른 구현예에서, 본 발명의 조성물은 단회 접종으로 투여되거나, 적당한 간격으로 2 회, 3 회, 4 회 또는 그 이상 투여될 수 있으나, 이에 제한되지 않는다. 예를 들면, 스트렙토코커스 뉴모니애에 의해서 발생하는 침습성 질환에 대해 유아 및 갓난아이를 대상으로 한 정기접종 계획은 생후 2, 4, 6 및 12 내지 15 개월일 수 있다.In another embodiment of the present invention, the composition of the present invention may be administered in a single inoculation, or two, three, four or more times at appropriate intervals, but is not limited thereto. For example, routine inoculation plans for infants and newborns for invasive diseases caused by Streptococcus pneumoniae can be 2, 4, 6 and 12 to 15 months of age.
또한, 상기 조성물은 스트렙토코커스 뉴모니애 유래의 하나 이상의 단백질을 더 포함할 수 있다. 포함시키기에 적당한 스트렙토코커스 뉴모니애 단백질의 예에는 제WO2002/053761호에 기술된 단백질뿐만 아니라, 제WO2002/083855호에서 동정된 단백질도 모두 본 발명의 범위에 포함될 수 있다.In addition, the composition may further comprise one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include not only the proteins described in WO2002 / 053761, but also the proteins identified in WO2002 / 083855, all within the scope of the present invention.
본 발명의 구체적인 일 실시예에서는 총 0.5 mL 중 4.4 ㎍/mL의 각 다당류, 단 6B는 8.8 ㎍/mL; 약 29.3 ㎍의 CRM197 운반 단백질; 0.5 mg의 알루미늄 원소 (2 mg 알루미늄 포스페이트) 애쥬번트; 염화나트륨 약 4.25 mg(보존제 미포함의 경우) 혹은 약 3.5 mg (보존제 포함의 경우); 석시네이트 완충액 약 295 ㎍; 2-페녹시에탄올 약 3 mg 및 포름알데히드 약 60 ㎍을 혼합하여 (보존제 포함 시), 13 가 ('LBVE013'으로 명명) 및 17 가 ('LBVE017'로 명명)의 폐렴구균병의 예방을 위한 백신 조성물을 제조하였다. 또한 상기 조성물의 함량을 100 %로 하고, 일부 혈청형인 6A, 6B, 14, 19A 및 19F의 함량을 조절한 백신 조성물을 별도로 제조하였다.In a specific embodiment of the present invention, 4.4 μg / mL of each polysaccharide in a total of 0.5 mL, provided that 6B is 8.8 μg / mL; About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 μg succinate buffer; About 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde were mixed (with preservatives) to prevent pneumococcal disease of the 13-valent (named 'LBVE013') and 17-valent (named 'LBVE017') Vaccine compositions were prepared. In addition, the content of the composition was 100%, and a vaccine composition was prepared separately by adjusting the content of some serotypes 6A, 6B, 14, 19A and 19F.
본 발명의 구체적인 다른 일 실시예에서는 상기 LBVE013 백신 조성물을 접종한 래빗의 혈청에서, 프리베나13® 보다 높은 수준의 혈청형 특이적 IgG 농도를 확인하였으나 (표 1), 다른 혈청형 유래 협막 다당류를 추가하여 17 가로 조성물을 제조한 경우에는 각 혈청형에 대한 IgG 역가가 현저히 감소하는 것을 확인하였다 (도 1). 이에 대하여, 혈청형 6A, 6B, 14, 19A 및 19F 접합체의 투여양을 동시에 감소시켜 전체 혈청형에 대한 IgG 역가를 재 측정한 결과 가수 증가에 따라 감소하였던 IgG 역가가 다시 회복되는 현상을 확인하였으며, 특히 상기 5 종의 접합체를 40 % 내지 95 % 함량으로 감소시켰을 때 이와 같은 면역원성 증가 현상을 보이는 것을 확인하였다 (도 2 및 3). 나아가, 이와 같은 결과는 17 가 조성물에서만 나타나는 현상으로 13 가의 경우 오히려 상기 5 종의 접합체 함량을 감소시킴에 따라 전반적으로 혈청형에 대한 IgG 역가가 감소되는 양상을 보임을 확인하였다 (도 4). In another specific embodiment of the present invention, serum levels of rabbits inoculated with the LBVE013 vaccine composition confirmed higher serotype-specific IgG concentrations than prevena13 ® (Table 1), but other serotype-derived capsular polysaccharides were identified. In addition, when the 17 transverse composition was prepared, it was confirmed that the IgG titer for each serotype was significantly decreased (FIG. 1). On the other hand, by reducing the dose of serotypes 6A, 6B, 14, 19A and 19F conjugates at the same time and re-measuring the IgG titer for the entire serotype, it was confirmed that the IgG titer that decreased with the increase of the valence was restored again. In particular, when the five conjugates were reduced to 40% to 95% content, it was confirmed that such an immunogenicity was increased (FIGS. 2 and 3). Furthermore, this result was confirmed that only the 17-valent composition showed a decrease in IgG titer on the serotype as a whole by decreasing the content of the five conjugates in the case of 13-valent (FIG. 4).
본 발명의 다른 양태는 협막 다당류 -운반 단백질 접합체를 포함하는 폐렴구균에 대한 면역원성 조성물이다.Another aspect of the invention is an immunogenic composition for pneumococci comprising capsular polysaccharide-carrying protein conjugates.
접합체 및 폐렴구균은 상기 설명한 바와 같다.The conjugate and pneumococcus are as described above.
본 발명의 협막 다당류-운반 단백질 접합체를 포함하는 조성물은 15 종 혹은 17 종의 각기 다른 혈청형을 갖는 스트렙토코커스 뉴모니애 유래 협막 다당류를 포함하며, 이를 체내에 투여할 경우 항원으로 인식하여 이에 대한 항체를 생산할 수 있도록 면역반응을 일으키는바, 폐렴구균에 대한 면역원성 조성물로 이용될 수 있다.The composition comprising the capsular polysaccharide-carrying protein conjugate of the present invention comprises Streptococcus pneumoniae-derived capsular polysaccharide having 15 or 17 different serotypes, and when administered to the body, it is recognized as an antigen It can be used as an immunogenic composition against pneumococcal to cause an immune response to produce an antibody.
본 발명의 다른 양태는 상기 백신 조성물 혹은 면역원성 조성물을 이를 필요로 하는 개체에 투여하여 폐렴구균병을 예방하는 방법이다.Another aspect of the invention is a method of preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.
본 발명의 다른 양태는 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한, 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F 및 23F 유래의 15 종의 협막 다당류 각각이 운반 단백질에 공유결합적으로 접합된 협막 다당류-운반체 단백질 접합체 15 종의 용도를 제공하는 것이다.Another aspect of the invention is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B for use in the preparation of vaccine compositions for the prevention of pneumococcal disease 15 capsular polysaccharides derived from, 18C, 19A, 19F, and 23F each provide a use of 15 capsular polysaccharide-carrier protein conjugates covalently conjugated to a carrier protein.
본 발명의 또 다른 양태는 폐렴구균병의 예방을 위한 백신 조성물의 제조에 사용하기 위한, 상술한 15 종의 접합체에 더하여 스트렙토 코커스 뉴모니애 혈청형 22F 및 33F 유래의 협막 다당류 각각이 운반 단백질에 공유결합적으로 접합된 2 종의 접합체를 더 포함하는 협막 다당류-운반체 단백질 접합체 17 종의 용도를 제공하는 것이다.Another aspect of the present invention provides a capsular polysaccharide derived from Streptococcus pneumoniae serotypes 22F and 33F, in addition to the aforementioned 15 conjugates, for use in the preparation of a vaccine composition for the prevention of pneumococcal disease. It is to provide the use of 17 kinds of capsular polysaccharide-carrier protein conjugate further comprising two conjugates covalently conjugated.
