WO2001000233A1 - Compositions vaccinales comprenant des antigenes encapsules dans des microspheres d'alginate destinees a l'administration orale et procedes de preparation de ces compositions - Google Patents

Compositions vaccinales comprenant des antigenes encapsules dans des microspheres d'alginate destinees a l'administration orale et procedes de preparation de ces compositions Download PDF

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WO2001000233A1
WO2001000233A1 PCT/KR1999/000466 KR9900466W WO0100233A1 WO 2001000233 A1 WO2001000233 A1 WO 2001000233A1 KR 9900466 W KR9900466 W KR 9900466W WO 0100233 A1 WO0100233 A1 WO 0100233A1
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antigen
protein
microspheres
alginate
psaa
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PCT/KR1999/000466
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English (en)
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Seo Young Jeong
Ick Chan Kwon
Joo Ae Park
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Korea Institute Of Science And Technology
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Priority to AU55295/99A priority Critical patent/AU5529599A/en
Publication of WO2001000233A1 publication Critical patent/WO2001000233A1/fr

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    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41B—SHIRTS; UNDERWEAR; BABY LINEN; HANDKERCHIEFS
    • A41B11/00—Hosiery; Panti-hose
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41B—SHIRTS; UNDERWEAR; BABY LINEN; HANDKERCHIEFS
    • A41B11/00—Hosiery; Panti-hose
    • A41B11/004—Hosiery with separated toe sections
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41B—SHIRTS; UNDERWEAR; BABY LINEN; HANDKERCHIEFS
    • A41B2400/00—Functions or special features of shirts, underwear, baby linen or handkerchiefs not provided for in other groups of this subclass
    • A41B2400/32—Therapeutic use
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41B—SHIRTS; UNDERWEAR; BABY LINEN; HANDKERCHIEFS
    • A41B2400/00—Functions or special features of shirts, underwear, baby linen or handkerchiefs not provided for in other groups of this subclass
    • A41B2400/60—Moisture handling or wicking function

Definitions

  • the present invention relates to vaccine compositions for oral administration consisting of an protein antigen encapsulated in biodegradable alginate microspheres by the diffusion-controlled interfacial gelation technique which produces microspheres having less than 5 ⁇ m of diameter, and to preparation process thereof .
  • Mucosal immune system is capable of responding to invading pathogens in the mucosal surfaces by producing pathogen specific secretory IgA antibodies.
  • the local secretory IgA has been known to prevent both the colonization of pathogens at the mucosal tissues and the spread into the systemic circulation more efficiently compared to the systemic antibodies (Service, 1994).
  • the mucosal tissues and secretory glands represent the largest residence of T cells, B cells, and plasma cells in the body. Additionally, secretory IgA, comprising more than 60% of all immunoglobulins in the body, is produced in these tissues. Stimulation of mucosal immunity is believed to arise at specialized aggregates of lymphoid tissues collectively termed the bronchus-associated lymphoid tissue (hereinafter, referred to as "BALT”) and gut-associated lymphoid tissue (hereinafter, referred to as "GALT”) .
  • BALT bronchus-associated lymphoid tissue
  • GALT gut-associated lymphoid tissue
  • Unique characteristics of BALT and GALT include the ability to communicate immunogenic information arising at one mucosal surface to other mucosal surfaces in the body. Furthermore, it has been reported that, if properly modulated, GALT and BALT could confer systemic immunity as well as mucosal immunity against diverse toxins and pathogens (Shalaby, 1995).
  • Mucosal immunity is characterized in the predominance of dimeric IgA in external secretions and the preponderance of IgA secreting plasma cells beneath secretory epithelia.
  • Mechanisms of antibacterial function of secretory IgA are based on its opsonizing properties and its ability to interfere with the adherence of bacterial antigens to mucosal surfaces (Abraham, 1994), thereby limiting bacterial colonization and enhancing elimination of bacteria.
  • Other mechanism is reported that the secretory IgA may lyse bacteria in the presence of lysozymes and complements.
  • the production of IgA is initiated at specialized lymphoid tissues, in particular those in the upper respiratory and gastrointestinal tracts.
  • GALT tissue consist of Peyer' s patches (hereinafter, referred to as "PP"), appendix and solitary lymphoid nodules.
  • PP Peyer' s patches
  • BALT whose most prominent follicles are the palatine and pharyngeal tonsils, is the principal mucosal lymphoid tissue of the respiratory tract. It should be noted however, that there are some differences in functions of BALT and GALT.
  • M cells In the dome region of PP, there is a specialized epithelium known as microfold or M cells, which pick up antigens and transport them into underlying lymphoid tissues. Antigens are processed and stimulate B and T cells in the germinal centers of follicles located beneath the dome. These cells leave the PP via the efferent lymphatics and enter the systemic circulation through the thoracic duct (see Fig.l). Especially, the B cells expand clonally in the mesenteric lymph node(MLN) and become mature IgA plasma cells, which subsequently produce secretory IgA in external secretions. This cellular distribution system comprises the common mucosal immune system of the body.
  • MN mesenteric lymph node
  • CT cholera toxin
  • E . col i heat-labile endotoxin muramyldipeptide or phorbol ester
  • a powerful adjuvant such as cholera toxin (CT)
  • E . col i heat-labile endotoxin muramyldipeptide or phorbol ester
  • mucosal adjuvants seem to work by modulating the signal transduction pathway of immune cells or by stimulating the immune responses of microenvironment .
