WO2020009462A1 - Composition immunogène multivalente comprenant des conjugués polysaccharide-protéine et vaccin la comprenant pour prévenir une maladie provoquée par streptococcus pneumoniae - Google Patents

Composition immunogène multivalente comprenant des conjugués polysaccharide-protéine et vaccin la comprenant pour prévenir une maladie provoquée par streptococcus pneumoniae Download PDF

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WO2020009462A1
WO2020009462A1 PCT/KR2019/008113 KR2019008113W WO2020009462A1 WO 2020009462 A1 WO2020009462 A1 WO 2020009462A1 KR 2019008113 W KR2019008113 W KR 2019008113W WO 2020009462 A1 WO2020009462 A1 WO 2020009462A1
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polysaccharide
immunogenic composition
multivalent immunogenic
streptococcus pneumoniae
protein
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PCT/KR2019/008113
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Korean (ko)
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최석근
이찬규
김락기
김희연
이성균
황윤희
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주식회사 유바이오로직스
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Priority claimed from KR1020190079797A external-priority patent/KR20200005458A/ko
Publication of WO2020009462A1 publication Critical patent/WO2020009462A1/fr

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

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  • the present invention relates to immunogenic compositions comprising multivalent pneumococcal polysaccharide-protein conjugates, and pharmaceutical compositions comprising the same.
  • Streptococcus pneumoniae is a major causative agent of pneumonia. According to the National Statistical Office, the mortality rate from pneumonia in 2010 was 14.9 per 100,000, one of the top 10 causes of death, an 82.9% increase from 2000. In addition, in 2012, according to the WHO, 476,000 HIV-negative children under the age of five worldwide died from infections caused by Streptococcus pneumoniae in 2008, and 5% of all children under 5 years of age were caused by the bacteria. Died.
  • Dr. The 14-valent polysaccharide vaccine was developed by Robert Austrian and then developed into a 23-valent polysaccharide vaccine.
  • Multivalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high risk patients. However, infants and children are not immune to most pneumococcal polysaccharides due to T-cell independent immune response.
  • the valent pneumococcal conjugate vaccine (Prevnar®) contains capsular polysaccharides from the seven most common serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.
  • Prevna covers approximately 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease in the United States, Europe, and other parts of the world, respectively.
  • Surveillance data accumulated over the years following the introduction of Prevna clearly predicted a reduction in invasive pneumococcal disease caused by serotypes included in Prevna in the United States.
  • serotype coverage was a limit to the serotype coverage and increased invasive pneumococcal disease caused by serotypes not included in Prevna, especially 19A.
  • PCV-13 contains six additional serotypes (1, 3, 5, 6A, 7F, 19A) in addition to the seven serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) included in Prevna. Pneumococcal conjugate vaccine. According to the U.S.
  • ABSs Active Bacterial Core surveillance
  • PCV Physicalcoccal conjugate vaccine
  • pneumococcal conjugate vaccine Current on the market is an invasive disease caused by pneumococcal (serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, etc.) It is used as a vaccine to prevent.
  • the pneumococcal vaccine uses CRM 197 , a non-toxic variant of diphtheria toxoid, as a transport protein, and chemical binding to polysaccharides of each serotype is carried out through reductive amination and activating hydroxyls of the polysaccharides. Induces covalent binding of proteins.
  • chemical bonding using reductive amination may change the epitope of the polysaccharide because the procedure is rather complicated.
  • Polysaccharide vaccines with altered epitopes show differences in immune responses, which affect the immunogenicity of the vaccine.
  • serotype replacement has been shown by some serotypes showing antibiotic resistance and multi-tolerance.
  • interregional variations in serotype distributions have raised regional limits for prebes or coverage. Therefore, there was a need to further expand the scope by adding serotypes from existing pneumococcal conjugate vaccines.
  • the present invention provides a multivalent immunogenic composition which can induce excellent serum IgG titers and which can be usefully used to prevent diseases caused by pneumococci in infants, children, and adults.
  • the multivalent immunogenic composition comprises a physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates, wherein each polysaccharide-protein conjugate is of a different serotype conjugated to a carrier protein.
  • a physiologically acceptable vehicle and 15 different polysaccharide-carrying protein conjugates, wherein each polysaccharide-protein conjugate is of a different serotype conjugated to a carrier protein.
  • Capsular polysaccharide derived from Streptococcus pneumoniae, the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A , 19F, 22F and 23F.
