WO2000004922A1 - Vaccins contre l'infection due a l'escherichia coli o157 - Google Patents

Vaccins contre l'infection due a l'escherichia coli o157 Download PDF

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Publication number
WO2000004922A1
WO2000004922A1 PCT/US1998/014976 US9814976W WO0004922A1 WO 2000004922 A1 WO2000004922 A1 WO 2000004922A1 US 9814976 W US9814976 W US 9814976W WO 0004922 A1 WO0004922 A1 WO 0004922A1
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WIPO (PCT)
Prior art keywords
coli
composition
serum
specific polysaccharide
shiga toxin
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PCT/US1998/014976
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English (en)
Inventor
Shousun C. Szu
John B. Robbins
Edward Konadu
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
KONADU, Yvonne, Ageyman
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Application filed by The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services, KONADU, Yvonne, Ageyman filed Critical The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority to BR9815953-4A priority Critical patent/BR9815953A/pt
Priority to PCT/US1998/014976 priority patent/WO2000004922A1/fr
Priority to US09/744,289 priority patent/US6858211B1/en
Priority to CA2714833A priority patent/CA2714833A1/fr
Priority to AU85758/98A priority patent/AU767047B2/en
Priority to CA2338093A priority patent/CA2338093C/fr
Publication of WO2000004922A1 publication Critical patent/WO2000004922A1/fr
Priority to US10/987,428 priority patent/US7553490B2/en
Priority to US11/015,436 priority patent/US7247307B2/en
Priority to US12/471,049 priority patent/US8168195B2/en

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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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0283—Shigella
    • 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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to conjugates ofthe O-specif ⁇ c polysaccharide of Shiga toxin-producing bacteria, such as E. coli 0157, with a carrier, and compositions thereof, and to methods of using of these conjugates and/or compositions thereof for eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity of, bacterial infections. More particularly it relates to the use of polysaccharides containing the tetrasaccharide repeat unit:
  • conjugates thereof to induce serum antibodies having bactericidal (killing) activity against E. coli, in particular E. coli 0157.
  • the conjugates, and compositions thereof, are useful as vaccines to induce serum antibodies which have bactericidal or bacteriostatic activity against against E. coli, in particular E. coli 0157, and are useful to prevent and/or treat illnesses caused by E. coli 0157.
  • the invention further relates to the antibodies which immunoreact with the O-specific polysaccharide of E. coli 0157 and/or the carrier, that are induced by these conjugates and/or compositions thereof.
  • the invention also relates to methods and kits for detection, identification, and/or diagnosis of E. coli 0157, using one or more ofthe polysaccharides, conjugates or antibodies described above.
  • carbohydrate-based, antibacterial vaccines The basis of using carbohydrates as vaccine components is that the capsular polysaccharides and the O- specific polysaccharides on the surface of pathogenic bacteria are both protective antigens and essential virulence factors.
  • the first saccharide-based vaccines contained capsular polysaccharides of Pneumococci: in the United States a 14-valent vaccine was licensed in 1978 followed by a 23-valent vaccine in 1983.
  • Other capsular polysaccharides licensed for human use include a tetravalent meningococcal vaccine and the Vi polysaccharide of Salmonella typhi for typhoid fever.
  • Escherichia coli O157:H7 an emerging infectious agent, was first recognized as a human pathogen in 1983 [6]. Diseases caused by this pathogen have subsequently been recognized worldwide [7]. Infection with E. coli 0157 causes a spectrum of illnesses with high morbidity and mortality, ranging from watery diarrhea to hemorrhagic colitis and the extraintestinal complication of hemolytic-uremic syndrome (HUS). HUS can lead to acute renal failure requiring dialysis, and in children and infants this complication has a considerable mortality. In some studies, E. coli 0157 was the most common cause of dysentery in patients seen in hospital clinics [8].
  • E. coli strains associated with HUS produce at least one toxin identical to the exotoxin of Shigella dysenteriae serotype 1, referred to herein as Shiga toxin 1 (Stxl).
  • This toxin has been variously referred to in the literature as Vero cytotoxin 1 (VT1), Shiga-like toxin 1 (SLT-I), and Shiga toxin l(Stx-I or Stxl).
  • VT1 Vero cytotoxin 1
  • SLT-I Shiga-like toxin 1
  • Shiga toxin l(Stx-I or Stxl) Shiga toxin l(Stx-I or Stxl).