  • CT is the most potent mucosal adjuvant identified up to now.
  • Its immunomodulating effects include an enhanced antigen presentation by a variety of cell types, promotion of isotype differentiation in B cells leading to an increased IgA formation and complex stimulatory as well as inhibitory- effects on T-cell proliferation and lymphokine production (Holmgren et al . , 1993) .
  • antigens can be incorporated into liposome with micrometer or nanometer size, microemulsion, microspheres or nanospheres by microencapsulation method using colloidal microparticle .
  • Microencapsulation is defined as a technique whereby chemical or biological materials such as drugs, enzymes, toxins, bacteria and viruses are encapsulated within polymer matrices, which has advantages preventing the encapsulated materials from being inactivated by water, heat or oxidation, or enhancing the biocompatibility of the materials.
  • antigens can be incorporated into microcapsules that protect antigens from denaturation at low pH and hydrolytic degradation by enzymes in the gastrointestinal tract.
  • antigen sequestration in microcapsules may provide a mechanism to evade neutralization by maternal antibody.
  • Biodegradable and biocompatible microspheres have been used for antigen delivery into the GI tract(Muir et al . , 1994).
  • microspheres Most of the methods to produce microspheres, however, include the steps of using organic solvent and/or heating at high temperature. Antigens may denature under these harsh conditions.
  • the production of microspheres using gelatin, poly (D, L-lactic acid), poly (glycolic acid) or albumin, for example, may involve heat as high as 130 °C or the use of organic solvents such as formaldehyde, glutaraldehyde, methylene chloride and hexafluoroacetone sesquihydrate, to dissolve or to cross-link the polymer (Jepon et al . , 1993, Maloy et al . , 1994).
  • the alginate microspheres can be prepared easily in aqueous solutions at room temperature and, therefore, are very useful in encapsulating antigens.
  • Alginate is an anionic copolymer of 1 , 4-linked- ⁇ - D-mannuronic acid and ⁇ -L-guluronic acid, and has been used as a food additive, compressed tablets disintegrator and gelation agent. Alginate also has been used widely applied to immobilize cells, to hybrid artificial organs and as delivery system for drugs and antigens due to its non-toxicity and biocompatibility (K.Park, 1993) .
  • Alginate has an unique property of forming a gel in the presence of divalent cations such as Ca + . Such a gel formation occurs mainly at the junctions between ions and homopolymeric blocks of guluronic acid. Since calcium-alginate gel produced through this process has bridge formed by ion bond, it can make hard hydrogel . Alginate gel can be prepared easily in aqueous solutions and also swelled and gradually disintegrated in a living body. Therefore, alginate has been widely used for the polymer matrix of microspheres owing to the physiochemial properties required for polymer matrix.
  • microspheres at the small intestine are restricted to microspheres of less than 10 ⁇ m in diameter (Jepson, 1993).
  • Kinetic studies on the fate of the microspheres within the GALT showed that the microspheres small than 5 ⁇ m in diameter were transported through the efferent lymphatics, while those larger than 5 ⁇ m in diameter remained in PP.
  • Streptococcus pneumoniae is a major respiratory mucosal pathogen that causes meningitis, otitis media, bacteremia, and pneumonia. It has been well established that specific antibodies to capsular polysaccharides can elicit a protective immune response against pneumococcal infection in adults (Macleod, 1945). In the case of children ( ⁇ 2 years), elderly, and those with immunodeficiencies, however, the polysaccharide vaccines do not elicit effectively pneumococcus- specific protective antibody responses (Anderson, 1977, Peters, 1994) and children can have repeated infections involving strains of the same capsular serotype.
  • Protein polysaccharide conjugates or pneumococcal proteins alone have been considered as an alternative means to induce protective immunity in infants and children against pneumococcal infection (Yamamoto, 1997).
  • the immunogenic nature of proteins makes them prime targets for new vaccine strategies.
  • Pneumococcal proteins being investigated as potential vaccine candidates include pneumolysin, neuraminidase, autolysin, pneumococcal surface adhesion A(PsaA), and pneumococcal surface protein A(PspA) (McDaniel, 1996). These proteins could become more efficacious vaccines since they would offer a broader range of protection against a greater number of serotypes and could elicit T cell dependent immune response and also provide a memory response.
  • a novel vaccine compositions for oral administration consisting of a protein antigen encapsulated in biodegradable alginate microspheres by the diffusion-controlled interfacial gelation technique and preparation process thereof.
  • Alginate-encapsulated antigen of this invention shows higher levels of antigenicity than the naked protein antigen, and are considered as a proper carrier for an efficient delivery of antigens by the Peyer' s patch and by concomitant transport through lymphatics because of having less than 5 ⁇ m of diameter.
  • It is also an object of this invention to provide a process for preparing the vaccine compositions comprising the steps of: a) Mixing aliquot of a protein antigen or a protein antigen including immune adjuvant with a alginate aqueous solution; b) Homogenizing by adding a mixture of the alginate and a protein antigen of step a) to n- octanol containing an emulsifier; c) Spraying n-octanol solution containing CaCl, into the emulsion while stirring the whole emulsion slowly; d) Adding additional CaCl 2 solution to saturate the emulsion, and curing microspheres; e) Dehydrating microspheres by addition of dehydrating solvent; f) Collecting the microspheres on membrane filters and washing with alcohol, and then drying in vacuo .