구체적으로 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비, 혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고, 혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하의 함량비를 가질 수 있다.Specifically, the capsular polysaccharide derived from serotypes 6A, 14, 19A, and 19F of the serotype 1-derived capsular polysaccharide in the composition has a content ratio of more than 0.25 and 0.95 or less, and the capsular polysaccharide derived from serotype 6B is 0.5. It may exceed and have a content ratio of 1.9 or less.
더욱 구체적으로 상기 조성물에서 혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 존재할 수 있다.More specifically, in the composition, the capsular polysaccharide derived from serotype 22F and the capsular polysaccharide derived from 33F may be present in the composition in the same amount as the capsular polysaccharide derived from serotype 1.
백신 조성물, 면역원성 조성물 및 폐렴구균병의 예방 등에 대해서는 상기 설명한 바와 같다.Vaccine compositions, immunogenic compositions, and prevention of pneumococcal disease are as described above.
본 발명에서는 15 종 이상의 각기 다른 혈청형을 갖는 폐렴구균 유래의 협막 다당류, 구체적으로 17종의 각기 다른 혈청형을 갖는 폐렴구균 유래의 협막 다당류를 포함하는 조성물을 제공하며, 특히 상기 조성물은 각 협막 다당류가 최적의 함량으로 포함되어, 우수한 혈청 IgG 역가와 기능적 항체 활성을 유도할 수 있다. 따라서, 본 발명에 따른 백신 조성물 및 면역원성 조성물은 영·유아, 소아, 및 성인의 폐렴구균에 의한 질환을 예방하는데 유용하게 사용될 수 있다.The present invention provides a composition comprising pneumococcal capsular polysaccharides having 15 or more different serotypes, specifically, capsular polysaccharides derived from pneumococcal having 17 different serotypes, and in particular, the composition comprises Optimal amounts of polysaccharides can be included to induce good serum IgG titers and functional antibody activity. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be usefully used to prevent diseases caused by pneumococci in infants, infants, children, and adults.
도 1은 13가(LBVE013)에서 17가(LBVE017)로 가수가 늘어나면서 전반적으로 대부분 혈청형에서 IgG 역가 저하 현상이 일어나며 이때 상대적으로 13가에서 IgG 역가가 높은 6A, 6B, 14, 19A, 19F의 접합체 투여 용량을 동시에 50%로 줄일 경우, 전반적으로 IgG 역가 상승(회복) 현상이 일어남을 보여 주고있다. 22F, 33F의 경우 사용된 기준 (standard)이 나머지 혈청형과 다르므로 다른 혈청형과 분리하여 별도로 도면화 하였다.FIG. 1 shows that IgG titers are generally lowered in most serotypes as the valence increases from 13-valent (LBVE013) to 17-valent (LBVE017), and 6A, 6B, 14, 19A, and 19F have high IgG titers at 13-valent. Increasing the conjugate's dose to 50% simultaneously suggests an overall increase in IgG titer (recovery). In the case of 22F and 33F, the standard used is different from the rest of the serotypes, so it is drawn separately from other serotypes.
도 2는 17가 조성물(LBVE017)에서 혈청형 6A, 6B, 14, 19A, 19F 접합체의 양을 동시에 100%, 90%, 75%, 50%, 25%, 10%, 1%로 변화 시켰을 경우 17가 혈청형 각각의 전체적인 변역원성 변화 패턴을 보여준다. 22F, 33F의 경우 기준 (standard)이 나머지 혈청형과 다르므로 다른 혈청형과 분리하여 별도로 도면화 하였다Figure 2 shows the amount of serotypes 6A, 6B, 14, 19A, 19F conjugates in the 17-valent composition (LBVE017) simultaneously changed to 100%, 90%, 75%, 50%, 25%, 10%, 1% 17 shows serotypic patterns of change in each of the serotypes. In the case of 22F and 33F, the standard is different from the rest of the serotypes.
도 3은 17가 조성물(LBVE017)에서 혈청형 6A, 6B, 14, 19A, 19F 접합체 각각의 용량 임계점 설정에 관한 도면으로써, 17가(LBVE017) 모든 혈청형을 용량 줄임 없이 다 넣어 주었을 때 (100%)의 6A, 6B, 14, 19A, 및 19F 혈청형의 각 GMT값을 각각의 (하한)기준으로 잡을 경우, 기준보다 높은 면역원성을 보여줄 수 있는 상기 각 5 종의 접합체양의 범위가 40 내지 95 % 라는 것을 보여준다.FIG. 3 is a diagram of dose threshold setting of serotypes 6A, 6B, 14, 19A, and 19F conjugates in 17-valent composition (LBVE017), when all 17-valent (LBVE017) serotypes were all received without dose reduction (100 When each GMT value of the 6A, 6B, 14, 19A, and 19F serotypes of%) is taken as the (lower limit), the range of conjugate amounts of each of the five conjugates above 40 can show higher immunogenicity than the reference. To 95%.
도 4는, 13가(LBVE013)의 경우, 17가 조성물과는 달리, 혈청형 6A, 6B, 14, 19A, 19F의 접합체 양을 동시에 감소시켰을 경우, 대부분의 혈청형에서 면역원성이 오히려 감소함을 보여준다. Figure 4 shows that in the case of 13-valent (LBVE013), unlike 17-valent compositions, immunogenicity is rather reduced in most serotypes when simultaneously reducing the amount of conjugates of serotypes 6A, 6B, 14, 19A, and 19F. Shows.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 국한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예Example 1:  One: 스트렙토코커스Streptococcus 뉴모니애의New Monica 협막 다당류 제조 Capsular Polysaccharide Manufacturing
1-1. 세포은행 제조1-1. Cell Bank Manufacturing
17 종의 각기 다른 혈청형 (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F)을 가진 스트렙토코커스 뉴모니애를 수탁기관인 미국 CDC (Center for Disease Control and Prevention,US)로부터 입수하였으며 하기와 같은 방법으로 세포 은행을 제조하였다.Streptococcus pneumoniae with 17 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) Obtained from the Center for Disease Control and Prevention (USC), a trustee, and prepared a cell bank in the following manner.
스트렙토코커스 뉴모니애 균주를 혈액 한천배지에 도말하여 단일 콜로니를 분리하였다. 10 개 이상의 단일 콜로니 중 성장이 좋은 단일 콜로니를 선정한 후 동물 유래의 성분을 포함하지 않은 액상배지에 접종하여 배양한 후 합성 글리세롤을 함유한 연구용 세포 은행 (Research Cell Bank, RCB)을 제조하였다. Streptococcus pneumoniae strains were plated on blood agar medium to isolate single colonies. Among the 10 or more single colonies, a single good growing colony was selected, inoculated and cultured in a liquid medium containing no animal-derived components, and a research cell bank (RCB) containing synthetic glycerol was prepared.
고유의 혈청형을 가진 다당류 발현이 확인 된 연구용 세포 은행 중 바이알 1 개를 꺼내어 동물 유래의 성분을 포함하지 않은 액상배지에 세포를 증식시킨 후 합성 글리세롤을 추가하여 마스터 세포은행을 제조하였으며, 마스터 세포 은행 중 바이알 1 개를 꺼내어 동물 유래의 성분들을 포함하지 않은 액상배지에 세포를 증식시킨 후 합성 글리세롤을 추가하여 제조용 세포 은행을 제조하였다. 제조된 세포 은행은 다음 단계에서 사용하기 위해 -70 ℃ 이하에서 보관하여 사용하였다.Master cell bank was prepared by taking out one vial from the cell bank for which the expression of polysaccharides with unique serotypes 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 to proliferate cells in a liquid medium containing no animal-derived components, and then synthetic glycerol was added to prepare a cell bank for production. The prepared cell bank was stored at -70 ° C or below for use in the next step.