  • VT2 toxin (variously referred to as VT2, SLT-II, Stx-II, or Stx2), structurally and functionally related to Stxl and having a cross-reactive A subunit, is also produced.
  • E. coli O157:H7 is a common serotype that produces these toxins.
  • strains of E. coli 0157 without Stx have been isolated from patients with hemorrhagic colitis.
  • E. coli 0157 has been compared to that of Shigella dysenteriae type 1 [9, 10]. Both E. coli 0157 and S. dysenteriae type 1 secrete almost identical exotoxins (Stxl or Stx2) and cause bloody diarrhea, with its complications, only in humans. Antibiotic treatment does not ameliorate the course of enteritis caused by E. coli 0157, and it may in fact increase the incidence of HUS caused by E. coli and S. dysenteriae type 1 [11,12]. Unlike S. dysenteriae type 1, which is confined to humans, E. coli O157:H7 lives in cattle and in other domesticated animals without causing symptoms. The feces of infected animals serve as a source of E. coli 0157 infection in humans, through contamination of drinking water and meat.
  • the immunogenicity of saccharides, alone or as protein conjugates, is related to several variables: 1) species and the age ofthe recipient; 2) molecular weight ofthe saccharide; 3) density ofthe saccharide on the protein; 4) configuration ofthe conjugate (single vs. multiple point attachment); and 5) the immunologic properties ofthe protein.
  • polysaccharides can induce the synthesis of antibodies from B-cells alone, they are described as T-independent antigens.
  • Three properties of polysaccharides are associated with T-independence; 1) their repetitive polymeric nature, which results in one molecule having multiple identical epitopes; 2) a minimum molecular weight that is related to their ability to adhere to and cross-link membrane-bound IgM receptors, resulting in signal transduction and antibody synthesis; and 3) resistance to degradation by mammalian enzymes.
  • Most capsular polysaccharides are of comparatively high molecular weight (>150 kD), and elicit antibodies in older children and in adults but not in infants and young children.
  • O-SPs are of lower molecular weight ( ⁇ 100 kD), and may be considered to be haptens because they combine with antibody (are antigenic) but do not elicit antibody synthesis (are not immunogenic).
  • the immunogenicity of O-SPs as conjugates may be explained by two factors: 1) the increase in molecular weight that allows the O-SP to adhere to a greater number of membrane-bound IgM and induce signal transduction to the B-cell; and 2) their protein component, which is catabolized by the O-SP stimulated B cell resulting in a peptide-histocompatibility II antigen signal to T cells.
  • LPS is not suitable for parenteral administration to humans because of toxicity mediated by the lipid A domain.
  • O-SP is prepared by treatment of LPS with either acid or hydrazine in order to remove fatty acids from lipid A.
  • the resultant products retain the core region and the O-SP with its heterogeneous range of molecular weights (M r ).
  • Conjugates are prepared by schemes that bind the carrier to the O-SP at multiple sites along the O-SP, or attempt to activate one residue ofthe core region.
  • conjugates having polysaccharides containing the tetrasaccharide repeat unit: ( ⁇ 3)- ⁇ -£ ) -Gak7NAc-(l ⁇ 2)- ⁇ -Z)-PerpNAc-(l ⁇ 3)- ⁇ -Z-Fucp-(l ⁇ 4)- ⁇ -j9-Glc ⁇ -(l ⁇ ), and compositions thereof, to induce serum antibodies having bactericidal (killing) activity against E. coli, in particular E. coli 0157.
  • the conjugates, and compositions thereof are useful as vaccines to induce serum antibodies which have bactericidal or bacteriostatic activity against against E. coli, in particular E. coli 0157, and are useful to prevent and/or treat illnesses caused by E. coli 0157.
  • These conjugates have the advantage of inducing both (1) serum IgG anti-O157-LPS with bactericidal activity, and (2) neutralizing antibodies to Shiga toxin 1 or Shiga toxin 2 (Stxl or Stx2)[l 9,20,21].
  • Such antibodies may be isolated, or may be provided in the form of serum containing these antibodies.
  • the invention also provides methods and kits for identifying, detecting, and/or diagnosing E. coli 0157 infection or colonization using the antibodies which immunoreact with the O-specific polysaccharide of E. coli.
  • the invention also relates to methods and kits for identifying, detecting and/or diagnosing the presence of Shiga toxins 1 or 2.
