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WO1998033521A1 - Conjugue polysaccharidique pneumococcique et vaccins conjugue polysaccharidique antipneumococcique a sous-unite b de toxine cholerique - Google Patents

Conjugue polysaccharidique pneumococcique et vaccins conjugue polysaccharidique antipneumococcique a sous-unite b de toxine cholerique Download PDF

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Publication number
WO1998033521A1
WO1998033521A1 PCT/KR1998/000007 KR9800007W WO9833521A1 WO 1998033521 A1 WO1998033521 A1 WO 1998033521A1 KR 9800007 W KR9800007 W KR 9800007W WO 9833521 A1 WO9833521 A1 WO 9833521A1
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conjugate
ctb
acid
mol
poly
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PCT/KR1998/000007
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English (en)
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Seo Young Jeong
Ick-Chan Kwon
Yong-Hee Kim
Seung-Yong Seong
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Korea Institute Of Science And Technology
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Priority to AU56812/98A priority Critical patent/AU5681298A/en
Publication of WO1998033521A1 publication Critical patent/WO1998033521A1/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
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a novel microencapsulated conjugate and conjugate vaccines. More particularly, the present invention relates to a microencapsulated pneumococcal capsular polysaccharide conjugate vaccine for oral administration and a pneumococcal capsular polysaccharide conjugate vaccine for intranasal administration, wherein the vaccines evoke mucosal as well as systemic antibody response to antigen conjugates and provide protection against challenges by Streptococcus pneumoniae.
  • Acute respiratory infection by S.pneumoniae results in more than one million deaths per year worldwide. It is believed that pneumococcus are the leading cause of pneumonia, meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis and conjunctivitis.
  • the S.pneumoniae has been known to colonize in the nasopharynx and to invade into the blood stream after an inflammatory activation of lining cells.
  • a major entry site of pneumococci into the human body is through the mucosal surfaces. Immunization against pneumococci at the mucosal surfaces is most effective because the mucosal immune system is capable of responding to the invading pathogens in the gastrointestinal, respiratory and urogenital tracts by producing pathogen-specific secretory IgA (slgA) and long term serum IgG antibodies.
  • the local slgA has been known to prevent both the colonization at the mucosal tissues and the spread into the systemic circulation more efficiently compared to systemic antibodies. Higher induction of slgA responses could often be achieved through a direct immunization via gut associated lymphoid tissue (GALT), specifically through the Peyer's Patches (PP) of the gastrointestinal (Gl) tract.
  • GALT gut associated lymphoi
  • CT cholera toxin
  • biodegradable and biocompatible microspheres have been used to deliver antigens into the Gl tract.
  • Gelatin, poly ⁇ Alactice), poly glycolic acid and albumin have been used for fabrications of the microspheres.
  • the harsh conditions of preparing the microspheres such as the organic solvent used or the high temperature can denature the antigens.
  • Alginate microspheres (AM) have been preferably used as a carrier of antigens because these microspheres can be prepared in aqueous solutions and at room temperature.
  • AM of less than 10 ⁇ m in diameter can be uptaken at the small intestine, preferably less than 5 ⁇ m in diameter.
  • Microspheres of less than 5 ⁇ m in diameter are transported through the efferent lymphatics, while those larger than 5 ⁇ m in diameter remain in the Peyer's Patches; therefore, the size of the microspheres are an important consideration, i.e., the smaller in diameter the more effectively can the AM be uptaken in the Peyer's Patches.
  • pneumococcal respiratory infections such as meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.
  • a Streptococcus pneumoniae capsular polysaccharide conjugate to be administered intranasally which is prepared by: dissolving a serotype S.pneumococcal capsular polysaccharide (PS) in deionized water and activating the PS; coupling the activated PS with a spacer molecule (SM) under gentle stirring; adding 1 -ethyl-3(3-dimethylaminoproyl)-carbodiimide (EDC) slowly into a solution containing the PS and a cholera toxin B subunit; stopping the reaction, and thus forming crude conjugates; centrifuging the crude conjugates, and dialyzing a supernatant; and, removing unreacted proteins.