  • polysaccharide-carrying protein conjugate may be conjugated with ethylene glycol dihydrazide or a derivative thereof represented by the following Chemical Formula 1.
  • the carrier protein may be CRM 197 .
  • the multivalent immunogenic composition may further include an adjuvant.
  • the adjuvant may be selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
  • a pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of the multivalent immunogenic composition.
  • the polyvalent immunogenic composition is 2 to 5 ⁇ g of each sugar; 30-60 ⁇ g CRM 197 transport protein; 0.1 to 0.3 mg of aluminum adjuvant; And a single 0.5 mL dose formulated to contain sodium chloride as excipient.
  • a physiologically acceptable vehicle and fifteen different polysaccharide-carrying protein conjugates, each polysaccharide-carrier protein conjugate, each of the different serotypes of Streptococcus pneumoniae conjugated to a carrier protein
  • Capsular polysaccharide derived from Streptococcus pneumoniae the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F
  • administering a pharmaceutical composition comprising an immunologically effective amount of a multivalent immunogenic composition prepared from 23F.
  • a physiologically acceptable vehicle and fifteen different polysaccharide-carrying protein conjugates, each polysaccharide-carrier protein conjugate, each of the different serotypes of Streptococcus pneumoniae conjugated to a carrier protein
  • Capsular polysaccharide derived from Streptococcus pneumoniae the capsular polysaccharide is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F
  • the multivalent immunogenic composition prepared from 23F, for the prevention of diseases caused by pneumococci.
  • Multivalent immunogenic compositions according to the invention comprise capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F pneumococcal serotypes This can lead to good serum IgG titers and functional antibody activity.
  • the multivalent immunogenic composition according to the present invention due to the added serotypes 11A and 22F, not only the immunogenicity of the remaining 13-valent serotypes does not appear, It can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and especially in vaccines suitable for infant vaccination.
  • Figure 2 is a graph showing the immunogenic OPA results for the rabbit experiment according to Test Example 1.
  • the present invention includes a physiologically acceptable vehicle, and 15 different polysaccharide-carrying protein conjugates, wherein each polysaccharide-protein conjugate is a different serotype of Streptococcus pneumoniae conjugated to a carrier protein.
  • Derived capsular polysaccharide wherein the capsular polysaccharide is prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F.
  • Multivalent immunogenic compositions are provided.
  • a pharmaceutical composition for inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate comprising an immunologically effective amount of a multivalent immunogenic composition.
  • Serotype replacement has been shown by some serotypes showing antibiotic resistance and multi-tolerance.
  • interregional variations in serotype distributions have raised regional limits for prebes or coverage. Therefore, there was a need to broaden the scope of application by adding serotypes rather than removing serotypes from existing pneumococcal conjugate vaccines.
  • Capsular polysaccharides can be prepared by standard techniques known to those skilled in the art, and capsular polysaccharides can be reduced in size to reduce viscosity or to increase the solubility of activated capsular polysaccharides.
  • capsular polysaccharides can be prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F and 23F of Streptococcus pneumoniae.
  • pneumococcal conjugates are prepared by separate procedures and formulated into a single dosage form.
  • each pneumococcal polysaccharide serotype is grown in soy-based medium, and the individual polysaccharides are then purified by centrifugation, precipitation, ultrafiltration.
  • the carrier protein is preferably a nontoxic, nonreactogenic, obtainable protein in sufficient quantity and purity.
  • the carrier protein should be appropriate for standard conjugation methods.
  • the carrier protein may be CRM 197 .
  • CRM 197 is a non-toxic variant (toxoid) of diphtheria toxin isolated from the culture of Corynebacterium diphtheria strains grown in cassanoic acid and yeast extract-based medium. CRM 197 is purified via ultrafiltration, ammonium sulphate precipitation and ion exchange chromatography.
  • diphtheria toxoids can also be used as carrier proteins.
  • suitable carrier proteins include exotoxin A from tetanus toxoid, pertussis toxoid, cholera toxoid (WO 2004/083251), E. coli LT, E. coli ST, and Pseudomonas aeruginosa. Such inactivated bacterial toxins are included.
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group C5a peptidase from B streptococci, or Haemophilus influenzae protein D may also be used.
  • Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivatives of tuberculin (PPD) can also be used as carrier proteins.
  • Variants of diphtheria toxins such as CRM 173 , CRM 228 and CRM 45 can also be used as carrier proteins.
  • Purified polysaccharides are chemically activated to produce sugars that can react with the carrier protein. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein can be performed by known methods.