  • the invention provides conjugates of an E. coli 0157 O-specific polysaccharide covalently bound, either directly or through a linker, to a carrier, and compositions thereof.
  • the present invention also encompasses mixtures of such conjugates and compositions thereof.
  • the carrier is the non-toxic B subunit of Shiga toxin 1 or 2 (StxBl, StxB2), or a non-toxic mutant of Stxl or Stx2 holotoxin.
  • the particular E. coli 0157-Stx conjugate is part of a composition containing O-SP-carrier conjugates from other E. coli strains that commonly cause HUS, to form a multivalent vaccine for broad coverage against HUS.
  • Hyperimmune plasma containing both anti-LPS and neutralizing antibodies to Stxs are expected to provide protective and therapeutic effects in at-risk individuals and in patients during outbreaks.
  • the invention also provides methods of using these conjugates or compositions thereof to induce in mammals, in particular, humans, the production of antibodies which immunoreact with the O-specific polysaccharide of E. coli 0157.
  • antibodies which immunoreact with Shiga toxin 1 or Shiga toxin 2 are also produced.
  • the antibodies which immunoreact with the O-specific polysaccharide of E. coli 0157 are useful for the identification, detection, and/or diagnosis of E. coli 0157 colonization and/or infection.
  • Antibodies which have bactericidal or bacteriostatic activity against E. coli 0157 are useful to prevent and/or treat illnesses caused by E. coli 0157.
  • Antibodies which immunoreact with Shiga toxins 1 and 2 are useful to neutralize Shiga toxins 1 and 2, and either decrease the incidence and/or severity of hemolytic-uremic syndrome, or prevent the increase of its incidence and/or severity, in established infections.
  • compositions of this invention are capable, upon injection into a human of an amount containing 25 ⁇ g of E. coli 0157 O-specific polysaccharide, of inducing in the serum bactericidal activity against E. coli 0157, such that the serum kills, in the presence of complement, 50% or more of E. coli 0157 at a serum dilution of 1300: 1 or more.
  • Preferred compositions can induce serum bactericidal activity against E. coli 0157 such that the serum kills 50% or more of E. coli 0157 at a serum dilution of 32,000:1 or more, and the most preferred compositions can induce serum bactericidal activity against E. coli 0157 such that the serum kills 50% or more of E.
  • the O-SP conjugate vaccines of this invention are designed to induce serum IgG antibodies that will inactivate an inoculum of E. coli 0157 at the entrance ofthe jejunum before an infection is established.
  • the invention also provides a saccharide-based vaccine, which is intended for active immunization for prevention of E. coli 0157 infection, and for preparation of immune antibodies as a therapy, preferably for established infections.
  • the vaccines of this invention are designed to confer specific preventative immunity against infection with E. coli 0157, and to induce antibodies specific to E. coli 0157 O-SP and LPS.
  • the E. coli 0157 vaccine is composed of non-toxic bacterial components, suitable for infants, children of all ages, and adults.
  • compositions including but not limited to, mammalian serum, plasma, and immunoglobulin fractions, which contain antibodies which are immunoreactive with E. coli 0157 O-SP, and which preferably also contain antibodies which are immunoreactive with Shiga toxins 1 or 2, in particular with the B subunit of Shiga toxins 1 or 2.
  • These compositions in the presence of complement, have bacteriostatic or bactericidal activity against E. coli 0157.
  • These antibodies and antibody compositions are useful to prevent, treat, or ameliorate infection and disease caused by the microorganism.
  • the invention also provides such antibodies in isolated form.
  • High titer anti-0157 sera, or antibodies isolated therefrom, could be used for therapeutic treatment for patients with E. coli 0157 infection or hemolytic- uremic syndrome (HUS).
  • Antibodies elicited by the O-SP conjugates of this invention may be used for the treatment of established E. coli 0157 infections, and are also useful in providing passive protection to an individual exposed to E. coli 0157.
  • the present invention also provides diagnostic tests and/or kits for E. coli 0157 infection and/or colonization, using the conjugates and/or antibodies ofthe present invention, or compositions thereof.
  • the present invention also provides an improved method for synthesizing an O-SP peptide conjugate, particularly the E. coli 0157 O-SP conjugated to the B subunit of Shiga toxin 1 or 2 (Stxl or Stx2), or to a mutant, nontoxic Stxl or Stx2 holotoxin.