  • PS serotype S.pneumococcal capsular polysaccharide
  • SM spacer molecule
  • EDC 1 -ethyl-3(3-dimethylaminoproyl)-carbodiimide
  • Figures 1 (a-c) are scanning electron micrographs of alginate microspheres formed with different alginate concentrations: (a) 1 % (w/v); (b) 3% (w/v); and, (c) 5% (w/v).
  • Figures 2(a-c) are scanning electron micrographs of alginate microspheres formed with various concentration of CaCI 2 in n-octanol: (a) 2% (w/v); (b) 4% (w/v); and, (c) 8% (w/v).
  • Figures 3(a-b) are scanning electron micrographs of alginate microspheres formed with various surfactant: (a) Span 80; and, (b) HCO-10.
  • Figure 4 is a scanning electron micrograph of the alginate microspheres containing PS19-CTB.
  • Figure 5 is a cumulative release profile of an entrapped PS19-CTB from alginate microspheres at 37° C in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Figure 6 is a graph illustrating the antigenicity and binding capacity of PS19-CTB to GM1-gangiioside after microencapsulation.
  • Figure 7 is a graph illustrating an intestinal anti-PS19 IgA responses and serum IgM responses following peroral immunization with various doses of PS19-CTB entrapped in the alginate microspheres.
  • Figures 8(a-c) are confocal laser scanned images of alginate microspheres (AM) with FITC-dextran uptaken in the Peyer's Patch. Wherein Fig. 8(a) illustrates the uptake of the microspheres at hour three (3), and Figs. 8(b) and 8(c) at hour five (5), respectively. Figure 8(c) is magnified at x1890.
  • Figures 9(a-c) are respectively a Sepharcyl S-300 chromatography elution profile of an nonconjugated PS and CTB, a Sepharcyl S-300 chromatography elution profile of a crude conjugate of PS and CTB in the void fraction, and a picture taken by a scanning electron microscope of the microspheres entrapping the PS-CTB conjugates.
  • Figure 10 shows several graphs illustrating serum anti-PS IgM, IgG, and IgA, and as well as bronchoalveolar IgA and intestinal IgA antibody response of mice two weeks after the 3rd oral vaccination.
  • Figure 1 1 is a graph showing an anti-CTB antibody response of the mice after an oral immunization with vaccines according to the present invention.
  • Figure 12 shows several graphs illustrating clearance of pneumococci from the bronchoalveoli (top) and from the blood (bottom) of the mice immunized with vaccines according to the present invention.
  • the present invention provides compositions which are useful for immunization against pneumococcal respiratory infections, and which are effective against other types of pneumococcal infections such as meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.
  • PS pneumococcal capsular polysaccharide
  • CTB cholera toxin B subunit
  • AM(PS-CTB) conjugate vaccine i.e., AM(PS-CTB)
  • intranasal administration of a PS-CTB conjugate vaccine inhibit challenges by live S.pneumoniae.
  • the PS-CTB conjugate vaccines administered intranasally may or may not be administered together with the AM(PS-CTB) conjugate vaccine.
  • composition according to the present invention have demonstrated that oral administration of the AM(PS-CTB) and intranasal administration of the PS-CTB, induced significant increase in levels of serum IgM, serum IgG, serum IgA, bronchoalveolar IgA and intestinal IgA.
  • the AM(PS-CTB) induced bronchoalveolar and intestinal IgA, and prominent serum IgG and serum IgA responses.
  • alginate microspheres were considered as a proper carrier for the efficient delivery of antigens to the Peyer's Patch (PP) and a concomitant transport through the lymphatics, see FIGS 8(a-c).
  • alginate microspheres of less than 5 ⁇ m in diameter can be uptaken at the small intestine; however, the current technology for making the alginate microspheres is still restricted to a size limit of 5 ⁇ m in diameter or larger.