1-2. 발효 및 다당류 분리1-2. Fermentation and Polysaccharide Separation
제조용 세포 은행 중 바이알 1 개를 해동하여 동물 유래의 성분을 포함하지 않은 액상배지에 접종하여 종 발효를 시작하였다. 일정 균체 농도 (Optical Density, OD600)에 도달하여 중간-지수 증식기의 종말점에 도달할 때까지 무교반 상태로 37 ± 2 ℃에서 종배양을 실시 하였다. 종배양에서 얻은 배양액을 동물 유래의 성분을 포함하지 않은 액상배지를 함유하는 발효기에 접종하여 주 발효(main fermentation)를 시작하였다. One vial in the cell bank for preparation was thawed and seeded in a liquid medium containing no animal-derived component to start species fermentation. Species were cultured at 37 ± 2 ° C. in an unstirred state until reaching a certain cell concentration (Optical Density, OD 600 ) and reaching the end point of the mid-exponential growth phase. The culture broth obtained from the species culture was inoculated into a fermentor containing a liquid medium containing no animal-derived components to start main fermentation.
그 다음 37 ± 2 ℃에서 수산화 칼륨 용액으로 배지의 pH를 조절하면서 본배양을 실시하였다. 2 시간 마다 최적의 세포 밀도와 배지에 포함된 글루코스 농도를 측정하였다. 발효는 배지 내 글루코스가 고갈되었을 때 종료하였다. This culture was then carried out while adjusting the pH of the medium with potassium hydroxide solution at 37 ± 2 ° C. Every two hours, the optimal cell density and glucose concentration in the medium were measured. Fermentation was terminated when glucose in the medium was depleted.
발효가 종료된 후, 12 % 소듐 데옥시콜레이트 (sodium deoxycholate)를 최종 0.12 %가 되도록 배양물에 1시간 동안 첨가하여 세포를 용해시키고 세포에 결합된 다당류를 유리시켰다.After the fermentation was completed, 12% sodium deoxycholate was added to the culture for 1 hour to a final 0.12% to lyse the cells and release the polysaccharide bound to the cells.
1-3. 협막 다당류의 정제1-3. Purification of Capsular Polysaccharides
소듐 데옥시콜레이트가 처리된 샘플에 인산을 가한 뒤, 원심 분리를 통해 상층액을 회수하였다. 회수한 상층액을 뎁쓰 필터 (depth filter)에 통과시킨 다음, 농축 및 인산 완충용액으로 버퍼 교환을 진행하였다. 버퍼 교환 후, 샘플을 탄소활성탄 필터 (active carbon filter)에 통과시킨 다음, 하기와 같은 두 가지 방법으로 불순물 제거를 진행하였다.Phosphoric acid was added to the sample treated with sodium deoxycholate, and the supernatant was recovered by centrifugation. The recovered supernatant was passed through a depth filter and then buffer exchanged with concentrated and phosphate buffer. After the buffer exchange, the sample was passed through an active carbon filter, and then impurities were removed by the following two methods.
1) 14 개의 혈청형 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F 및 23F의 경우, CTAB (cetyltrimethylammonium bromide)와 이온 결합이 가능하므로 CTAB 공정을 진행하였다. CTAB 처리, 원심 분리, 염화나트륨 (NaCl) 및 요오드화 나트륨 (NaI) 처리, 및 원심 분리 과정을 수행하였다.1) For 14 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F, 22F, and 23F, the CTAB process is possible because of the ionic bond with CTAB (cetyltrimethylammonium bromide). Proceeded. CTAB treatment, centrifugation, sodium chloride (NaCl) and sodium iodide (NaI) treatment, and centrifugation were performed.
2) CTAB와 반응하지 않는 3 개의 혈청형 7F, 14 및 33F는 인산알루미늄겔 (Algel) 용액을 첨가하여 반응시킨 다음, 원심 분리를 통해 얻어진 상층액을 사용하였다.2) Three serotypes 7F, 14, and 33F that do not react with CTAB were reacted with the addition of an aluminum phosphate gel (Algel) solution, and then the supernatant obtained through centrifugation was used.
상기 두 가지 형태의 불순물 제거 공정을 완료한 샘플은 뎁쓰필터 (Depth filter) 및 한외여과 (UF/DF) 공정을 거친 다음, 에탄올과 염화나트륨의 양을 조절하며 원말 형태로 만들어 보관 하였다.After completing the two types of impurities removal process, the sample was subjected to a depth filter and ultrafiltration (UF / DF), and then the amount of ethanol and sodium chloride was adjusted and stored in a raw form.
실시예Example 2:  2: 스트렙토코커스Streptococcus 뉴모니애New Monica 협막  Capsular 폴리사카라이드Polysaccharides -단백질 접합체 제조Protein Conjugate Preparation
2-1: 환원적 2-1: Reductive 아미노화법과Amination and 시아닐화법의Cyanylated 접합방식 비교 Joining method comparison
혈청형 9V의 정제 다당류와 CRM197 캐리어 단백질을 이용하여, 환원적 아미노화법 (reductive amination) 및 시아닐화법 (cyanylation)의 접합방식을 시도하여 두 접합방식의 접합수율을 비교하였다. 구체적인 접합 과정은 다음과 같다.Using the serotype 9V purified polysaccharide and CRM197 carrier protein, the conjugation yield of the two conjugation methods was compared by attempting conjugation method of reductive amination and cyanylation. Specific bonding process is as follows.
(1) 환원적 아미노화법 (1) reductive amination
9V 다당류 원액에 11.7 mg의 과요오드산염 (Sodium periodate)를 투입하여 21 ~ 25 ℃에서 교반하면서 다당류를 활성화시켰다. 산화된 다당류를 100 kDa의 한외여과 필터와 WFI를 이용하여 농축 및 투석 여과 시킨 후 남은 잔류 다당류를 CRM197과 당류/CRM197=0.5 비율로 섞어 동결건조시켰다. 동결 건조된 복합체를 해동시키고 21 ~ 25 ℃에서 안정화 (평형화)하였다. 평형화된 복합체를 당류 20 g 당 0.1 M의 비율로 인산나트륨 (Na3PO4) 완충액 용액에서 항온처리 (37 ± 2 ℃)하여 용해 한 후, 시아노보로하이드라이드 (100 mg/mL)를 투입함으로써 접합반응을 시작하였다. 37 ± 2 ℃에서 약 44 ~ 52 시간 동안 항온 처리한 후, 온도를 23 ± 2 ℃로 낮추고 0.9 % NaCl 용액 1 mL을 반응기에 첨가하였다. 당류 1 몰 당 나트륨 보로하이드라이드 1.8 내지 2.2 몰 당량이 되도록 나트륨 보로하이드라이드 용액 (100 mg/mL)을 첨가하고, 혼합물을 23 ± 2 ℃에서 교반하며 항온 처리 하여 당류에 존재하는 반응하지 않은 임의의 알데히드를 환원시켰다. 0.9 % 염화나트륨 수용액 5 mL을 사용하여 혼합물을 희석시키고 희석된 접합 혼합물을 100 kDa MWCO 멤브레인을 이용하여 투석 여과시켰다. 11.7 mg of sodium periodate was added to a 9V polysaccharide stock solution to activate the polysaccharides while stirring at 21 to 25 ° C. Oxidized polysaccharides were concentrated and diafiltered using a 100 kDa ultrafiltration filter and WFI, and the remaining polysaccharides were lyophilized by mixing CRM197 with saccharides / CRM197 = 0.5. Lyophilized complexes were thawed and stabilized (equilibrated) at 21-25 ° C. The equilibrated complex was dissolved by incubation (37 ± 2 ° C) in sodium phosphate (Na 3 PO 4 ) buffer solution at a rate of 0.1 M per 20 g of saccharides, and then cyanoborohydride (100 mg / mL) was added. The conjugation reaction was started by doing so. After incubation at 37 ± 2 ° C. for about 44-52 hours, the temperature was lowered to 23 ± 2 ° C. and 1 mL of 0.9% NaCl solution was added to the reactor. Sodium borohydride solution (100 mg / mL) is added to 1.8 to 2.2 molar equivalents of sodium borohydride per mole of saccharide, and the mixture is incubated with stirring at 23 ± 2 ° C. to any unreacted saccharide present in the saccharide. Aldehydes were reduced. The mixture was diluted with 5 mL of 0.9% aqueous sodium chloride solution and the diluted conjugate mixture was diafiltered using a 100 kDa MWCO membrane.