  • the present invention provides vaccine composition wherein the protein antigen is selected from the group consisting of bacterial surface proteins, endotoxin or exotoxin antigens, autoantigens , and allergens.
  • the protein antigen is selected from the group consisting of bacterial surface proteins, endotoxin or exotoxin antigens, autoantigens , and allergens.
  • various protein antigens can be used in vaccine composition of this invention; i.e., pnemococcal antigen, Hemophll us parainfl uenza antigen, diphtheria antigen, pertussis antigen, tetanus antigen, enterotoxigenic E .
  • col i antigen dysentery antigen, cholera antigen, gonococcus antigen, influenza virus antigen, B type hepatitis antigen, measles antigen, smallpox virus antigen, and rubella antigen.
  • it can be selected from pneumococcal proteins such as pneumolysin, neuraminidase, autolysin, pneumococcal surface adhesion A(PsaA) and pneumococcal surface protein A (PspA) .
  • This invention also provides the vaccine composition including additionally immnune adjuvants, such as cholera toxin, cholera toxin B subunit, E . coli heat-labile enterotoxin, muramyldipeptide or phorbolester , added to inside or outside of microspheres .
  • this invention provides a process for preparing the vaccine composition, wherein the diffusion-controlled interfacial technique is employed.
  • Fig. 1 shows circulation of lymphocytes within the mucosa-associated lymphoid system.
  • Fig. 2a shows the structures of alginate monomers, ⁇ -D-mannuronic acid(M) and ⁇ -L-guluronic acid(G) .
  • Fig. 2b shows a proposed structural model for the chelation of ions by GG residues and the gel formation by binding of divalent cation such as Ca 2+ .
  • Fig. 3 shows fabrication method to prepare alginate microspheres.
  • Fig. 4 shows construction process of plasmid pCYB4-PsaA.
  • Fig. 5 shows the result confirming expression of recombinant protein by 7 clones using western blotting.
  • Fig. 6 shows nucleotide sequence of PsaA gene.
  • Fig. 7 shows growth curve of transformed XL j -Blue at different temperatures.
  • Fig. 8 shows differences in band patterns in pellets and supernatants of cell lysates at various culture temperatures by western blotting.
  • Fig. 9a shows the identification of the purified PsaA(35 kDa) protein on SDS-PAGE.
  • Fig. 9b shows the identification of the purified PsaA(35 kDa) protein by western blotting.
  • Fig. 10 shows in vi tro release profile of the entrapped PsaA from alginate microspheres .
  • Fig. 11 shows immunogenicity of recombinant PsaA introduced by subcutaneous immunization.
  • Fig. 12 shows standard curve for the quantification of antibodies determined by a sandwich ELISA.
  • Fig. 13 shows antibody responses induced by oral immunization with encapsulated PsaA [hereinafter, referred to as E(PsaA)] and naked PsaA [hereinafter, referred to as N(PsaA)].
  • Fig. 14 is an electromicroscopic photograph which shows that alginate microspheres of the present invention are smaller than 5 ⁇ m in diameter.
  • vaccine composition for oral administration may be prepared through process that protein antigen is encapsulated in biodegradable alginate microspheres by the diffusion-controlled interfacial gelation technique which produces microspheres having less than 5 ⁇ m of diameter.
  • the present invention provides vaccine composition wherein protein antigen is selected from the group consisting of bacterial surface protein antigens, endotoxin or exotoxin antigen, autoantigens, and allergens.
  • protein antigens can be used in vaccine composition of this invention; i.e., pnemococcal antigen, Hemophilus parainfluenza antigen, diphtheria antigen, pertussis antigen, tetanus antigen, enterotoxigenic E . coli antigen, dysentery antigen, cholera antigen, gonococcus antigen, influenza virus antigen, B type hepatitis antigen, measles antigen, smallpox virus antigen, and rubella antigen.
  • it can be selected from pneumococcal proteins such as pneumolysin, neuraminidase, autolysin, pneumococcal surface adhesion A(PsaA), and pneumococcal surface protein A(PspA) .
  • pneumococcal proteins such as pneumolysin, neuraminidase, autolysin, pneumococcal surface adhesion A(PsaA), and pneumococcal surface protein A(PspA) .
  • pneumococcal surface adhesion A(PsaA) proteins of Streptococcus pneumoniae is preferred as antigen encapsulated in alginate microspheres, and can be obtained by process comprising the steps of; a) Amplifying the gene encoding PsaA by the polymerase chain reaction (PCR) using the chromosome DNA of S .pneumoniae as template; b) Cloning the PsaA gene into pCYB4 vector; c) Transforming Escherichia coli with the recombinant vector; d) Incubating the Escherichia coli transformant; and e) Isolating and purifying the recombinant PsaA
  • PCR polymerase chain reaction
  • Alginate microspheres including protein antigens can be prepared easily in aqueous solutions at room temperature, and have advantages such as non-toxicity and biocompatibility . Therefore, alginate, an anionic copolymer of 1 , 4-linked- ⁇ -D-mannuronic acid and ⁇ -L- guluronic acid, is of great use for encapsulating antigens .
  • the vaccine composition of this invention includes additionally immnune adjuvants, such as cholera toxin, cholera toxin B subunit, E . coli Heat labile enterotoxin, muramyldipeptide or phorbolester , which are added to inside or outside of microspheres to increase the immunogenicity.