  • a method for preparing the alginate microspheres of less than 5 ⁇ m in diameter to be used as a carrier for an oral mucosal delivery was developed for the present invention.
  • the alginate concentration range can vary from 1 % (w/v) to 10% (w/v), more preferably between l %-6% (w/v), but most preferably less than 5% (w/v).
  • n-octanol is used as the organic solvent in which CaCI 2 is dissolved and slowly diffused into an aqueous phase containing the alginate for a diffusion controlled interfacial gelation.
  • the stirring rate is important to form microemulsion droplets with the desired diameter.
  • the stirring rate range is between 5,000 rpm and 10,000 rpm, preferably at a stirring rate of 8,000 rpm for one (1 ) hour.
  • further continuous stirring at a low speed is required until the gelation is completed to ensure that the hardened microspheres are isolated as discrete particles.
  • the particle size is also dependent upon the concentration of CaCI 2 in the n-octanol and the rate of the addition into the medium.
  • the smallest microspheres( ⁇ 5 ⁇ m) are formed when 1 % (w/v) of CaCI 2 in n-octanol is used.
  • the size of the microspheres could be further reduced by spraying CaCI 2 solution onto the emulsion rather than by adding dropwise using a syringe.
  • PS pneumococcal capsular polysaccharide
  • 11A, 11 B 1 1 C, 1 1 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43,
  • the activated PS is coupled to a spacer molecule (SM), such as 6-aminocaproic acid (AC), or molecules of C4 to C18, such as but not limited to glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, ⁇ -alanine, poly-L-lysine, alanine, sistine, homocysteine etc., at a ratio between 1 :10 to 1 :1000, preferably at 1 :750 (mol/mol) under gentle stirring at room temperature for more than 4 hours but less than 15 hours at a pH of 7.2.
  • the reaction product is dialyzed against three changes of deionized
  • the conjugation between the PS-SM and CTB is performed using the EDC method as described in, Peeter, C.C. et al.
  • the PS-CTB is conjugated by slowly adding 5-50 mg, more preferably 5-35 mg, but most preferably 20 mg, of the PC-CTB to a final concentration of 0.1 M 1 -ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) into 0.5 to 10 ml, more preferably 0.5 to 3 ml, but most preferably 1 ml solution containing equal amounts (5-50 mg, more preferably 5-20 mg, most preferably 10 mg) of the PS-SM and cholera toxin B subunit (CTB), and incubated.
  • the pH is maintained at 4.7 with the addition of 0.1 M HCI acid.
  • the reaction is stopped by the addition of 4.8 mg of ethanolamine, or any base that can increase the pH, such as but not limited to NaOH, KOH, etc., after 4 hours, for thus forming crude conjugates.
  • the crude conjugates are centrifuged, and the supernatant is removed and dialyzed against a phosphate buffer saline (PBS) solution. Unreacted proteins are removed by using a Sephacryl S-300 column chromatography in the PBS. Fractions that contain both the protein and PS are pooled and concentrated by using an Am icon Centriprep ® .
  • a l t h o u g h a n a l g i n ate of a n i o n i c copo lym e rs of 1 ,4-linked- ⁇ -D-mannuronic acid and ⁇ -L-guluronic acid is used as the microspheres
  • various kinds of polymers could be substituted as the alginate microspheres, such as but not limited to ethylene-vinyl acetate, polyiminocarbonate, poly (DL-lactide-co-glycolides), a polysaccharide such as dextran, puliuran, chitosan, cellulose, modified cellulose, a natural polyamide such as collagen, gelatin, albumin, synthetic polymers such as polylactide, polyglycolide and copolymers of polylactides and glycolides, polyorthoesters, polyanhidride, polyvinylpyrrolidone
  • the alginate microspheres are prepared by a modified water in oil
  • the w/o microemulsions are prepared by homogenizing the mixtures for variable times and at various rpms with a homogenizer. Then, n-octanol containing CaCI 2 1.0 - 8.0 (w/v) is added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres are cured after mixing with addition of 5.0 ml of 1-8% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol. The microspheres are collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m, or any hyrophobic filter membrane.