(2) 시아닐화법 (2) cyanylation
가수분해 처리과정 없이 준비된 9V 다당류 원액에 염화나트륨 분말을 첨가하여 2 M NaCl 다당류 용액을 제조하였다. 다당을 활성화 시키기 위하여 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 다당 대비 0.5 w/w% 비율로 용해하고 9V 다당류 용액에 첨가한 후, 15 분 동안 교반하여 다당 활성화 반응을 유도하였다. 그 후 수산화 나트륨 용액을 pH가 9.5 ± 0.1 ℃가 될 때까지 상승시킨 후, 다당류의 하이드록실기가 CDAP에 의해 충분히 활성화될 수 있도록 3 분 동안 교반하였다. 다당 활성화 과정을 거친 다당류 용액에 다당 대비 CRM197 1.0 w/w%의 비율로 CRM197을 첨가하여 상온에서 1 시간 동안 접합반응을 진행하였다. 접합반응은 2 M 글리신 (glycine) 용액을 CDAP 1 몰 당량 대비 3 몰 당량으로 첨가하고 pH를 9.0으로 조정하여 상온에서 밤새 인큐베이션함으로써 반응을 종결하였다. 반응 종결된 접합체는 0.9 % 염화나트륨을 포함한 완충액을 통해 한외여과필터에 농축 및 투석 여과시켰다.A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to a 9V polysaccharide stock solution prepared without hydrolysis. In order to activate polysaccharide, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a ratio of 0.5 w / w% to polysaccharide, added to a 9V polysaccharide solution, and stirred for 15 minutes to induce a polysaccharide activation reaction. The sodium hydroxide solution was then raised until the pH was 9.5 ± 0.1 ° C. and then stirred for 3 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP. CRM197 was added at a ratio of CRM197 1.0 w / w% to polysaccharide in the polysaccharide solution that had undergone polysaccharide activation, and the conjugation reaction was performed at room temperature for 1 hour. The conjugation reaction was terminated by adding 2 M glycine solution in 3 molar equivalents to 1 molar equivalent of CDAP and incubating overnight at room temperature with pH adjusted to 9.0. The terminated conjugate was concentrated and diafiltered into an ultrafiltration filter through a buffer containing 0.9% sodium chloride.
그 결과, 시아닐화 방법에 의해 제조된 접합체는 환원적 아민화 방법에 의해 제조된 접합체에 비해 그 수율이 4 배 이상인 것을 확인할 수 있었다. 따라서, 본 발명자들은 협막 다당류 및 CRM197로 시아닐화 방법을 이용하여 접합체를 제조하였다.As a result, it was confirmed that the conjugate produced by the cyanation method was 4 times or more in yield compared to the conjugate produced by the reductive amination method. Thus, we prepared conjugates using the capsular polysaccharide and the cyanylation method with CRM197.
2-2. 접합 및 접합체의 정제2-2. Conjugation and Purification of Conjugates
스트렙토코커스 뉴모니애의 협막 다당류 및 CRM197의 접합체 제조는 하기 6 단계의 공정을 거쳐 수행되었다.Preparation of the conjugate of the capsular polysaccharide and CRM197 of Streptococcus pneumoniae was carried out through the following six steps.
단계 1. 협막 다당류의 용해 및 가수분해 Step 1. Dissolution and Hydrolysis of Capsular Polysaccharides
각각의 혈청형에서 유래한 협막 다당류 원말을 최종 농도 범위가 아래 기술된 범위가 되도록 주사용수에 용해하고 0.45 ㎛ 필터를 통해 여과시켰다. The original capsular polysaccharide originated 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 μm filter.
세부적으로 혈청형 1, 3 및 4의 경우 0.8 - 2.0 mg/㎖의 범위로 용해하였고, 혈청형 5, 6B, 9V, 18C 및 19F 의 경우 4 - 8 mg/㎖, 혈청형 6A, 12F 및 19A는 8 - 12 mg/㎖, 혈청형 7F, 14 및 23F의 경우 2 - 4 mg/㎖로 용해하여 여과를 진행하였다. 또한, 혈청형 15B, 22F, 33F의 경우 2 - 5 mg/㎖의 범위로 용해하여 여과를 진행하였다Specifically dissolved in the range 0.8-2.0 mg / ml for serotypes 1, 3 and 4, 4-8 mg / ml for serotypes 5, 6B, 9V, 18C and 19F, serotypes 6A, 12F and 19A Was dissolved at 8-12 mg / ml, and 2-4 mg / ml for serotypes 7F, 14 and 23F. In addition, in the case of serotypes 15B, 22F, and 33F, the solution was dissolved in the range of 2-5 mg / ml and filtered.
혈청형 별로 아래 기술된 pH 및 온도 범위에서 용액을 항온 처리하여 진행하였다. 세부적으로 혈청형 1, 3, 5, 6B, 7F, 14 및 23F의 경우 밤새 70 - 80 ℃, 혈청형6A 및 19F 경우 1 - 4시간 동안 70 - 80 ℃, 혈청형 9V 및 18C의 경우, 인산용액을 이용하여 1 - 3 시간 동안 pH 2.0, 65 - 80 ℃ 에서 항온처리 과정을 수행하였다. 혈청형 22F, 33F의 경우 밤새 75 - 85 ℃, 혈청형 12F는 인산용액을 이용하여 1 - 3 시간 동안 pH 2.0, 75 - 85 ℃ 에서 항온처리 과정을 수행하였으며 Each serotype was run by incubating the solution in the pH and temperature ranges described below. Specifically, phosphoric acid at 70-80 ° C. overnight for serotypes 1, 3, 5, 6B, 7F, 14 and 23F, 70-80 ° C. for 1-4 hours for serotypes 6A and 19F, for serotypes 9V and 18C. The solution was incubated at pH 2.0, 65-80 ° C. for 1-3 hours. Serotypes 22F and 33F were incubated overnight at 75-85 ℃ and serotype 12F at pH 2.0, 75-85 ℃ for 1-3 hours using phosphate solution.
혈청형 4, 15B, 19A의 경우 가수분해를 진행하지 않았다. 그 다음 21 ℃ 내지 24 ℃로 냉각시키고 6.0 ± 1.0의 목표 pH로 수산화 나트륨을 첨가함으로써 가수분해를 중지시켰다.In the case of serotypes 4, 15B, and 19A, no hydrolysis proceeded. The hydrolysis was then stopped by cooling to 21 ° C.-24 ° C. and adding sodium hydroxide to a target pH of 6.0 ± 1.0.