  • immnune adjuvants such as cholera toxin, cholera toxin B subunit, E . coli Heat labile enterotoxin, muramyldipeptide or phorbolester , which are added to inside or outside of microspheres to increase the immunogenicity.
  • the preferred size of the alginate microspheres is less than 5 ⁇ m of diameter to effectively deliver antigens.
  • a process for preparing the vaccine compositions comprising the steps of: a) Mixing aliquot of a protein antigen or a protein antigen including immune adjuvant with a alginate aqueous solution; b) Homogenizing by adding a mixture of the alginate and a protein antigen of step a) to n-octanol containing an emulsifier; c) Spraying n-octanol solution containing CaCl 2 into the emulsion while stirring the whole emulsion slowly; d) Adding additional CaCl 2 solution to saturate the emulsion, and curing microspheres; e) Dehydrating microspheres by addition of dehydrating solvent; f) Collecting the microspheres on membrane filters and washing with alcohol, and then drying in vacuo .
  • the alginate microspheres solution of step a) is used preferably in the concentration of between 1-5 weight% .
  • the ratio of the alginate aqueous solution to total solvent volume between 1:1 to 1:10 is preferred, which controls the size and the gelation of microspheres .
  • HCO hydrogenated castor oil
  • HCO- 60 and Span-80 are preferred of which the concentration is between 1-10 weight% .
  • the n-octanol containing CaCl 2 of step c) is used preferably in the concentration of between 0.5-2 weight%, and the CaCl 2 solution of step d) between 5-10 weight% .
  • methanol, ethanol, isopropanol or acetone is preferred.
  • the filter of step f) is preferably a polyvinylidene difluoride (PVDF) membrane filter with a pore size of between 0.10-0.44 ⁇ m.
  • PVDF polyvinylidene difluoride
  • the effect of surfactant is also important. As the polarity of the surfactant increases, by increasing the polyethylene oxide (PEO) units of hydrogenated castor oil (HCO) series, the size of microspheres decreases.
  • PEO polyethylene oxide
  • HCO hydrogenated castor oil
  • the initial concentration of alginate in the aqueous phase is one of these parameters .
  • concentration higher than 5% it is difficult to produce small and homogeneous microspheres since the viscosity of the alginate solution is too high.
  • concentration lower than 5% agglomerated microspheres are obtained.
  • the choice of the organic solvent is found to be another important factor in determining the particle size and its distribution. Throughout the distribution polymerization studies, it was shown that the particle size decreased as the solvent polarity increased (Lok and Ober, 1985, Paine, 1990) .
  • n- octanol is used as an organic solvent in which CaCl 2 is dissolved and the solution is slowly diffused into the aqueous phase containing alginate for the diffusion- controlled interfacial gelation.
  • the stirring rate is also an important factor in forming emulsion droplet with a desired diameter.
  • the emulsification was performed at 8,000 rpm for one hour. When the stirring speed was lower than 5,000 rpm, heterogeneous microspheres with diameter larger than 10 ⁇ m are obtained. On the other hand, a high speed stirring above 10,000 rpm resulted in the formulation of agglomerated particles with a low yield.
  • a possible the stirring rate is about 5,000 rpm to about 10,000 rpm, and is 7,000 to 8,000 rpm preferably.
  • further continuous stirring at a low speed is required until the gelation is completed so that the hardened microspheres could be isolated as discrete particles.
  • the particle size is also dependent on the concentration of CaCl 2 in n-octanol and the rate of addition into the medium.
  • the size of the microspheres could be further reduced by spraying CaCl 2 solution onto the emulsion rather than by dropwise addition using a syringe.
  • microspheres used for an in vi tro release test in a preferred embodiment of this invention are prepared by 5% (w/v) alginate solution emulsified in a n-octanol solution containing 5% HCO-60, followed by curing with 1% CaCl 2 in n-octanol.
  • microspheres loaded with antigen are observed by the scanning electron microscopy.
  • the microspheres of this invention has a smooth surface without noticeable defects, with the diameter in the range of 0.5 to 5 ⁇ m.
  • the Alginate microspheres of this invention may be administered orally to human, used as the preparations of various drug forms .
  • the preparations for oral administration includes compressed tablets, pills, pulves, granules, capsules et al . , and may contain appropriate one or more excipients such as starch, calcium carbonate, sucrose, lactose or gelatin. Besides additives, lubricants such as a magnesium stearate, talc may be added.
  • the effective dose of protein antigen is 20-400 ⁇ g/dose, and preferably 40-100 ⁇ g/dose.
  • S .pneumoniae was cultured in BHI and 10% FBS media by anaerobic incubation at 37°C overnight.
  • Cultured S . pneumoniae ( 1.5ml) was harvested by centrifugation for 2 min at 13,000rpm in a microcentrifuge . The pellet was resuspended in 340 ⁇ l TE buffer containing 227 ⁇ l of lysozyme(10 mg/ml) and incubated at room temperature for 30 min.
  • pCYB4(6.8kb, New England Biolabs, USA) contains the IPTG-inducible Ptac promotor and a multiple cloning site(MCS) .
  • the vector was designed to produce a fusion of the C-terminus of the target protein and the N- terminus of the intein.
  • the DNA encoding chitin binding domain (CBD) is present at the C-terminus of the intein for affinity purification.