  • an O-SP peptide conjugate particularly the E. coli 0157 O-SP conjugated to the B subunit of Shiga toxin 1 or 2 (Stxl or Stx2), or to a mutant, nontoxic Stxl or Stx2 holotoxin.
  • E. coli LPS-protein conjugates of this invention are expected to be useful for several purposes, including but not limited to:
  • the invention is intended to be included in the routine immunization schedule of infants and children, and in individuals at risk for E. coli 0157 infection. It is also planned to be used for intervention in epidemics caused by E. coli 0157. Additionally, it is may be used as a component of a multivalent vaccine for E. coli 0157 and other enteric pathogens, useful for example for the routine immunization of infants.
  • the invention is also intended to prepare antibodies with bacteriostatic bactericidal activity toward E. coli 0157, for therapy of established infection.
  • the invention is also intended to provide a diagnostic test for E. coli 0157 infection and/or colonization. Definitions
  • Gd ⁇ p galactosaminopyranosyl
  • Peip perosaminopyranosyl
  • Fucp fucopyranosyl
  • G cp glucopyranosyl.
  • O-SP when used alone refers generically to O-specific polysaccharide, whether produced by acidolysis or hydrazinolysis of lipopolysaccharide.
  • O-SP-rEPA O-specific polysaccharide produced by acidolysis
  • DeA-LPS O-specific polysaccharide produced by hydrazinolysis
  • the terms “immunoreact” and “immunoreactivity” refer to specific binding between an antigen or antigenic determinant-containing molecule and a molecule having an antibody combining site, such as a whole antibody molecule or a portion thereof.
  • antibody refers to immunoglobulin molecules and immuno logically active portions of immunoglobulin molecules.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), as well as chimeric antibody molecules.
  • Polymeric carriers are those portions known in the art as Fab, Fab', F(ab') 2 and F(v), as well as chimeric antibody molecules.
  • Carriers are chosen to increase the immunogenicity ofthe polysaccharide and/or to raise antibodies against the carrier which are medically beneficial. Carriers that fulfill these criteria are described in the art [22, 23, 24, 25].
  • a polymeric carrier can be a natural or a synthetic material containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, ' or carboxyl groups.
  • the carrier can be water soluble or insoluble.
  • Water soluble peptide carriers are preferred, and include but are not limited to natural or synthetic polypeptides or proteins, such as bovine serum albumin, and bacterial or viral proteins or non-toxic mutants or polypeptide fragments thereof, e.g., tetanus toxin or toxoid, diphtheria toxin or toxoid, Pseudomonas aeruginosa exotoxin or toxoid, recombinant Pseudomonas aeruginosa exoprotein A, pertussis toxin or toxoid, Clostridium perfringens and Clostridium welchii exotoxins or toxoids, mutant non-toxic Shiga toxin holotoxin, Shiga toxins 1 and 2, the B subunit of Shiga toxins 1 and 2, and hepatitis B surface antigen and core antigen.
  • natural or synthetic polypeptides or proteins such as bovine serum albumin, and bacterial
  • water insoluble carriers examples include, but are not limited to, aminoalkyl SEPHAROSE, e. g., aminopropyl or aminohexyl SEPHAROSE (Pharmacia Inc., Piscataway, NJ), aminopropyl glass, and the like.
  • Other carriers may be used when an amino or carboxyl group is added, for example through covalent linkage with a linker molecule.
  • a polysaccharide containing at least one carboxyl group may be thiolated with cystamine, or aminated with adipic dihydrazide, diaminoesters, ethylenediamine and the like.
  • Groups which can be introduced by such known methods include thiols, hydrazides, amines and carboxylic acids.
  • Thiolated and aminated intermediates are stable, and may be freeze dried and stored cold.
  • Thiolated intermediates may be covalently linked to a polymeric carrier containing a sulfhydryl group, such as a 2-pyridyldithio group.
  • Aminated intermediates may be covalently linked to a polymeric carrier containing a carboxyl group through carbodiimide condensation. See for example reference [26], where 3 different methods for conjugating Shigella O-SP to tetanus toxoid are exemplified. Because the methods ofthe present invention better preserve the native structure ofthe antigen, they are preferred over methods which oxidize the polysaccharide with periodate [18].
  • the polysaccharide can be covalently bound to a carrier with or without a linking molecule.