단계 2. 협막 다당류와 CRM197의 접합 반응 공정Step 2. Conjugation Reaction Process of Capsular Polysaccharide and CRM197
모든 혈청형에 염화나트륨 분말을 첨가하여 2 M NaCl 다당류 용액을 제조하였다. 각 혈청 별로 적절한 CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)를 50/50 아세토니트릴/주사용수 (v/v) 용액 100 ㎖ 당 CDAP 1 g의 비율로 용해하였다. 세부적으로 혈청형 6A, 14 및 33F의 경우 다당 대비 CDAP 1 w/w%를, 혈청형 4의 경우 2 w/w%를, 혈청형 1, 3, 6B, 7F 15B 및 19A의 경우 3 w/w%를, 혈청형 5, 9V, 18C, 19F, 22F 및 23F의 경우 4 w/w%, 그리고 혈청형 12F의 경우 5 w/w%의 무게비율로 용해하고 각각의 다당류 용액에 첨가하였다. 이어, 1 내지 3 분 후 수산화 나트륨 용액을 첨가하여 pH 9.4 내지 9.7로 상승시킨 후 다당류의 하이드록실기가 CDAP에 의해 충분히 활성화될 수 있도록 3 내지 7 분 동안 교반하였다. 다당 대비 CRM197 0.5 - 1.0 w/w%를 각 혈청형 다당 용액에 첨가하여 1 시간 내지 4 시간 동안 접합반응을 진행하였으며 HPLC-SEC을 이용하여 반응 전환율을 측정하였고 필요에 따라 CDAP를 추가 투입하였다. Sodium chloride powder was added to all serotypes to prepare a 2 M NaCl polysaccharide solution. For each serum, the appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) was dissolved at a rate of 1 g of CDAP per 100 ml of 50/50 acetonitrile / water for injection (v / v) solution. Specifically, 1 w / w% of CDAP relative to polysaccharide for serotypes 6A, 14, and 33F, 2 w / w% for serotype 4, and 3 w / for serotypes 1, 3, 6B, 7F 15B, and 19A. w% was dissolved at a weight ratio of 4 w / w% for serotypes 5, 9V, 18C, 19F, 22F and 23F, and 5 w / w% for serotype 12F and added to each polysaccharide solution. After 1 to 3 minutes, sodium hydroxide solution was added to raise the pH to 9.4 to 9.7, followed by stirring for 3 to 7 minutes so that the hydroxyl groups of the polysaccharides could be sufficiently activated by CDAP. Conjugation reaction was performed for 1 hour to 4 hours by adding CRM197 0.5-1.0 w / w% to polysaccharide to each serotype polysaccharide solution. The reaction conversion was measured using HPLC-SEC, and CDAP was added as needed.
단계3. 접합 반응 종결 Step 3. Termination of conjugation reaction
모든 혈청형에 대해 첨가한 CDAP 1 몰 당량 대비 3 내지 6 몰 당량의 글리신 (glycine) 용액을 첨가하고 pH를 9.0으로 조정하여 반응을 종결하였다. 접합 용액을 21 내지 24 ℃에서 1 시간 교반한 후 2 내지 8 ℃ 저온에서 밤새 보관하였다.The reaction was terminated by adding 3-6 molar equivalents of glycine solution relative to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0. The conjugation solution was stirred at 21-24 ° C. for 1 hour and then stored overnight at 2-8 ° C. low temperature.
단계 4: 한외여과Step 4: Ultrafiltration
희석된 접합 혼합물을 최소 20 용적의 완충액을 사용하여 한외여과필터에 농축 및 투석 여과시켰다. 여기서 완충액은 pH 5.5 내지 6.5의 범위를 유지하며, 0.9 % 염화나트륨을 포함한 완충액을 사용하였다. 한외여과필터의 분획 분자량은 모든 혈청형에서 300 kDa을 사용하여 실시하였고, 투과액은 폐기하였다. The diluted conjugate mixture was concentrated and diafiltered into an ultrafiltration filter using at least 20 volumes of buffer. Here the buffer was maintained in the range of pH 5.5 to 6.5, and a buffer containing 0.9% sodium chloride was used. Fractional molecular weight of the ultrafiltration filter was carried out using 300 kDa in all serotypes, and the permeate was discarded.
단계 5: 제균 여과Step 5: Sterile Filtration
투석 여과 후의 잔류액을 완충액을 이용하여 다당함량 농도 기준으로 0.4 g/L 미만이 되도록 희석하여 0.22 ㎛ 필터를 통해서 여과시켰다. 여과된 산물에 대해 제조과정 중 제어 (당류 함량, 잔류 DMAP)를 실시하였다. 여과시킨 잔류액에 대해 제조과정 중 제어를 실시하여 추가적인 농축, 투석 여과 및/또는 희석이 필요한지의 여부를 결정하였다.The residue after diafiltration was diluted to less than 0.4 g / L based on polysaccharide concentration using a buffer and filtered through a 0.22 μm filter. The filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were performed on the filtered residue to determine if further concentration, diafiltration and / or dilution were needed.
단계 6: 흡착Step 6: adsorption
제균 여과액에 알루미늄 염 (주로 인산 알루미늄)을 최종 농도가 알루미늄 이온 기준으로 1 mg/mL이 되도록 첨가하여 흡착시키며, 5.5 내지 6.5의 pH 범위를 유지할 수 있도록 여분의 염을 추가하였다. 흡착이 완료된 원액은 품질 검사를 실시하여 품질 적합성 여부를 확인하고, 사용하기 전까지 2 내지 8 ℃에 냉장 보관하였다.Aluminum salts (mainly aluminum phosphate) were added to the sterile filtrate so that the final concentration was 1 mg / mL based on aluminum ions, and extra salts were added to maintain a pH range of 5.5 to 6.5. After the adsorption, the stock solution was subjected to quality inspection to confirm quality suitability, and refrigerated at 2 to 8 ° C. until use.
실시예 3. 다가 폐렴구균 접합체 백신의 제제화Example 3 Formulation of a Multivalent Pneumococcal Conjugate Vaccine
배치 용적 (batch volume) 및 벌크 다당류 농도를 기준으로 하여 최종 벌크 농축액의 필요량을 계산하였다. 필요량의 0.85 % 염화나트륨, 석시네이트 완충액, 2-페녹시에탄올 및 포름알데히드를 미리 라벨링한 제제화 용기에 첨가한 후에, 벌크 농축액을 첨가하였다. 그 다음, 충분히 혼합하고 0.22 ㎛ 필터를 통해서 여과시켰다. 제제화된 벌크액을 서서히 혼합한 뒤, 벌크 알루미늄 포스페이트를 첨가하고 충분히 혼합하였다. pH를 체크하고 필요한 경우에 조절하였다. 제제화된 벌크 제품을 2 내지 8 ℃에서 보관하였다. 얻어진 백신 조성물은 총 0.5 mL 중에 4.4㎍/mL의 각 다당류 (13 가 (이하, 'LBVE013')의 경우 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F 및 23F; 17 가 (이하, 'LBVE017')의 경우 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F), 단 6B는 8.8 ㎍/mL; 약 29.3 ㎍의 CRM197 운반 단백질; 0.5 mg의 알루미늄 원소 (2 mg 알루미늄 포스페이트) 애쥬번트; 염화나트륨 약 4.25 mg; 석시네이트 완충액 약 295 ㎍; *2-페녹시에탄올 약 3 mg 및 *포름알데히드 약 60 ㎍을 함유한다 (* 보존제를 첨가할 경우에 해당됨). 상기 함량을 100 %의 기준량으로 하고, 하기 실시예에서는 필요에 따라 일부 혈청형의 함량을 조절(예컨대, 90 %, 75 %, 50 %, 25 %, 10 %, 1 %의 함량)하여 추가 분석을 수행하였다.The required amount of final bulk concentrate was calculated based on the batch volume and bulk polysaccharide concentration. The required amount of 0.85% sodium chloride, succinate buffer, 2-phenoxyethanol and formaldehyde were added to a previously labeled formulation vessel, followed by the bulk concentrate. Then it was thoroughly mixed and filtered through a 0.22 μm filter. The formulated bulk liquid was slowly mixed, then bulk aluminum phosphate was added and mixed well. pH was checked and adjusted if necessary. The formulated bulk product was stored at 2-8 ° C. The resulting vaccine composition contains 4.4 μg / mL of each polysaccharide (13, hereinafter, 'LBVE013') in 0.5 mL total, 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23F. For 17 is (hereinafter 'LBVE017') 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F), except that 6B is 8.8 μg / mL; About 29.3 μg CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride; About 295 μg succinate buffer; It contains about 3 mg of 2-phenoxyethanol and about 60 μg of formaldehyde (applicable when preservatives are added). The content is set as the reference amount of 100%, and in the following examples, the content of some serotypes is adjusted as needed (eg, 90%, 75%, 50%, 25%, 10%, 1%) for further analysis. Was performed.