  • a hydrophilic conjugate may be prepared by synthesizing an ethylene glycol group in a hydrazide linker. That is, the polysaccharide-carrying protein conjugate may provide an immunogenic composition, wherein the polysaccharide-conjugated protein conjugate is conjugated with ethylene glycol dihydrazide and a derivative thereof.
  • n may be preferably 1 to 10, more preferably, may be 1 to 5, most preferably, may be 1 to 3, but is not limited thereto. .
  • the stability of the high aqueous solution of the produced vaccine can be preserved, thereby improving pneumococcal vaccine efficacy.
  • CDAP Cyanodiaminopyridinium tetrafluoroborate
  • EDAC 1-ethyl-3- (3-dimethylaminno propyl) -carbodimide
  • CDAP Cyanodiaminopyridinium tetrafluoroborate
  • EDAC 1-ethyl-3- (3-dimethylaminno propyl) -carbodimide
  • CDAP Cyanodiaminopyridinium tetrafluoroborate
  • EDAC 1-ethyl-3- (3-dimethylaminno propyl) -carbodimide
  • the obtained polysaccharide-protein conjugate can be purified by various methods. Examples of these methods include concentration / diafiltration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed together to formulate into the immunogenic compositions of the invention and used as vaccines. Formulations of immunogenic compositions of the invention can be carried out using methods known in the art. For example, 15 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to make the composition.
  • Examples of such vehicles include, but are not limited to, water, buffered saline, polyols such as glycerol, propylene glycol, liquid polyethylene glycols, and dextrose solutions.
  • the immunogenic composition of the present invention comprises one or more adjuvants.
  • an "adjuvant” is a substance used to increase the immunogenicity of an immunogenic composition of the present invention.
  • adjuvants are often provided to boost the immune response and are well known to those skilled in the art.
  • Adjuvants suitable for increasing the effectiveness of the composition may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, but are not limited thereto.
  • aluminum salts are used as the adjuvant.
  • 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 (III), aluminum hydroxyphosphate adjuvant (APA) ), Amorphous alumina, and the like, but is not limited thereto.
  • APA is a suspension of aluminum hydroxyphosphate. Mixing aluminum chloride and sodium phosphate in a ratio of 1: 1 precipitates aluminum hydroxyphosphate sulfate.
  • a high shear mixer is used to prepare the precipitate in 2-8 ⁇ m, followed by dialysis with physiological saline and sterilization.
  • commercially available Al (OH) 3 eg alhydrogel or Superfos
  • Al (OH) 3 eg alhydrogel or Superfos
  • 50-200 g of protein can be adsorbed per mg of aluminum hydroxide, and the ratio depends on the protein's pi and the pH of the solvent.
  • Low pI proteins bind more strongly than proteins with high pi.
  • Aluminum salts form antigen reservoirs that slowly release antigens for two to three weeks, nonspecifically activating macrophages, complement and innate immune mechanisms.
  • the present invention provides a pharmaceutical composition (eg, a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition.
  • a pharmaceutical composition eg, a vaccine formulation
  • Vaccine preparations according to the invention can be used to protect or treat a person susceptible to pneumococcal by administration via the systemic or mucosal route.
  • an "effective amount" refers to a dosage required to elicit an antibody that is capable of significantly reducing the likelihood of becoming infected with Streptococcus pneumoniae or the severity of the infection.
  • Administration includes injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract.
  • intranasal administration is used for the treatment of pneumonia or otitis media, because it can more effectively prevent nasopharyngeal carriers of pneumococcus, thereby weakening the infection at an early stage.
  • the amount of the conjugate at each vaccine dose is chosen to be an amount that induces an immunoprotective response without significant side effects. This amount may vary depending on the serotype of pneumococcal. In general, each dose will comprise 0.1 to 100 ⁇ g, preferably 0.1 to 10 ⁇ g, more preferably 1 to 5 ⁇ g polysaccharide.
  • Optimal amounts of ingredients for a particular vaccine can be identified by standard studies involving the observation of an appropriate immune response in a subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose for humans. Dosage can also be determined empirically.
  • the vaccine compositions of the invention are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, respectively, conjugated to CRM 197 , And sterile liquid formulation of 23F pneumococcal capsular polysaccharide.
  • each polysaccharide At each 0.5 mL dose of 2 to 5 ⁇ g of each polysaccharide; 30-60 ⁇ g CRM 197 carrier protein; 0.1 to 0.3 mg of aluminum adjuvant; And sodium chloride as excipients.