  • a carboxyl- group-containing polysaccharide and an amino-group-containing carrier are mixed in the presence of a carboxyl activating agent, such as a carbodiimide, in a choice of solvent appropriate for both the polysaccharide and the carrier, as is known in the art [25].
  • the polysaccharide is often conjugated to a carrier using a linking molecule.
  • a linker or crosslinking agent, as used in the present invention is preferably a small linear molecule having a molecular weight of about 500 or less, and is non-pyrogenic and non-toxic in the final product form, for example as disclosed in references [22 - 25].
  • linkers or crosslinking agents are homobifunctional or heterobifunctional molecules, e.g., adipic dihydrazide, ethylenediamine, cystamine, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-N-(2-iodoacetyl)- ⁇ -alaninate-propionate (SIAP), succinimidyl 4-(N-maleimido-methyl)cyclohexane- 1-carboxylate (SMCC), 3,3'-dithiodipropionic acid, and the like.
  • SMCC N-maleimido-methylcyclohexane- 1-carboxylate
  • SMCC 3,3'-dithiodipropionic acid
  • attachment ofthe E. coli 0157 O-specific polysaccharide to a protein carrier can be accomplished by methods known to the art.
  • the attachment is accomplished by first cyanating the O- specific polysaccharide with a cyanylation reagent, such as cyanogen bromide, N- cyano-N,N,N-triethylammonium tetrafluoroborate, l-cyano-4-(N,N- dimethylamino)pyridine tetrafluoroborate, or the like.
  • a cyanylation reagent such as cyanogen bromide, N- cyano-N,N,N-triethylammonium tetrafluoroborate, l-cyano-4-(N,N- dimethylamino)pyridine tetrafluoroborate, or the like.
  • cyanylation reagents are known to those skilled in the art [27].
  • coli 0157 O-specific polysaccharide may then be reacted with a linker, such as a dicarboxylic acid dihydrazide, preferably adipic acid dihydrazide, so as to form a hydrazide-functionalized polysaccharide.
  • a linker such as a dicarboxylic acid dihydrazide, preferably adipic acid dihydrazide
  • This hydrazide-functionalized polysaccharide is then coupled to the carrier protein by treatment with a peptide coupling agent, preferably a water-soluble carbodiimide such as l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the like.
  • the cyanated E. coli 0157 O-specific polysaccharide is directly reacted with the carrier protein, without introduction of a linker. It has been found, surprisingly, that, in the exemplified conjugates, elimination ofthe customary linker provides a more effective immunogen in the case ofthe E. coli 0157 O-specific polysaccharide.
  • the unbound materials are removed by routine physicochemical methods, such as for example gel filtration or ion exchange column chromatography, depending on the materials to be separated.
  • the final conjugate consists ofthe polysaccharide and the carrier bound directly or through a linker.
  • the present inoculum contains an effective, immunogenic amount of a polysaccharide-carrier conjugate of this invention.
  • the effective amount of polysaccharide-carrier conjugate per unit dose sufficient to induce an immune response to E. coli 0157 depends, among other things, on the species of mammal inoculated, the body weight ofthe mammal, and the chosen inoculation regimen, as is well known in the art.
  • Inocula typically contain polysaccharide-carrier conjugates with concentrations of polysaccharide from about 1 micrograms to about 10 milligrams per inoculation (dose), preferably about 3 micrograms to about 100 micrograms per dose, and most preferably about 5 micrograms to 50 micrograms per dose.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (polysaccharide) calculated to produce the desired immunogenic effect in association with the required diluent.
  • Inocula are typically prepared as solutions in physiologically tolerable (acceptable) diluents such as water, saline, phosphate-buffered saline, or the like, to form an aqueous pharmaceutical composition.
  • physiologically tolerable (acceptable) diluents such as water, saline, phosphate-buffered saline, or the like
  • Adjuvants such as aluminum hydroxide, may also be included in the compositions.
  • the route of inoculation may be intramuscular, subcutaneous or the like, which results in eliciting antibodies protective against E. coli 0157.
  • a second or booster dose may be administered approximately 4 to 6 weeks after the initial injection. Subsequent doses may be administered as indicated herein, or as desired by the practitioner.
  • An antibody ofthe present invention in one embodiment is characterized as comprising antibody molecules that immunoreact with E. coli 0157 O-SP or LPS.