실시예Example 4. 용량을 조절한 다가 폐렴구균 접합체 백신의 면역원성 평가 4. Immunogenicity Evaluation of Dose-Adjusted Multivalent Pneumococcal Conjugate Vaccine
(1) 평가 방법(1) Evaluation method
상기 실시예 3에서 제조한 각각의 다가 폐렴구균 백신 조성물이 토끼에서 면역 반응을 유도하는 능력을 가지는지를 평가하기 위한 연구를 수행하였다. 이러한 면역원성은, 항원-특이적 ELISA를 통해 혈청 IgG 농도를 측정하여 확인하였다. A study was conducted to evaluate whether each multivalent pneumococcal vaccine composition prepared in Example 3 has the ability to induce an immune response in rabbits. This immunogenicity was confirmed by measuring serum IgG concentration via antigen-specific ELISA.
제제화된 각각의 다가 폐렴구균 백신 조성물 혹은 대조군인 프리베나13®을 계획된 사람 임상 용량 (각 다당류 4.4 ㎍/mL, 예외: 6B 8.8 ㎍/mL인 경우를 100 %로 하여 필요에 따라 일부 혈청형의 함량을 조절)으로 0 주차, 2 주차 및 4 주차에 뉴질랜드 화이트 (New Zealand White) 토끼의 근육 내로 면역 접종시키고, 접종 후 2 주 간격으로 각각의 혈청을 채취하였다. 채취된 혈청에 대하여 멀티플렉스 비드 어세이(Multiplex bead assay)를 이용하여 혈청형 특이적 IgG 농도를 측정하였다. 이를 상세히 설명하면 다음과 같다.Planned to formulate each of the multivalent pneumococcal vaccine compositions or control the Prevenar 13 ® human clinical dose (for each polysaccharide 4.4 ㎍ / mL, exception: in some serotypes along the 6B 8.8 ㎍ / mL in case needed to make 100% The content was adjusted) and immunized into the muscles of New Zealand White rabbits at parking 0, 2 and 4, and each serum was taken at 2 week intervals after inoculation. Serum-type specific IgG concentration was measured on the collected serum using a multiplex bead assay. This will be described in detail as follows.
자성을 가지는 비드(magnetic bead)에 13 혹은 17종의 다당항원을 접합시킨 용액을 96-웰 플레이트에 넣고 부착시켰다. 각 개체 별 혈청은 비특이적 항원-항체반응을 최소화 하기 위해 CWPS 다중 용액(CWPS multi®, Statens Serum Institute) 1 mg/mL과 상온에서 30 분간 반응시켜 흡착 시킨 후, Tween 20이 포함된 항체 희석용 완충액을 이용하여 적당한 희석 배수로 희석하였다. 13 혹은 17종의 다당항원 접합 자성 비드가 부착된 플레이트를 세척용 완충액으로 2회 세척하고, 미리 흡착 및 희석한 혈청 50 ㎕를 플레이트에 넣은 후 실온에서 30분간 반응시켰다. A solution in which 13 or 17 polysaccharide antigens were conjugated to magnetic beads was attached to a 96-well plate. Each individual serum was adsorbed by reacting with 1 mg / mL CWPS multi solution (CWPS multi ® , Statens Serum Institute) for 30 minutes at room temperature to minimize non-specific antigen-antibody reactions, and then buffered for antibody dilution containing Tween 20. Was diluted to a suitable dilution multiple using. Plates with 13 or 17 polysaccharide antigen-conjugated magnetic beads were washed twice with a wash buffer, and 50 μl of previously adsorbed and diluted serum was added to the plates and allowed to react at room temperature for 30 minutes.
반응시킨 플레이트를 같은 방법으로 3 회 세척하고, 각 웰에 R-PE 고우트 항-래빗 IgG(R-Phycoerythrin goat anti-Rabbit IgG)(1:500)를 넣은 후 실온에서 30 분간 반응시켰다. 플레이트를 위와 같은 방법으로 3회 세척하고 각 웰에 완충액을 80 ㎕씩 넣은 후, 멀티플렉스 리더(Multiplex Reader)를 이용해 형광을 측정하였다. The reacted plate was washed three times in the same manner, and each well was added with R-PE Ghycoerythrin goat anti-Rabbit IgG (1: 500), followed by reaction at room temperature for 30 minutes. The plate was washed three times in the same manner as above, and 80 μl of the buffer solution was added to each well, and fluorescence was measured using a multiplex reader.
객관적인 면역원성 평가를 위해 대조군으로 프리베나13®을 면역하여 채취한 혈액 샘플을 같이 분석하였다.For objective immunogenicity evaluation, blood samples obtained by immunizing prevena 13 ® as a control were analyzed together.
(2) 평가 결과(2) evaluation result
먼저 13 가 백신 제제에 있어서, 상기 실시예 3의 방법을 통해 제조한 조성물 (LBVE013)과 프리베나13®의 면역원성을 비교 평가하였다. 그 결과, 본 발명의 실시예를 통해 제조된 13 가 면역원성 조성물 (LBVE013)은 프리베나13®에 비해 모든 혈청형에서 프리베나13® 보다 높은 수준의 IgG 농도 수준이 확인되었다. 특히, 혈청형 1, 6B, 7F, 9V 14 및 19F에서는 프리베나13® 보다 2 ~ 6 배의 우수한 효과를 보였다 (표 1).First, in the trivalent vaccine formulation, the immunogenicity of the composition prepared by the method of Example 3 (LBVE013) and Prevena 13 ® was evaluated. As a result, Example 13 is an immunogenic composition (LBVE013) prepared in the present invention are all relative to Prevenar 13 ® IgG concentration levels higher than prevena 13 ® were identified in the serotype. In particular, serotypes 1, 6B, 7F, 9V 14 and 19F showed two to six times better effects than prevena 13 ® (Table 1).