  • the liquid may be filled into a single dose syringe or glass vial without preservatives. After shaking, the vaccine is a homogeneous white suspension that can be administered intramuscularly immediately.
  • the compositions of the invention may be administered in a single inoculation.
  • the vaccine composition of the present invention may be administered twice, three times, four times or more at appropriate intervals, for example at 1, 2, 3, 4, 5, or 6 month intervals, or It can be administered according to any combination thereof.
  • the immunization plan can follow the one specified for Prevna.
  • routine vaccination plans for infants and babies are 2, 4, 6 and 12 to 15 months of age.
  • the composition is inoculated four times at 2, 4, 6 and 12 to 15 months of age.
  • Compositions of the present invention may also comprise one or more proteins from Streptococcus pneumoniae.
  • Streptococcus pneumoniae proteins suitable for inclusion include not only the proteins described in WO 02/053761, but also the proteins identified in WO 02/083855.
  • compositions of the present invention can be administered to a subject by one or more routes of administration, parenteral, transdermal or mucosal, intranasal, intramuscular, intraperitoneal, intradermal, intravenous or subcutaneous routes known to those skilled in the art, and can be formulated accordingly.
  • the composition of the present invention is a liquid formulation comprising injection via intramuscular, intraperitoneal, epidermal, vein, arterial or subcutaneous routes; Or by respiratory mucosal injection.
  • Liquid preparations for injection include solutions or the like.
  • Streptococcus pneumoniae culture and polysaccharide purification were performed by the process development method developed by our company.
  • Streptococcus pneumoniae Each serotype of Streptococcus pneumoniae was obtained from the Culture Collection University of Goteborg (CCUG). The obtained methods and criteria for the characterization of Streptococcus pneumoniae were referred to CDC's Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis , Streptococcus pneumoniae , and Hemophilus influenzae , Chapter 8: Identification and Characterization of Streptococcus pneumoniae .
  • cryopreserved 1 vial cryopreserved was thawed and seeded in soy-based medium and cultured at 37 ° C. and 5% CO 2 .
  • target absorbance OD 600nm 0.6 ⁇ 1.2 was reached inoculated in this culture fermenter was adjusted to maintain 37 °C, pH 7.0 ⁇ 7.2, 60 ⁇ 90rpm, glucose 1.5 ⁇ 2.0%.
  • Polysaccharides of 15 different serotypes were sized in the target molecular weight range by mechanical methods. Polysaccharides of the target molecular weight were chemically activated and unreacted by ultrafiltration / diafiltration. The activated polysaccharide was conjugated while forming an amide bond by CRM 197 and EDAC, purified using ultrafiltration / diafiltration, and finally filtered through a 0.2 ⁇ m disinfection filter.
  • Each serotype polysaccharide solution was prepared with a final concentration range of 5.0-10.0 mg / mL.
  • serotypes 1, 4, 5, 7F, 11A, 14, 19A, 19F, 22F, and 23F either omitted target molecular weight control or performed one or three target molecular weight control processes at 5000 to 15000 psi pressure. .
  • Serotypes 6A, 6B, 9V, 18C performed the target molecular weight adjustment process 1-3 times at 15000-25000 psi pressure in the same manner.
  • Serotype 3 was subjected to the target molecular weight adjustment process 1-3 times at a pressure of 25000-30000 psi in the same manner.
  • the polysaccharide molecular weight and viscosity of each serotype were determined for the degree of application of the process, and it was confirmed that the target molecular weight range was reached using SEC-MALS (in-process test).
  • Table 1 below shows the low molecular weight device conditions and target molecular weight range for each serotype.
  • the mixed solution of which the activation reaction is completed is subjected to a buffer solution exchange and concentration of activated polysaccharide at 10 to 30 kDa MWCO ultrafiltration filter with water for injection at least 20 to 40 multiples of the process solution.
  • the permeate was discarded and the residue was filtered through a 0.45 ⁇ m filter.
  • the ADH (%) content of the filtered process solution was measured using TNBSA, and the residual DMAP amount was checked using RP-HPLC (in-process inspection).
  • a transport protein CRM 197 is used in the conjugation reaction of 10 to 30 kDa MWCO ultrafiltration using a filtration filter buffer solution of 0.1M MES (2-Morpholinoethanesulfonic acid monohydrate ) Buffer (pH 6.0 ⁇ 0.2) to 10 to 20 of the process solution CRM 197 Buffer exchange and concentration were carried out above the drainage. The permeate was discarded and the residue was filtered through a 0.45 ⁇ m filter.