  • Ncol and Smal restriction enzyme (Promega, USA) sites are present at MCS .
  • Recombinant plasmid was transformed into E. coli XLj-Blue (Novagen, USA) by electroporation, and plasmid DNA was prepared according to the standard protocol to give the genomic DNA of S . pneumoniae (Maniatis, 1989).
  • DNA sequence for PsaA was retrieved from DNA database as shown in Fig. 6.
  • the gene PsaA encodes a
  • the amplified region by PCR in the total 1330bp gene is between position 232 and 1112. Signal peptide and stop codon were removed from each terminus .
  • Reaction mixture 50 ⁇ l ) contained 2 units of Taq polymerase, 400 nmol of a forward primer(SEQ ID No;l), 400 nmol of reverse primer(SEQ ID No;2), 1 ⁇ g of genomic DNA as a template and 200 ⁇ M of each dNTP. Primers for PCR were synthesized by Oligos Etc. Inc. (Willsonile, USA).
  • Thermal cycle for amplification was : 1 cycle of 94 ° C for 3 min; 32 cycles of 94 ° C for 15 sec, 55 ° C for 15 sec, 72 ° C for 1 min; final extension at 72 ° C for 5 min and holding at 4 ° C.
  • the reaction mixture was electrophoresed and then eluted from an agarose gel by QIA quick Gel Extraction Kit.
  • the resulting DNA pellet was dissolved in distilled water.
  • the DNA was incubated at 45 ° C for 15 min in a Klenow enzyme (2 unit/1 ⁇ l) buffer in twice the volume of the DNA solution and followed by adding 1/10 volume of total reaction volume for 0.125 mM dNTP at 37 ° C for 30 min.
  • the mixture was treated with TE-saturated phenol followed by chloroform: isoamylalcohol (24 : 1 by vol), and DNA was precipitated with ethanol . Finally, the eluted DNA was dissolved in distilled water.
  • Amplified insert DNA and pCYB4 vector were digested first by Smal at 25 C and secondly by Ncol at 31 ° C. Digested DNA was electrophoresed, eluted by QIAquick Gel Extraction Kit, and was treated with TE- saturated phenol and chloroform: isoamylalcohol (24 : 1 ) , followed by the ethanol precipitation. Purified DNA was redissolved in distilled water.
  • the vector and insert DNA were mixed thoroughly by pippetting and incubated at 45 C for 15 min to solubilize DNA, followed by storage on ice for 5-10 min.
  • T4 ligase and 1 mM ATP were added into the mixture in a ligase buffer. Ligation reaction was performed at 4 C overnight (see Fig.4).
  • E . coli XL X -Blue were inoculated in 10 ml SOB (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 and 10 mM MgS0 4 ) media supplemented with tetracycline (50 ug/ml) and were cultured overnight.
  • 10 ml SOB 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 and 10 mM MgS0 4
  • tetracycline 50 ug/ml
  • One microliter of culture cells (1.6X10 9 cell/ml) was transferred to 100 ml of fresh SOB/TC and grown at 37 C in a shaking incubator. When OD 600 reached 0.5, the culture was put on ice for 20-30 min.
  • the cells were harvested by centrifugation (Sovall SS-34 rotor: 3,000 rpm, 15 min, 4 ° C) . After discarding the supernatant, the pellet was resuspended in 10 ml of ice-cold RF1[100 mM RbCl, 50 mM MnCl 2 , 30 mM potassium acetate, 10 mM CaCl 2 and 15% (w/v) glycerol (pH5.8 ) ] solution, and placed on ice for additional 15 min.
  • RF1 ice-cold RF1[100 mM RbCl, 50 mM MnCl 2 , 30 mM potassium acetate, 10 mM CaCl 2 and 15% (w/v) glycerol (pH5.8 )
  • Cells were harvested as above, and the pellet was resuspended in 8 ml of ice-cold RF2[10 mM MOPS, 10 mM RbCl, 75 mM CaCl 2 and 15 % (w/v) glycerol (pH6.8 ) ] solution.
  • the cell suspension was aliquoted ( 100 ⁇ l per tube) and each aliquot was mixed with 4 ⁇ l of ligation mixture obtained from the step (1-3) .
  • the DNA-cell mixture was chilled on ice for 1 hr and incubated at 42 C for 2.5 min, followed by 2 min incubation on ice again.
  • plasmids were purified from each colony and digested with restriction enzyme.
  • the control colony was a transformed XL X -Blue cell harboring only pCYB plasmid.
  • 32 colonies were selected out of total 178 colonies. Some colonies from the selected 32 colonies were randomly picked up, and was inoculated in LB media containing ampicillin.
  • Each plasmid was purified from the culture as described in step (1-2) . Purified plasmids were double-digested by Kpnl and Ncol at 31 C into pCYB4 vector and PsaA insert.
  • PVDF polyvinylidenefluoride
  • Anti-PsaA rabbit serum was diluted 100 folds in PBS and used as a primary antibody.
  • Peroxidase-conjugated goat anti-rabbit immunoglobulin antibodies (Organon Teknika Corp, USA) were also diluted in PBS ( 1 : 10 , 000) and used as secondary antibodies.
  • washed membrane was incubated in a 4-chloro-l-naphtol (Sigma chemical Co., USA) . As a result, 4 clones expressed either 90 or 35 kDa proteins.