  • An antibody ofthe present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing an E. coli 0157 polysaccharide-protein carrier conjugate to induce, in the mammal, antibody molecules having immunospecificity for the immunizing polysaccharide.
  • Antibody molecules having immunospecificity for the protein carrier such as the B subunit of Shiga toxins 1 or 2, will also be produced.
  • the antibody molecules may be collected from the mammal and, optionally, isolated and purified by methods known in the art.
  • Human or humanized monoclonal antibodies are preferred, including those made by phage display technology, by hybridomas, or by mice with human immune systems.
  • the antibody molecules ofthe present invention may be polyclonal or monoclonal.
  • Monoclonal antibodies may be produced by methods known in the art.
  • Portions of immunoglobulin molecules, such as Fabs, may also be produced by methods known in the art.
  • the antibody ofthe present invention may be contained in blood plasma, serum, hybridoma supernatants and the like.
  • Antibody-containing serum of this invention will be capable of killing, in the presence of complement, 50% of E. coli Ol 57at a serum dilution of 1300: 1 or more, preferably will do so at a dilution of 32,000:1 or more, and most preferably will be capable of killing 50% of E. coli 0157 at a dilution of 64,000:1 or more.
  • the antibodies ofthe present invention are isolated to the extent desired by well known techniques such as, for example, ion chromatography or affinity chromatography.
  • the antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgGi, IgG 2 , IgG 3 , IgG 4 and the like.
  • Antibodies ofthe IgG class are preferred for purposes of passive protection.
  • the antibodies ofthe present invention have a number of diagnostic and therapeutic uses.
  • the antibodies can be used as an in vitro diagnostic agents to test for the presence of E. coli 0157 in biological samples or in meat and meat products, in standard immunoassay protocols.
  • Such assays include, but are not limited to, agglutination assays, radioimmunoassays, enzyme-linked immunosorbent assays, fluorescence assays, Western blots and the like.
  • the biological sample is contacted with first antibodies ofthe present invention, and a labeled second antibody is used to detect the presence of E. coli 0157 to which the first antibodies have bound.
  • Such assays may be, for example, of direct format (where the labeled first antibody is reactive with the antigen), an indirect format (where a labeled second antibody is reactive with the first antibody), a competitive format (such as the addition of a labeled antigen), or a sandwich format (where both labeled and unlabelled antibody are utilized), as well as other formats described in the art.
  • the antibodies ofthe present invention are also useful in prevention and treatment of infections and diseases caused by E. coli 0157.
  • the dosage of administered antibodies will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
  • the recipient In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 1 mg/kg to about 10 mg/kg body weight ofthe mammal, although a lower or higher dose may be administered.
  • the antibodies ofthe present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, or lessen or attenuate the severity, extent or duration ofthe infection by E. coli 0157.
  • Antibodies which immunoreact with Shiga toxin 1 or 2 are intended to be provided to the recipient subject in an amount sufficient to prevent, or lessen or attenuate the severity, extent or duration ofthe infection by Shigatoxin producing organisms, such as E. coli strains 0157, Ol l l, 026, and 017.
  • the administration ofthe agents ofthe invention may be for either "prophylactic” or "therapeutic" purpose.
  • the agents are provided in advance of any symptom.
  • the prophylactic administration ofthe agent serves to prevent or ameliorate any subsequent infection.
  • the agent is provided at (or shortly after) the onset of a symptom of infection.
  • the agent ofthe present invention may, thus, be provided prior to the anticipated exposure to E. coli 0157 (or other Shiga toxin producing bacteria), so as to attenuate the anticipated severity, duration or extent of an infection and disease symptoms, after exposure or suspected exposure to these bacteria, or after the actual initiation of an infection.
  • polysaccharide-carrier conjugates of this invention as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used.
  • 0157 LPS were detoxified by hydrolysis with acetic acid (designated O-SP) or with hydrazine (designated DeA-LPS) and then covalently bound to Clostridium welchii exotoxin C (Pig Bel toxoid [CW]), Pseudomonas aeruginosa recombinant exoprotein A (rEPA), or bovine serum albumin (BSA) [8].
  • O-SP acetic acid
  • DeA-LPS hydrazine
  • rEPA Pseudomonas aeruginosa recombinant exoprotein A
  • BSA bovine serum albumin
  • DeA-LPS-rEPA 2 Mice were immunized with these conjugate compositions containing 2.5ug of polysaccharide, with booster injections, and the determination of antibody levels and bactericidal antibody titers in mice were determined.