  • CRM 197 in 0.1 M MES Buffer was diluted to a concentration of 1.5 to 4.0 mg / mL using 0.1 M MES Buffer.
  • Activated polysaccharide was diluted with water for injection at a concentration of 2.5-5.0 mg / mL.
  • 1M MES Buffer was added to the activated polysaccharide to prepare activated polysaccharide in the 0.1M MES Buffer state.
  • Mixed with CRM 197 carrier protein in the same weight ratio of 0.1 M MES Buffer, 0.1 M MES Buffer was added so that the activated polysaccharide and delivery protein were in the concentration of 0.8 to 2.0 mg / mL in the final mixed solution, respectively.
  • CRM 197 was added to the activated polysaccharide and mixed at 19-25 ° C. for 3-5 minutes.
  • a 1-5 M solution (in 0.1 M MES Buffer) of 1-ethyl-3-(-3dimethylaminopropyl) carbodiimide hydrochloride) coupling reagent was prepared and added to a final EDAC concentration of 0.05 to 0.25 M. After mixing, the mixture was mixed for 3 to 5 minutes at 19 to 25 ° C. The conjugation reaction was carried out at 2 to 8 ° C. for 14 to 16 hours.
  • the conjugation reaction solution was mixed with a 100 kDa MWCO ultrafiltration filter, and the buffer solution was exchanged and concentrated at a concentration of 20 to 40 or more of 1X PBS (Sodium phosphate) Buffer (pH 7.2 ⁇ 0.5).
  • 1X PBS sodium phosphate
  • Buffer pH 7.2 ⁇ 0.5
  • the final conjugate was diluted to 200-600 ⁇ g / mL concentration with 1 ⁇ PBS Buffer and filtered through a 0.22 ⁇ m filter.
  • the final conjugate stock was refrigerated at 2-8 ° C.
  • the conjugated polysaccharide and protein (polysaccharide-protein) contents were analyzed by anthrone assay and lowry assay, respectively.
  • Free polysaccharide unbound with protein was precipitated polysaccharide-protein conjugate with DOC (Deoxycholic acid), then obtained by the free polysaccharide of the supernatant layer and analyzed by anthrone assay (%). Calculated.
  • the free polysaccharide ratio was 40% or less, and the polysaccharide / CRM197 conjugate ratio was 0.3-2.0, as shown in Table 2 below. It can be seen that the distribution.
  • Serotype Free polysaccharide content (%) Polysaccharide / protein splicing ratio One 10 to 30% 0.5 to 1.2 3 15 to 40% 0.5 to 1.2 4 10 to 30% 0.5 to 1.2 5 15 to 40% 0.7 to 1.5 6A 10 to 30% 0.5 to 1.2 6B 10 to 30% 0.5 to 1.2 7F 15 to 40% 0.5 to 1.2 9 V 10 to 30% 0.8 to 1.5 11A 10 to 30% 0.5 to 1.2 14 15 to 40% 0.8 to 1.5 18C 15 to 40% 0.3 to 1.0 19A 15 to 40% 0.5 to 1.2 19F 15 to 40% 0.5 to 1.2 22F 10 to 30% 0.5 to 1.2 23F 10 to 30% 0.5 to 1.2
  • the dose of conjugate included in each vaccine was chosen in such a way that there were no side effects and induce an immune response. This amount may vary depending on the serotype of pneumococcal. Based on the results of animal experiments, the vaccination dose in humans can be determined.
  • the required amount of final bulk concentrate was calculated based on the bulk saccharide concentration.
  • Each of the 15 polysaccharides was mixed into a concentrate, followed by the slow mixing of aluminum phosphate.
  • the formulated bulk product was stored at 2-8 ° C.
  • the resulting vaccine composition contained 2.2 ug of each polysaccharide, 4.4 ug for 6B, 30-60 ug of CRM 197 carrier protein and 0.125 mg of aluminum phosphate adjuvant in 0.5 mL total.
  • Test Example 1 Evaluation of immunogenicity of the multivalent pneumococcal conjugate
  • Prevnar13® R48621
  • Aluminum phosphate was used as the adjuvant and was prepared by adding the same amount as Prevnar13.
  • the antigen injection amount of rabbit was used 500uL of the same as humans, and the actual amount of polysaccharide of each antigen was as shown in Table 3.