  • PsaA gene sequence was confirmed by DNA sequencing. The first half of total 877 bases was determined by extension of Ptac promotor primer and the latter half by extension of the intein reverse primer. Sequencing was performed by dideoxy chain termination method using automatic fluorescence sequencer (Applied Biosystems, Model 373) (Sanger et al . , 1997). All procedures were done following the Manufacturer's instruction.
  • ⁇ Forward' reaction mixture (20 ⁇ l) contained 5 pmol 1 of Ptac promotor primer (SEQ ID No; 3), 1 ⁇ g of template plasmid and 8 ⁇ l of Thermo SequenaseTM dye terminator cycle sequencing pre-mix kit
  • Ptac promotor primer The condition for PCR was: 30 cycles of 96 ° C for 30 sec, 45 ° C for 15 sec, 60 ° C for 4 min. After PCR, the mixture was transferred to a fresh tube containing 2 ⁇ 1 of 3 M ammonium acetate and 50 ⁇ l of 95 % ethanol, then chilled at -70 ° C for 10 min followed by centrifugation . The pellet was washed with 70 % ethanol and was dried in a SpeedVac. Then
  • a fresh colony of clone selected from step (1-5) was inoculated into 10 ml LB media containing ampicillin and incubated at 37 ° C overnight. Cultured cells were seeded into 1 liter of fresh LB medium containing ampicillin and incubated at 37 C in a shaker at 600 rpm for 3 hrs. When OD 580 reached 0.5-0.6, culture was induced with 0.5 mM IPTG and incubated at 20 ° C for additional 18 hrs until OD 580 reached 2.0-2.1. The cells were harvested by centrifugation at 8,000 rpm for 30 min at 4 ° C, using Sovall SLA-1500 rotor. Cell pellet was stored at -70 ° C until the next experiments. Each pellet and supernatant were analyzed on SDS- PAGE and by western blotting(see Fig.8).
  • the pellet was resuspended in 25 ml ice-cold column buffer (Tris-HCl, pH 8.0, 500 mM NaCl, 0.1 mM EDTA and 0.1 % Triton X-100).
  • the cells were lysed by addition of 1.5 ml of lysozyme(10 mg/ml) and sonication.
  • the resulting lysate was centrifuged at 14,000 rpm for 30 min at 4 ° C, using Sovall SS-34 rotor. Supernatant was loaded onto the chitin beads affinity column pre- equilibrated with the same buffer.
  • the chitin beads have a binding capacity of about 2 mg protein/ml.
  • the purified PsaA protein was concentrated to 1.5 mg/ml using Amicon Centriprep 0 (molecular weight cut off: 10,000) . This concentrate was dialyzed to remove DTT against PBS or distilled water and used directly or as an encapsulated antigen, respectively. Antigens were stored at -20 ° C until use. The Protein content was determined by Bradford assay kit (Bio-Rad, USA) . The yield of pure PsaA was 10 mg per liter of bacterial culture. ⁇ Example 3> Microencapsulation
  • microspheres were prepared by a modified water in oil (w/o) emulsion technique (see Fig. 2a, 2b and 3) .
  • Alginate solution[5 ml, 5.0 %(w/v)] were added dropwise to 30 ml of n-octanol containing HCO-60 [hydrogenated castor oil, 5 % (w/v) ] as an emulsifier.
  • the w/o emulsion was prepared by homogenizing the mixtures for 1 hr in 8,000 rpm with homogenizer (Ultra-turrax ® disperser, IKA Werke, Germany) in a 100 ml baffled pyrex beaker (750mm in diameter x 900mm in height) . Then, n-octanol containing 1 % (w/v) calcium chloride was added into the emulsions by an air sprayer (Fuso Seiki Co. Ltd., Japan) while stirring the whole medium slowly with a magnetic stirrer. The microspheres were cured for 10 minutes after mixing additionally with 5 ml of 8% (w/v) CaCl 2 to saturate the mixture.
  • homogenizer Ultra-turrax ® disperser, IKA Werke, Germany
  • n-octanol containing 1 % (w/v) calcium chloride was added into the emulsions by an air sprayer (Fuso Seiki Co
  • microspheres were collected onto polytetrafluoroethylene (PTFE) membrane filters with pore of 0.44 ⁇ m(Alltech Associates, Inc. IL) .
  • PTFE polytetrafluoroethylene
  • the microspheres on the filter were washed with 20 ml of isopropyl alcohol and dried in vacuum for 18 hrs.
  • the size and surface morphology of the microspheres were examined using a scanning electron microscope (S- 2460N, Hitachi Ltd., Japan) as described elsewhere (Chun et al . , 1996) .
  • S- 2460N scanning electron microscope
  • step (3-1) Aliquots of antigens were added to 5 ml of 5% alginate aqueous solution to prepare the microspheres as described in step (3-1) .
  • the emulsion was prepared by homogenizing in 8,000 rpm for an hour.
  • HCO-60 Nakko Chemical Co. Ltd, Japan
  • CaCl 2 solution was used for the gelation.
  • the microspheres were stored in a sealed vial at 4 ° C until use.
  • the loading efficiency of antigens was measured by assaying the protein content. Total loaded amount of the antigens in the microspheres was determined by dissolving the microspheres completely in 0.1 M sodium citrate buffer at pH7.8 for 3 hours with magnetic stirring. After the dissolved solution was dialyzed against PBS, the total protein concentration was determined by the Bradford assay.