  • Geometric mean antibody level (ELISA units) and immunoglobulin class composition of LPS antibodies elicited by E. coli 0157-rEPA conjugates in mice are shown in Table 1.
  • DeA-LPS-rEPA 2 0.09 (0.06-0.11) 0.32 (0.06-1.53) 0.28 (0.21-0.45) a.
  • IgG and IgM components of the hyperimmune 0157 sera were used as standards and assigned a value of 100 ELISA U each. Injection of O-SP, DeA-LPS, or saline did not elicit detectable antibodies.
  • Vaccine Antibody titer Reciprocal bacterial
  • E. coli 0157:H7 is pooled hyperimmune sera from mice injected with heat-killed E. coli 0157. All other sera were from individual mice taken after the third conjugate injection. Serum dilutions were mixed with an equal volume of ⁇ 10 3 E. coli 0157:H7 organisms per ml and complement. b The reciprocal bactericidal titer is expressed as the highest serum dilution yielding 50% killing. Absorption with LPS or DeA-LPS removed all of the bactericidal activity from sera from conjugate-injected mice and 90% from the hyperimmune sera prepared by injection of heat-killed E. coli 0157.
  • Antibody levels are expressed as geometric means (GM). Levels below the sensitivity of ELISA were assigned the value of one-half of that level. Comparison of GM was performed with either the two-sided t-test, paired t-test or the Wilcoxon test where appropriate. Results - clinical responses
  • IgG Pre-vaccination GM IgG anti-LPS levels in the three groups were low and similar. One week after vaccination, 71/87 (82%) responded with a ⁇ 4-fold rise. Four weeks after vaccination, there were further rises in GM levels in all three groups (p ⁇ .0001): all vaccinees responded with a >4-fold rise over the 1 week level. The GM levels in recipients of O-SP-rEPA were slightly higher than in those injected with either ofthe two DeA-LPS-rEPA conjugates (61.9 vs. 46.3 NS, 61.9 vs. 36.3, p ⁇ 0.05). At 26 weeks, the GM levels ofthe 3 groups were similar (32.8, 31.2, 33.1, NS).
  • coli 0157 at 1 week had IgG anti-LPS levels at pre-immunization, 1-, 4-, and 26-week post-immunization of 0.81, 1.15, 7.73 and 7.01 respectively, that are lower than the GM of all 3 groups.
  • IgM Each conjugate elicited a significant rise in IgM anti-LPS at the 4 and 26 weeks intervals (p ⁇ 0.0001). O-SP-rEPA elicited the highest level at each post vaccination interval but the difference was significant only at 4 weeks (32.8 vs. 18.1,19.1, p ⁇ 0.05). At the 4 week interval, there was a >4-fold rise in 61/87 (70%) and in 34/86 (39.5%) at 26 weeks compared to pre-vaccination levels. There was a significant decrease in serum IgM anti-LPS at 26 weeks in all ofthe three groups (p ⁇ 0.02) but there were no significant differences in the GM levels among the three groups. The volunteer who had a stool culture positive for E.
  • IgA Pre-vaccination levels of IgA anti-LPS were low. Similar to IgG and IgM anti-LPS, about 90% ofthe volunteers responded with >4-fold rise in IgA anti- LPS at one week, and 99% at four weeks (p ⁇ 0.001). IgA anti-LPS GM levels declined to about 70% ofthe levels at the 4 week interval.
  • Serum from high-responding volunteers (above the 75th percentile) was diluted serially and the diluted samples were analyzed for their ability to kill E. coli O157:H7. Pre-vaccination sera had no detectable bactericidal activity against E. coli O157:H7. The three conjugates elicited serum bactericidal activity that roughly correlated with the serum IgG and IgM anti-LPS antibody levels.
  • Table 4 The results in Table 4 are those for serum from high-responding volunteers.
  • the bactericidal titer (reciprocal dilution) for 50% killing ranged from about 2400 to about 32000.
  • E. coli 0157 O-SP was prepared by treatment of LPS with acetic acid as previously described [8, 9].
  • the B-subunit of Shigella toxin I (StxBl) was synthesized by Vibrio cholerae strain 0395-N1 (pSBC32 containing the StxBl gene) and purified by affinity chromatography [20, 21].
  • SDS 7% PAGE of StxBl showed one major band at 9 kDa and a faint band with slightly higher molecular weight.