  • PVDF polyvinylidene difluroide
  • microspheres on the filters are washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours. See FIGS. 5 and 9(a-c), and wherein the anthrone reaction (solid line) and BCA protein assay (dotted line) were used to monitor the PS and protein contents before the conjugation.
  • the arrow denotes the void volume.
  • the GM1 -ELISA was used after the conjugation with anti-PS antibody (solid line) or with anti-CTB antibody (dotted line) as the primary antibody.
  • the conjug tion of the PS nd the TB w s confirmed Seph cc I S-300 elution profiles.
  • the completion of the conjugation was also confirmed by using an enzyme-linked immunosorbent assay with a GM1 -ganglioside (GMIg)-coated plate (GM1 -ELISA).
  • GMIg GM1 -ganglioside
  • GM1 -ELISA enzyme-linked immunosorbent assay with a GM1 -ganglioside (GMIg)-coated plate
  • the released conjugate was neutralized (rectangle) or was not neutralized (circle) before applying into wells, respectively.
  • the conjugate bound to the wells coated with GM1 g was detected by anti-PS antibody (filled symbol) and anti-CTB antibody (open symbol).
  • the nonspecific binding of the conjugate was evaluated by using wells coated with bovine serum albumin as a control (triangle).
  • a solution containing the PS-CTB was applied to wells coated with GM1g and bound conjugates were detected with anti-CTB antibodies or with anti-PS antibodies.
  • the conjugates were preincubated with a soluble GM1 g (3 ⁇ M) before adding to the GM1 g coated wells.
  • BSA bovine serum albumin
  • the released conjugate showed binding affinities to GM1g and to anti-PS19 antibodies as well as to anti-CTB antibodies.
  • the conjugates did not bind to GM1g if it was preincubated with the soluble GM1 g.
  • the GM1 g neutralized the binding capacity of the conjugates completely. There was no specific binding of the conjugate to the BSA. This suggests that the binding activity of the conjugates toward GM1g and the immunoreactivity to the anti-PS19 antibodies or to the anti-CTB antibodies were maintained after the encapsulation.
  • mice Groups of 10 female Balb/c mice (Korea Advanced Institute of Science and Technology, Daeduk, Korea), 6-8 weeks old, were respectively orally administered three times at an interval of two weeks with mock microspheres, naked PS-CTB (i.e., without AM), AM(PS), AM(PS-CTB), and AM(PS-BSA).
  • CT cholera toxin
  • mice were kept under standardized conditions at ad libitum with pelletted food throughout the experiment. All the mice received 20 ⁇ g equivalent doses of the PS diluted in 500 ⁇ l of 0.1 M sodium bicarbonate buffer (pH 8.1) at a single time for peroral immunization. The doses were in the range of 20 - 500 ⁇ g/mouse for dose titration.
  • the mice were orally administered with microspheres via a blunt-tipped feeding needle inserted into the stomach.
  • Figure 11 The mice were orally administered with microspheres via a blunt-tipped feeding needle inserted into the stomach.
  • mice Two groups of the mice were vaccinated with three intranasal administrations of 20 ⁇ g PS (in 20 ⁇ l) in either encapsulated AM(PS-CTB) or naked form (PS-CTB).
  • a group of mice immunized intranasally with mock microspheres served as a control.
  • the carrier effect on the immunogenicity of the orally administered PS was compared with the immune responses of the mice vaccinated with the AM(PS), AM(PS-CTB) and AM(PS-BSA), as shown in FIG. 10.
  • the physical mixtures of the CTB and PS when microencapsulated and .used in immunization induced serum and mucosal immune responses not statistically different from those induced by the the AM(PS) (data not shown).
  • the AM (PS-CTB) induced prominent serum IgG, serum IgA and intestinal IgA responses among these groups (p ⁇ 0.05).