LBVE013LBVE013 Prevenar13Prevenar13
혈청형Serotype IgG(μg/mL)IgG (μg / mL) 혈청형Serotype IgG(μg/mL)IgG (μg / mL)
19F19F 82.482.4 6A6A 46.546.5
6A6A 64.764.7 6B6B 22.422.4
1414 61.161.1 23F23F 18.318.3
6B6B 59.559.5 19F19F 17.617.6
19A19A 26.726.7 19A19A 17.217.2
23F23F 21.821.8 1414 10.110.1
7F7F 13.413.4 55 8.48.4
55 11.411.4 44 6.76.7
44 9.19.1 7F7F 6.36.3
1One 5.35.3 18C18C 4.44.4
18C18C 4.74.7 33 2.72.7
33 3.03.0 1One 1.61.6
9V9 V 2.72.7 9V9 V 1.21.2
다음으로, 상기 LBVE013에 4 개 이상의 혈청형을 추가하여 제조한 LBVE017의 면역원성을 LBVE013과 비교하였다. 그 결과, 면역원성 조성물이 13 가에서 17 가로 가수가 늘어나면서 전반적인 IgG 역가 저하 현상이 일어나는 것을 확인하였다 (도 1). 다가면역원성 조성물의 면역원성 소실의 문제점을 극복하기 위하여, 본 발명자들은 상기 17 가 면역원성 조성물에 대하여 13가에서 상대적으로 IgG 역가가 높았던 6A, 6B, 14, 19A 및 19F 접합체 각각의 용량을 모두 50 % 수준으로 동시에 감소됨을 확인하였다. 그 결과, 전반적으로 면역원성 증가 현상이 일어나는 것을 확인할 수 있었다 (도 1). 특히 혈청형 5, 6A, 6B, 14, 18C, 19A의 경우에는 13 가 보다도 더 높은 수준의 IgG 역가가 확인되었다. Next, the immunogenicity of LBVE017 prepared by adding four or more serotypes to LBVE013 was compared with LBVE013. As a result, the immunogenic composition was confirmed that the decrease in the overall IgG titer occurs when the 17-valent valence is increased from 13 (FIG. 1). In order to overcome the problem of immunogenic loss of the polyimmunogenic composition, the inventors of the present invention have all administered the doses of each of the 6A, 6B, 14, 19A and 19F conjugates, which had relatively high IgG titers at 13 valent relative to the 17-valent immunogenic composition. It was confirmed that the reduction to the 50% level at the same time. As a result, it was confirmed that an overall increase in immunogenicity occurs (Fig. 1). Especially for serotypes 5, 6A, 6B, 14, 18C, and 19A, IgG titers at levels higher than 13 were found.
상기 결과에 기반하여 본 발명자들은 상기 특정 혈청형인 6A, 6B, 14, 19A 및 19F에 대한 투입 용량(input dose)을 동시에 감소시킴으로써 다가 폐렴구균 접합체 백신의 전반적인 혈청형 특이적 IgG 역가를 증가시킬 수 있을 것으로 판단하고, 상기 5 가지 접합체의 용량을 1 내지 90 % 수준의 투입 용량을 조절함으로써 상기 5가지 접합체의 유효한 투입 용량 범위를 확인하였다. 그 결과, 상기 5 가지 접합체의 투입 용량을 95 % 이하에서 25 % 초과 수준, 구체적으로 50 내지 90 % 수준으로 동시에 감소시킨 경우 (즉, 17가 90 %, 17가 75 %, 17가 50 %)에는 13가 면역원성 조성물과 비슷한 수준으로 전반적으로 감소된 면역원성이 회복되는 것을 확인할 수 있었다 (도 2 및 3). 따라서, 상기 시험 결과를 통해, 본 발명에서는 17 가 면역원성 조성물을 제조함에 있어서, 혈청형 6A, 6B, 14, 19A 및 19F의 접합체 용량을 동시에 일정 수준으로 감소시키는 경우 오히려 전체적인 면역원성이 회복되거나 증가되는 것을 확인하였다. 이에, 본 발명에 따른 면역원성 조성물은 백신의 전반적인 면역원성에 부정적인 영향을 줄 수 있는 가능한 면역 간섭현상을 극복하기 위한 실용적인 방안을 제공함으로써 폐렴구균 접합 백신을 개발하는데 매우 유용할 수 있다.Based on the results, we can increase the overall serotype specific IgG titer of the multivalent pneumococcal conjugate vaccine by simultaneously reducing the input doses for the specific serotypes 6A, 6B, 14, 19A and 19F. It was determined that there was, and the effective dosage range of the five conjugates was confirmed by adjusting the dosage of the five conjugates at a dosage level of 1 to 90%. As a result, when the input doses of the five conjugates were simultaneously reduced from 95% or less to more than 25%, specifically from 50 to 90% (i.e. 17% 90%, 17% 75%, 17% 50%) It was confirmed that the reduced immunogenicity is generally recovered to a level similar to the 13-valent immunogenic composition (FIGS. 2 and 3). Thus, through the above test results, in the present invention, in the preparation of the 17-valent immunogenic composition, when the conjugate doses of serotypes 6A, 6B, 14, 19A and 19F are simultaneously reduced to a certain level, the overall immunogenicity is restored or It was confirmed to increase. Thus, the immunogenic composition according to the present invention can be very useful for developing pneumococcal conjugate vaccines by providing a practical way to overcome possible immune interference which can negatively affect the overall immunogenicity of the vaccine.
비교예 1. 13 가에서 용량을 조절한 경우 면역원성의 평가 Comparative Example 1. Evaluation of immunogenicity when dose was adjusted at 13
본 발명자들은 17가 조성물에서 확인한 것과 같은 면역반응의 회복이 상기 결합체 각각의 투입 용량을 동시에 감소시킴으로써 13가 조성물에서도 유사하게 관찰될 수 있는지 확인하고자 하였다. 이에, 13 가에서도 상기 5 가지 혈청형의 투여 접합체양을 각각 50 % 혹은 25 %로 감소시켜 13 가 면역원성 조성물을 제조하고, 이의 면역원성을 본원발명의 100 %의 13 가 면역원성 조성물 (LBVE013) 및 프리베나 13®과 비교하였다. 그 결과, 13 가에서는 상기 17 가의 결과와는 달리 5 가지 혈청형의 용량을 감소시켰음에도 전체적인 면역원성이 회복되지 않았다 (오히려 감소하였다) (도 4). 이러한 결과는 본원발명의 17 가 면역원성 조성물에서 일부 혈청형의 용량을 줄여 확인한 결과가 당업자가 예측가능한 수준을 벗어나는 것이라는 것을 의미한다.The inventors have sought to see if recovery of the immune response as seen in the 17-valent composition can be similarly observed in the 13-valent composition by simultaneously reducing the dose of each of the conjugates. Accordingly, even at 13-valent, the amount of the conjugate conjugate of the five serotypes was reduced to 50% or 25%, respectively, to prepare a 13-valent immunogenic composition, and its immunogenicity was 100% of the 13-valent immunogenic composition of the present invention (LBVE013). ) And Prevena 13 ® . As a result, the total immunogenicity did not recover (rather, decreased) in the thirteen, even though the doses of the five serotypes were reduced, unlike the results of the seventeen. This result means that the result of reducing the dose of some serotypes in the 17-valent immunogenic composition of the present invention is beyond the level predictable by those skilled in the art.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (17)

17종의 협막 다당류 (capsular polysaccharide)-운반 단백질 접합체를 포함하고, Contains 17 capsular polysaccharide-carrying protein conjugates,
여기서 상기 접합체는 스트렙토코커스 뉴모니애 (Streptococcus pneumoniae) 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 (capsular polysaccharide) 각각이 각각의 운반 단백질에 공유결합적으로 접합된 것인, 폐렴구균병의 예방을 위한 백신 조성물.Wherein the conjugate is from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F A vaccine composition for preventing pneumococcal disease, wherein each of 17 capsular polysaccharides is covalently conjugated to respective carrier proteins.