  • the average efficiency for the encapsulation of PsaA protein [ (encapsulated protein/total protein in the solution) x 100] was 24% (42% for BSA protein).
  • the ratio of total PsaA protein loaded in the microspheres [ (weight of protein loaded/weight of microspheres) x 100] was approximately 0.4% (w/w) and that of BSA protein loaded was 0.8 % (w/w) .
  • the low encapsulation efficiency for PsaA protein may be due to the low protein concentration in the solution and a small size of PsaA protein.
  • the rate of the PsaA release from microspheres was determined as follows .
  • the known amount of microspheres 10 mg was placed in sealed vials containing PBS(1 ml) and shaken at 37 ° C. At various time points, aliqouts of supernatant were drawn after centrifugation at 1,000 g for 15 minutes and the protein content was determined by Bradford assay. The amount of protein in the aliquots was related to the total protein in the samples of microspheres and then, the cumulative percent release of protein was determined as a function of time.
  • FIG. 10 A typical in vi tro release profile of PsaA from the alginate microspheres in PBS is shown in Fig. 10.
  • PsaA was continuously released for 18 hours after having a burst of the release for the first hour. Although about one-half of the encapsulated antigen could be lost during the initial phase, 50% of antigens left in alginate micropheres after the initial burst could be released in a sustained fashion.
  • mice Female Balb/c mice, 6 to 8 weeks old, were obtained from DAEHAN Laboratory Animal Research Center Co. (Korea) . The animals were kept under standard specific pathogen free(SPF) conditions and given pelleted food and tap water throughout the experiment ad l ibi tum .
  • SPPF pathogen free
  • the emulsion of the antigens dissolved in PBS (200 ⁇ l) and complete Freund' s adjuvant (boosting was performed with incomplete Freund' s adjuvant) was injected subcutaneously (S . C . ) .
  • the doses based on the PsaA contents were 40 ⁇ g/mouse for immunization.
  • the mice were immunized in 2 week intervals for the second and third times.
  • Bronchoalveolar lavage was collected by pertracheal cannulation and gently washed with 0.7 ml of ice-cold PBS. About 0.5 ml of lung lavage was recovered from each mouse, and stored at -20 ° C after centrifugation 4 C at 3,000 xg for 5 min to remove debris. The blood contamination of the bronchoalveolar lavage fluid was assessed by detecting hemoglibin at O.D. 575nm . The murine blood diluted serially in PBS served as a standard. The mean blood contamination was 4 ⁇ 0.7 %. PsaA specific antibody responses at serum, gut wash and lung lavage were assayed according to the method of step (5-2) .
  • Anti-PsaA antibodies formed by immunization which was executed for the purpose of confirming the immunogenicity of recombinant PsaA is shown in Fig.11.
  • the levels of anti-PsaA antibodies of IgG and IgA increased generally, but the extent of increment was locally different.
  • the IgG response in intestine must result from a paracellular diffusion of serum-derived and locally produced IgG through epithelia.
  • the IgA responses generally lower than IgG responses, were induced by subcutaneous route. Antigenicity of recombinant PsaA protein could be confirmed by the results of these subcutaneous immunizations.
  • Stable peroxidase substrate buffer (PIERCE Chemical Co., USA) containing OPD(l mg/ml) was used to develop color. After the reaction was stopped by adding 100 ⁇ l of 2.0 N sulfuric acid, the optical density (0. D . ) at 492 nm was measured using a microplate reader (Molecular Device Corp, USA) . The sample was assayed in duplicate and a pair of blank well was included on each plate.
  • the quantity of the antibodies is expressed as geometric mean of concentraion of antibodies in the samples.
  • concentration of antibodies standard curves were made using sandwich ELISA. Micro-titer plates were coated with 50 ⁇ l of bicarbonate buffer containing 4 ⁇ g of goat anti-mouse immunoglobulin isotypes (IgM, IgG, IgA; Organon Teknika Corp. Durham, NC, USA) overnight at 4 ° C. After blocking with 5 % skim milk, purified mouse immunoglobuli (Organon Teknika Corp.) with known concentration of isotypes served as a standard.
  • Optical density was proportional to IgM antibody concentration from 20 to 120 ng/ml, to mouse IgG antibody at concentraions from 10 to 110 ng/ml and to mouse IgA antibody at concentraions from 10 to 100 ng/ml(see Fig.12).
  • the antigens were resuspened in PBS with 3 % Na 2 Co 3 immediately before peroral (P .0. ) immunization with 40 ug/mouse of E (PsaA) or N (PsaA) .
  • the mice were orally administered with the antigen solution (500 ⁇ l/dose) via blunt-tipped feeding needle inserted into the stomach .
  • PsaA specific antibody responses at serum, gut wash and lung lavage obtained using method of step (5-1) were assayed according to method of step (5-2) .
  • the effect of the carrier on the immunogenicity of orally-administered PsaA was investigated by comparing the immune responses of the mice immunized with E(PsaA) and N(PsaA) (see Fig.13).
  • CT cholera toxin
  • CTB Cholera toxin B subunit
  • microspheres of this invention are considered as a proper carrier for an efficient oral delivery of antigens because antigens can be incorporated into alginate microcapsules that protect antigens from denaturation at low pH and hydrolytic degradation by protease in gastrointestinal tract.