  • Each bronchoalveolar IgA responses induced by the AM(PS), AM(PS-CTB), and AM(PS-BSA) was significantly higher than of the PS-CTB (FIG. 10).
  • the serum IgA (240 + 41 ng/ml) and serum IgG (31 ⁇ 15 ng/ml) antibody responses of the mice immunized with the PS-CTB were significantly higher than those with mock microspheres (IgA: 25 ⁇ 16 ng/ml and IgG 4 ⁇ 1 ng/ml) and than those with the AM(PS-CTB) (IgA: 96 + 6 ng/ml and IgG 15 + 4 ng/ml).
  • Vaccination with oral administration of the AM(PS-CTB) or intranasal vaccination with the PS-CTB showed successful protection against challenges from live S.pneumoniae.
  • a control group of mice receiving mock AMs were highly susceptible to infection with S.pneumoniae, while immunized mice were protected.
  • Viable pneumococcus recovered from the lungs and blood of all groups of immunized mice showed significant reduction (>95%) in numbers as compared with the control (p ⁇ 0.05).
  • the PS-CTB conferred limited protection on mice when compared with the AM(PS-CTB). Seventy five percent of the colonizing bacteria were inhibited by the oral immunization with the PS-CTB.
  • a purified immunoglobulin with known concentrations of isotopes served as a standard.
  • the absorbance values of the standard immunoglobulin were determined by using a sandwich ELSIA.
  • Polystyrene microplates (Nunc Products, Roskilde, Denmark) were coated with PS19F by incubating 100 ⁇ l of the appropriate antigen solution (10 ⁇ g ml in PBA) per well for 2 hours at 37°C, and then over night at 4°C. Plates coated with the PS19F were washed six times with PBS containing 0.05% Tween 20 (PBST). The same washing was carried out after each subsequent antibody reaction. After washing, plates were coated with 5% skim milk for 1 hour at 37° C. Diluted specimens were added to paired wells and incubated for 2 hours at 37° C. After washing with PBST, 100 ⁇ l of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin
  • HRP horseradish peroxidase
  • the sera, intestinal and bronchoalveolar lavage were pooled from each group of mice and analyzed for the mice immunized with naked PS-CTB and AM(PS-CTB). Five ⁇ g of the CT was co-administered with antigens in two groups of mice. Anti-CTB antibodies were measured by ELISA. Cut-off values for the antibody titer were determined by analyzing the sample from the mice immunized with mock microspheres (0.004 for serum IgG, 0.135 for serum IgA, 0.0145 for lung IgA, and 0.045 for intestinal IgA). All antibody titers of the samples derived from mock-immunized mice were less than 1:16 by these cut-off values.
  • mice against intranasal challenge by live pneumococcus were assessed by recovering viable organism from lungs and blood of mice immunized with viable vaccines. Two weeks after the last immunization, the mice were anesthetized and challenged intranasally with 1 x 10 6 colony forming units of S.pneumoniae 19F in 20 ⁇ l of medium. The CFU of pneumococci in the bronchoalveolar lavage fluid and in the blood were counted 18 hours after the challenge. As shown in figure 12, the gray and white bars represent the CFU of pneumococci in specimens obtained from the mice after intranasal or the oral vaccination, respectively.
  • %inhibition representing 100 x (1-CFUtest/CFUcontrol).
  • PS19 pneumococcal capsular polysaccharide
  • AC 6-aminocaproic acid
  • the PS19-AM was conjugated by slowly adding 20 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma Chemical Co., St. Louis, MO) into 1 ml of a solution containing 10 mg of the PS19 and 10 mg of a cholera toxin B subunit (CTB) (List Biologicals Inc., Campbell, CA), and incubated for 4 hours. The pH was maintained at 4.7 by the addition of 0.1 M HCI. The reaction was stopped by the addition of 4.8 mg of ethanolamine after 4 hours, for thus forming crude conjugates.
  • EDC 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
  • CTB cholera toxin B subunit
  • the reaction was stopped by the addition of 4.8 mg of ethanolamine after 4 hours, for thus forming crude conjugates.