  • Group Serotypes and Antigen Levels Positive control group 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F 2.2ug, 6B 4.4ug (13-valent complex)
  • Test group 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 11A, 22F 2.2ug, 6B 4.4ug (15-valent complex)
  • the antigen injection schedule was boosted 2 weeks and 4 weeks after the first injection, and blood samples were collected before antigen injection to obtain serum samples of 0, 2, 4, 6 and 8 weeks.
  • Antibody production rate was measured by indirect ELISA method, and 15 polysaccharides sold by ATCC were standardized and coated at 100ng / well concentration, and 6 weeks of serum was 10 ⁇ g / mL of Pneumococcal Cell Wall Polysaccharide (CWPS Multi). Measured by diluting to 1/204800 ⁇ with dilution buffer added at 1/200 ⁇ . The absorbance values measured at 450 nm were plotted in a fourth-order equation, and the dilution factor of the value at which the absorbance was “1” was calculated to be an ELISA unit.
  • CWPS Multi Pneumococcal Cell Wall Polysaccharide
  • the single group injected with a single serotype was superior to all the serotypes compared to the positive control group, and was equal to or higher than the test group except for serotypes 14 and 23F.
  • Serotype ELISA unit Positive control group (13) Test group (15) Mono 13 common serotypes One 3051 8800 18174 3 2310 5326 4920 4 3500 5359 25000 5 1968 4519 13490 6A 3090 4777 18412 6B 3000 9350 9000 7F 19300 6744 76151 9 V 4196 6033 25825 14 1538 7321 2582 18C 6825 8205 30000 19A 2324 9756 7767 19F 889 9110 7586 23F 489 7896 968 2 additional serotypes 11A 3 5928 4854 22F 3 10536 5517
  • Serum functional antibody titer was evaluated by OPA assay. HL-60 cells were differentiated and incubated for 72 hours in 0.8% DMF. Each individual serum was diluted 1: 7 in opsonization buffer (Hanks' balanced salt solution containing Ca 2+ and Mg 2+ , 0.1% gelatin, 10% inactivated FBS). Pneumococci of each serotype stored at -80 ° C were dissolved and mixed with serum and stirred at 700 rpm at room temperature for 30 minutes.
  • the ratio of differentiated HL-60 cells and pneumococci was 400: 1 and Baby rabbit complement was mixed with 12.5%. After reacting for 45 minutes at 700 rpm shaking platform at 37 °C, 5% CO 2 condition was inoculated on 1.5% THY agar plate. After the plate was completely dried, 0.75% THY agar containing triphenyl tetrazolium chloride dye (TTC, 1mg / mL) was poured. Incubated at 37 ° C., 5% CO 2 . The colony of pneumococci was calculated using the NICE software and the opsonic index was measured using the Opsotiter3 program.
  • the multivalent immunogenic composition according to the present invention includes 15 capsular polysaccharides derived from 15 pneumococcal serotypes including serotypes 11A and 22F, thereby inducing excellent serum IgG titer and functional antibody activity. It can be seen that the multivalent immunogenic composition according to the present invention can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and in particular, it can be used in vaccines suitable for primary vaccination.
  • the multivalent immunogenic composition according to the present invention does not show the immunogenicity lowering of the remaining 13 valent serotypes due to the added serotypes 11A and 22F, It can be usefully used for preventing diseases caused by pneumococci in infants, children, and adults, and especially in vaccines suitable for infant vaccination.

Abstract

La présente invention concerne une composition immunogène multivalente comprenant des conjugués polysaccharide-protéine de Streptococcus pneumoniae et une composition pharmaceutique la comprenant. La composition immunogène multivalente selon un aspect de la présente invention comprend un véhicule physiologiquement acceptable et 15 conjugués polysaccharide-protéine porteuse différents, chacun des conjugués polysaccharide-protéine comprenant un polysaccharide capsulaire issu de Streptococcus pneumoniae d'un type de sérum différent fixé à une protéine porteuse, le polysaccharide capsulaire étant produit à partir de types sériques de 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F et 23F.
PCT/KR2019/008113 2018-07-06 2019-07-03 Composition immunogène multivalente comprenant des conjugués polysaccharide-protéine et vaccin la comprenant pour prévenir une maladie provoquée par streptococcus pneumoniae WO2020009462A1 (fr)

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KR1020190079797A KR20200005458A (ko) 2018-07-06 2019-07-03 다가 폐렴구균 다당체-단백질 접합체를 포함하는 면역원성 조성물, 및 이를 포함하는 약학 조성물

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