  • the microsphere of this invention is less than 5 ⁇ m in diameter, therefore, it can be effectively deliver the incoporated antigens to the Peyer' s patch and to lymphatics in systemic and local immune systems via concomitant transport pathways. Additionally, the microsphere of this invention not only shows higher levels of antigenicity than the naked protein antigen but is to enhance the level of serum and mucosal antibodies when co-administered orally with cholera toxin or cholera toxin B subunit, which are known to be powerful mucosal adjuvants.

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Abstract

La présente invention concerne des compositions vaccinales destinées à l'administration par voie orale ainsi que des procédés de préparation de ces dernières. Les compositions vaccinales sont principalement constituées d'un antigène protéique, selon une quantité efficace pour induire une réponse immunitaire dirigée contre ledit antigène, lui-même encapsulé dans des microsphères d'alginate. De manière plus particulière, cette invention concerne des compositions vaccinales destinées à l'administration orale qui sont constituées d'un antigène protéique encapsulé dans des microsphères d'alginate biodégradables au moyen d'une technique de gélification interfaciale contrôlée par la diffusion qui permet de produire des microsphères d'un diamètre inférieur à 5 νm; ainsi que des procédés de préparation associés.
PCT/KR1999/000466 1999-06-25 1999-08-19 Compositions vaccinales comprenant des antigenes encapsules dans des microspheres d'alginate destinees a l'administration orale et procedes de preparation de ces compositions WO2001000233A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2001078682A2 (fr) * 2000-04-17 2001-10-25 Ltt Institute Co., Ltd. Compositions pharmaceutiques a liberation prolongee
US6656470B2 (en) * 2000-05-12 2003-12-02 Pharmacia & Upjohn Company Vaccine composition, method of preparing the same, and method of vaccinating vertebrates
WO2006104486A1 (fr) * 2005-03-25 2006-10-05 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Sevices, Centers For Disease Control And Prevention Mise au point de la reaction en chaine de la polymerase en temps reel pour la detection d'adn pneumococcique et diagnostic de maladie pneumococcique
WO2012107095A1 (fr) 2011-02-10 2012-08-16 Alfa Biogene International B.V. Plante hsp70 pour utilisation dans le traitement d'une allergie alimentaire
CN105759036A (zh) * 2016-04-11 2016-07-13 新疆农业大学 用于微囊化大肠杆菌口服疫苗免疫抗体检测的检测方法及检测试剂盒
CN108478870A (zh) * 2017-07-12 2018-09-04 上海白衣缘生物工程有限公司 一种硬膜生物补片及其制备方法

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KR100406503B1 (ko) * 2001-09-25 2003-11-19 한국과학기술연구원 폐렴구균성 표면 접착단백질의 가용화용 조성물, 가용화용액상제형, 및 가용화용 분말제형, 이들 각각의 제조방법및 백신
KR102196572B1 (ko) * 2019-10-10 2020-12-29 대한민국 알긴산나트륨 캡슐을 포함하는 어류용 백신 조성물 및 이의 제조방법

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WO1998018346A2 (fr) * 1996-10-25 1998-05-07 Privates Institut Bioserv Gmbh Microcapsules de gout neutre, leur procede de production et leur utilisation
EP0873752A2 (fr) * 1997-04-23 1998-10-28 Pfizer Inc. Encapsulation de vaccin sous-unitaire contre le virus de l'herpès bovin type-1 basée sur un solvant aqueux
US5900238A (en) * 1995-07-27 1999-05-04 Immunex Corporation Vaccine delivery system

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US5900238A (en) * 1995-07-27 1999-05-04 Immunex Corporation Vaccine delivery system
WO1998018346A2 (fr) * 1996-10-25 1998-05-07 Privates Institut Bioserv Gmbh Microcapsules de gout neutre, leur procede de production et leur utilisation
EP0873752A2 (fr) * 1997-04-23 1998-10-28 Pfizer Inc. Encapsulation de vaccin sous-unitaire contre le virus de l'herpès bovin type-1 basée sur un solvant aqueux

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001078682A2 (fr) * 2000-04-17 2001-10-25 Ltt Institute Co., Ltd. Compositions pharmaceutiques a liberation prolongee
WO2001078682A3 (fr) * 2000-04-17 2002-04-18 Ltt Inst Co Ltd Compositions pharmaceutiques a liberation prolongee
US6656470B2 (en) * 2000-05-12 2003-12-02 Pharmacia & Upjohn Company Vaccine composition, method of preparing the same, and method of vaccinating vertebrates
US7160544B2 (en) 2000-05-12 2007-01-09 Pharmacia & Upjohn Company Method of vaccinating vertebrates
WO2006104486A1 (fr) * 2005-03-25 2006-10-05 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Sevices, Centers For Disease Control And Prevention Mise au point de la reaction en chaine de la polymerase en temps reel pour la detection d'adn pneumococcique et diagnostic de maladie pneumococcique
WO2012107095A1 (fr) 2011-02-10 2012-08-16 Alfa Biogene International B.V. Plante hsp70 pour utilisation dans le traitement d'une allergie alimentaire
CN105759036A (zh) * 2016-04-11 2016-07-13 新疆农业大学 用于微囊化大肠杆菌口服疫苗免疫抗体检测的检测方法及检测试剂盒
CN108478870A (zh) * 2017-07-12 2018-09-04 上海白衣缘生物工程有限公司 一种硬膜生物补片及其制备方法

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