  • the crude conjugates were centrifuged (30 min; 50,000 x g), and the supernatant was removed and dialyzed against phosphate buffer saline [PBS, 0.20g/L KCI, 0.20g/L KH 2 P0 4> 8.0g/L NaCI, 2.92 g/L Na 2 HP0 4 (12H 2 0)]. Unreacted proteins were removed by using a Sephacryl S-300 column (Pharmacia Biotech, Uppsala, Sweden) chromatography in PBS. Fractions that contained both the protein and PS was pooled and concentrated by using an Am icon Centriprep ® . Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique.
  • microspheres were cured after mixing by the addition of 5.0 ml of 8% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol (Oriental Chemical Industry, Seoul Korea).
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m (Alltech Associates, Inc., IL). Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation were prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 3.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier, see FIG. 1. The mixture was o homogenized. Then, n-octanol containing CaCI 2 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer.
  • HCO-60 hydrogenated caster oil 60
  • microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation were prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier, see FIG. 1. The mixture was homogenized. Then, n-octanol containing CaCI 2 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer.
  • HCO-60 hydrogenated caster oil 60
  • microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m.
  • PVDF polyvinylidene difluroide
  • the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours. Aliquot of alginate 5% (w/v) was dissolved in 5 ml of conjugate solution, for thereby completing the microencapsulation of the conjugate.
  • PS19-spacer conjugation and PS19-CTB conjugation were prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI 2 2% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2.
  • microspheres were cured after mixing by the addition of 5.0 ml of 2% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation were prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI 2 4% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2.
  • microspheres were cured after mixing by the addition of 5.0 ml of 4% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation were prepared in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI 2 8% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2.
  • microspheres were cured after mixing by the addition of 5.0 ml of 8% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing SPAN-80® 5% (w/v) (Nikko Chemical Co. Ltd., Tokyo, Japan) as an emulsifier, see FIG. 3. The mixture was homogenized. Then, n-octanol containing CaCI 2 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer.
  • n-octanol containing CaCI 2 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer.
  • microspheres were cured after mixing by the addition of 5.0 ml of 1 % (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 mi of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19-spacer conjugation and PS19-CTB conjugation prepared as in sample 1.
  • Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 10 (HCO-10) 5% (w/v) (Nikko Chemical Co. Ltd., Tokyo, Japan) as an emulsifier, see FIG. 3. The mixture was homogenized. Then, n-octanol containing CaCI 2 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer.
  • HCO-10 hydrogenated caster oil 10
  • microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI 2 followed by slow addition of 6 ml of isopropyl alcohol.
  • the microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 ⁇ m. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.
  • PVDF polyvinylidene difluroide
  • PS19 pneumococcal capsular polysaccharide
  • AC 6-aminocaproic acid
  • the PS19-CTB was conjugated by slowly adding 20 mg 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma Chemical Co., St. Louis, MO) into 1 ml of solution containing 10 mg of the PS19 and 10 mg of a cholera toxin B subunit (CTB) (List Biologicals Inc., Campbell, CA), and incubated for 4 hours.
  • EDC 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
  • CTB cholera toxin B subunit
  • the pH was maintained at 4.7 by the an addition of 0.1 M HCI.
  • the reaction was stopped by the addition of 4.8 mg of ethanolamine after 4 hours, for thus forming crude conjugates.
  • the crude conjugate was centrifuged (30 min; 50,000 x g), and the supernatant was removed and dialyzed against phosphate buffer saline [PBS, 0.20g/L KCI, 0.20g/L KH 2 P0 4 , 8.0g/L NaCI, 2.92 g/L Na 2 HP0 4 (12H 2 0)]. Unreacted proteins were removed by using a Sephacryl S-300 column (Pharmacia Biotech, Uppsala, Sweden) chromatography in PBS. Fractions that contained both the protein and PS was pooled and concentrated by using an Am icon Cent prep®. Thus, completing the conjugation of the PS19 and CTB.