제1항에 있어서, 상기 운반 단백질은 파상풍톡소이드, 백일해 톡소이드, 콜레라 톡소이드, 대장균 (E. coli) LT, 대장균 ST, 슈도모나스 애루지노사 (Pseudomonas aeruginosa) 유래의 외독소 A, 외막복합체 c (OMPC), 포린, 트랜스페린 결합 단백질, 뉴모리신, 폐렴구균 표면 단백질 A (PspA), 폐렴구균 어드헤신 (adhesin) 단백질 (PsaA), 그룹 A 또는 그룹 B 연쇄구균 유래의 C5a 펩티다제, 헤모필러스 인플루엔자(Haemophilus influenzae) 단백질 D, 오브알부민, 키홀 림펫 헤모시아닌 (KLH), 소 혈청 알부민 (BSA), 투베르쿨린의 정제된 단백질 유도체 (PPD), CRM173, CRM228, CRM45 및 CRM197로 이루어진 군에서 선택된, The method of claim 1, wherein the carrier protein is tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa , outer membrane complex c (OMPC), Porin, transferrin binding protein, pneumolysine, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase from group A or group B streptococci, haemophilus influenza ( Haemophilus influenzae ) protein D, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derivatives of tuberculin (PPD), CRM173, CRM228, CRM45 and CRM197,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제1항에 있어서, 상기 운반 단백질은 CRM197 단백질인, 폐렴구균병의 예방을 위한 백신 조성물.The vaccine composition of claim 1, wherein the carrier protein is CRM197 protein.
제3항에 있어서, 상기 접합체는 시아닐화 방법을 사용하여 협막 다당류 및 운반 단백질이 -O-C(NH)-NH- 기로 연결된 구조를 가지는 것인, 폐렴구균병의 예방을 위한 백신 조성물.The vaccine composition according to claim 3, wherein the conjugate has a structure in which the capsular polysaccharide and the carrier protein are connected by -O-C (NH) -NH- group using a cyanylation method.
제4항에 있어서, 상기 시아닐화 방법은, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) 또는 CNBr을 사용하여 수행되는 것인,The method of claim 4, wherein the cyanylation method is carried out using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제1항에 있어서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비,The method of claim 1, wherein compared to the capsular polysaccharide derived from serotype 1 in the composition,
혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고,Capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F each have a content ratio of greater than 0.25 and less than 0.95,
혈청형 6B 유래의 협막 다당류는 0.5를 초과하고, 1.9 이하의 함량비를 가지고,Capsular polysaccharides derived from serotype 6B are greater than 0.5 and have a content ratio of 1.9 or less,
혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 존재하는,Capsular polysaccharides from serotype 22F and capsular polysaccharides from 33F are present in the composition in equivalent amounts to capsular polysaccharides from serotype 1,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제1항에 있어서, 상기 조성물에서 혈청형 1 유래 협막 다당류 대비, The method of claim 1, wherein compared to serotype 1-derived capsular polysaccharide in the composition,
혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.4 내지 0.95 함량비이고,Capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F are 0.4 to 0.95, respectively,
혈청형 6B 유래의 협막 다당류는 0.8 내지 1.9 함량비이고,Capsular polysaccharide derived from serotype 6B has a content ratio of 0.8 to 1.9,
혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 존재하는,Capsular polysaccharides from serotype 22F and capsular polysaccharides from 33F are present in the composition in equivalent amounts to capsular polysaccharides from serotype 1,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제1항에 있어서, 상기 조성물은 애쥬번트, 보존제, 완충제, 냉동보호제, 염, 2가 양이온, 비이온성 세제 및 자유 라디칼 산화 억제제로 이루어진 군에서 선택된 하나 이상을 더 포함하는,The method of claim 1, wherein the composition further comprises one or more selected from the group consisting of adjuvant, preservative, buffer, cryoprotectant, salt, divalent cation, nonionic detergent and free radical oxidation inhibitor,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제8항에 있어서, 상기 애쥬번트는 알루미늄 포스페이트, 알류미늄 설페이트 및 알루미늄 하이드록사이드로 이루어진 군에서 선택된,The method of claim 8, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
제8항에 있어서, 상기 보존제는 페녹시에탄올 (2-phenoxyethanol), 포름알데히드 (formaldehyde), 또는 이들의 혼합물인, 폐렴구균병의 예방을 위한 백신 조성물. The vaccine composition of claim 8, wherein the preservative is phenoxyethanol, formaldehyde, or a mixture thereof.
제1항에 있어서, The method of claim 1,
상기 백신 조성물은 생리학적으로 허용되는 담체를 더 포함하는,The vaccine composition further comprises a physiologically acceptable carrier,
폐렴구균병의 예방을 위한 백신 조성물.Vaccine composition for the prevention of pneumococcal disease.
17종의 협막 다당류 (capsular polysaccharide)-운반 단백질 접합체를 포함하고, Contains 17 capsular polysaccharide-carrying protein conjugates,
여기서 상기 접합체는 스트렙토코커스 뉴모니애 혈청형 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F 및 33F 유래의 17 종의 협막 다당류 각각이 운반 단백질에 공유결합적으로 접합된 것인, 폐렴구균에 대한 면역원성 조성물.Wherein the conjugate comprises 17 types of capillaries from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. An immunogenic composition for pneumococcal, wherein each of the polysaccharides is covalently conjugated to a carrier protein.
제12항에 있어서, 상기 운반 단백질은 CRM197 단백질인, 폐렴 구균병에 대한 면역원성 조성물.The immunogenic composition of claim 12, wherein the carrier protein is CRM197 protein.
제13항에 있어서, 상기 접합체는 시아닐화 방법을 사용하여 협막 다당류 및 운반 단백질이 -O-C(NH)-NH- 기로 연결된 구조를 가지는 것인, 폐렴 구균병에 대한 역원성 조성물.The composition of claim 13, wherein the conjugate has a structure in which the capsular polysaccharide and the carrier protein are connected by a -O-C (NH) -NH- group using a cyanylation method.
제12항에 있어서, 상기 조성물에서 혈청형 1 유래의 협막 다당류 대비,The method of claim 12, wherein compared to the capsular polysaccharide derived from serotype 1 in the composition,
혈청형 6A, 14, 19A 및 19F는 각각 0.25를 초과하고, 0.95 이하인 함량비를 가지고,Serotypes 6A, 14, 19A and 19F each have a content ratio of greater than 0.25 and less than or equal to 0.95,
혈청형 6B는 0.5를 초과하고, 1.9 이하의 함량비를 가지고,Serotype 6B is greater than 0.5 and has a content ratio of 1.9 or less,
혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 존재하는, Capsular polysaccharides from serotype 22F and capsular polysaccharides from 33F are present in the composition in equivalent amounts to capsular polysaccharides from serotype 1,
폐렴구균에 대한 면역원성 조성물.Immunogenic Compositions against Pneumococci.
제12항에 있어서, 상기 조성물에서 혈청형 1 유래 협막 다당류 대비, The method of claim 12, wherein compared to serotype 1-derived capsular polysaccharide in the composition,
혈청형 6A, 14, 19A 및 19F 유래의 협막 다당류는 각각 0.4 내지 0.95 함량비이고,Capsular polysaccharides derived from serotypes 6A, 14, 19A and 19F are 0.4 to 0.95, respectively,
혈청형 6B 유래의 협막 다당류는 0.8 내지 1.9 함량비이고,Capsular polysaccharide derived from serotype 6B has a content ratio of 0.8 to 1.9,
혈청형 22F 유래의 협막 다당류 및 33F 유래의 협막 다당류는 혈청형 1 유래의 협막 다당류와 동량으로 조성물 내에 존재하는,Capsular polysaccharides from serotype 22F and capsular polysaccharides from 33F are present in the composition in equivalent amounts to capsular polysaccharides from serotype 1,
폐렴구균에 대한 면역원성 조성물.Immunogenic Compositions against Pneumococci.
제1항 또는 제12항의 조성물을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는, 폐렴구균을 예방 하는 방법.A method of preventing pneumococcal, comprising administering to a subject in need thereof the composition of claim 1.
PCT/KR2017/011959 2016-10-28 2017-10-27 Multivalent immunogenic composition having increased igg titer, and use thereof WO2018080213A1 (en)

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