  • microencapsulated AM(PS-CTB) and PS-CTB conjugates prepared as described herein can be used as vaccines against
  • compositions of the invention i.e., the vaccines
  • the vaccine can be administered orally, and in accordance with another embodiment the vaccine can be administered intranasally.

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Abstract

La présente invention concerne, d'une part un conjugué micro-encapsulé de polysaccharide capsulaire de Streptococcus pneumoniae et de sous-unité B de toxine cholérique, et d'autre part un vaccin conjugué polysaccharidique capsulaire de Streptococcus pneumoniae apportant une protection efficace contre les infections respiratoires pneumococciques telles que la méningite, l'otite moyenne, les bactériémies et les crises aiguës de bronchite chronique, de sinusite, d'arthrite ou de conjonctivite.
PCT/KR1998/000007 1997-01-31 1998-01-17 Conjugue polysaccharidique pneumococcique et vaccins conjugue polysaccharidique antipneumococcique a sous-unite b de toxine cholerique WO1998033521A1 (fr)

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AU56812/98A AU5681298A (en) 1997-01-31 1998-01-17 Pneumococcal polysaccharide conjugate and pneumococcal polysaccharide chol era toxin b subunit conjugate vaccines

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KR1997/3021 1997-01-31
KR1019970003021A KR19980067137A (ko) 1997-01-31 1997-01-31 알긴산염 초미세과립구를 이용한 폐렴구균 감연중에 대한 백신

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001026631A1 (fr) * 1999-10-08 2001-04-19 Korea Institute Of Science And Technology Microspheres modifiees en surface au moyen d'une sous-unite de la toxine b du cholera
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11116828B2 (en) 2017-12-06 2021-09-14 Merck Sharp & Dohme Corp. Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US11642406B2 (en) 2018-12-19 2023-05-09 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
EP3678654B1 (fr) 2017-09-07 2024-06-26 Merck Sharp & Dohme LLC Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse

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Publication number Priority date Publication date Assignee Title
WO1992014488A1 (fr) * 1991-02-15 1992-09-03 Uab Research Foundation Gene de structure de proteine de pneumocoque
WO1996039113A2 (fr) * 1995-06-02 1996-12-12 Uab Research Foundation Administration par voie orale d'antigenes pneumococciques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992014488A1 (fr) * 1991-02-15 1992-09-03 Uab Research Foundation Gene de structure de proteine de pneumocoque
WO1996039113A2 (fr) * 1995-06-02 1996-12-12 Uab Research Foundation Administration par voie orale d'antigenes pneumococciques

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001026631A1 (fr) * 1999-10-08 2001-04-19 Korea Institute Of Science And Technology Microspheres modifiees en surface au moyen d'une sous-unite de la toxine b du cholera
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9714283B2 (en) 2014-10-28 2017-07-25 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9815886B2 (en) 2014-10-28 2017-11-14 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9969793B2 (en) 2014-10-28 2018-05-15 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10683343B2 (en) 2014-10-28 2020-06-16 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US11339206B2 (en) 2014-10-28 2022-05-24 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US11780906B2 (en) 2014-10-28 2023-10-10 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US11897943B2 (en) 2017-03-15 2024-02-13 Adma Biomanufacturing, Llc Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11084870B2 (en) 2017-03-15 2021-08-10 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
EP3678654B1 (fr) 2017-09-07 2024-06-26 Merck Sharp & Dohme LLC Polysaccharides antipneumococciques et leur utilisation dans des conjugués immunogènes polysaccharide-protéine porteuse
US11116828B2 (en) 2017-12-06 2021-09-14 Merck Sharp & Dohme Corp. Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US11850278B2 (en) 2017-12-06 2023-12-26 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US12097250B2 (en) 2017-12-06 2024-09-24 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US12016914B2 (en) 2018-12-19 2024-06-25 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US11642406B2 (en) 2018-12-19 2023-05-09 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

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