  • adipic acid dihydrazide (ADH) was prepared as described previously [8, 30]. Briefly after addition of TEA in the above procedure, an equal volume of 0.8 M ADH in 0.5 M NaHCO 3 was added and the pH maintained at 8.0 to 8.5 for 2 hours. The reaction mixture was dialyzed against saline overnight at 4 °C and passed through a 2.5x31 cm P10 column in water. The void volume fractions were pooled, freeze-dried, and designated as OSP-AH. OSP-AH (10 mg), dissolved in 2 ml of saline, was added to an equal weight of StxBl and the pH brought to 5.1.
  • the reaction mixture was put on ice and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) was added to 0.05M and the pH maintained at 5.1 to 5.5 for 2 hours.
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • the reaction mixture was passed through a 1.5x90 cm Sepharose 6B column in 0.2 M NaCl, the void volume fractions collected and designated as OSP-AH-StxBl. Double immunodiffusion and ELISA were performed as described [8].
  • Toxin neutralization was determined by incubating dilutions of mouse serum with Stx-I or Stx-II at a final concentration of 100 pg/ml. The serum and toxin mixture was incubated at room temperature for 30 minutes and 0.1 ml was added to each well. Following incubation overnight, the surviving cells were determined spectro- photometrically using the crystal violet staining method of Gentry and Dalrymple[31]. Toxin neutralization was determined from a dose response curve of either Shiga toxin on each 96-well plate. Bactericidal activity was assayed as described [8, 10]. Results with 0157 O-SP — STXB1 conjugates
  • Derivatization of O-SP with adipic acid dihydrazide was 3.1% (wt/wt), similar to previous E. coli 0157 preparations [8].
  • the saccharide/protein ratios (wt/wt) were about 0.5 for both conjugates.
  • the yields, based on saccharide in the conjugates, were 2.3% for OSP-StxBl and 3.4% for OSP-AH-StxBl.
  • a single line of precipitation in double immunodiffusion was formed by rabbit anti-Stxl and mouse hyperimmune anti-0157 reacted against either conjugate.
  • the O-SP of E. coli 0157 LPS is a linear copolymer composed ofthe tetrasaccharide repeat unit: ( ⁇ 3)- ⁇ -£ ) -Gak7NAc-(l ⁇ 2)- ⁇ -D-Per )NAc-(l ⁇ 3)- ⁇ - Z-Fucp-(l- 4)- ⁇ -Z -Glcp-(l->). It is non-immunogenic, probably due to its comparatively low molecular weight. As with other polysaccharides, its immunogenicity is increased by binding it to proteins to form a conjugate.
  • the protective action of existing vaccines may be due to the induction of a critical level of specific IgG antibodies that, in many cases, inactivate the inoculum of the pathogen on epithelial surfaces including the intestine [39, 40].
  • serum IgG is a major immunoglobulin component of secretory fluids including that ofthe small intestine.
  • mice [8] all three conjugates induced IgG anti-LPS with bactericidal activity in the volunteers (Table 2).
  • Serum IgG anti-polysaccharide is the major, if not the sole host component, that confers immunity induced by these conjugates. Accordingly, it should be possible to standardize the potency of E. coli 0157 conjugates by chemical assay and by measurement of serum IgG anti-polysaccharide as has been done for Haemophilus influenzae type b conjugate vaccines.
  • the data show that the various E. coli 0157 LPS -protein conjugates disclosed herein will generate high antibody levels in humans (i.e., approximately 5- 10 times more IgG in humans than in mice) and high neutralization antibody titers in humans (i.e., 10 3 to 10 4 in humans as opposed to 10 2 in mice).
  • the data also show that the various E. coli 0157 LPS-protein conjugates disclosed herein will generate a greater than 4-fold rise in IgG antibody levels in about 80% of human subjects one week after a single injection and in all human subjects 4 weeks after a single injection.

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Abstract

Conjugués du polysaccharide à spécificité O de E. coli O157 avec un véhicule, et compositions à base desdits conjugués, procédés d'utilisation de ces conjugués et/ou compositions pour induire une réponse immunogène chez des mammifères, dont des réponses qui fournissent une protection contre des infections bactériennes ou en réduisent la gravité. La présente invention concerne plus particulièrement l'utilisation de polysaccharides contenant l'unité de répétition tétrasaccharide (→3)-α-D-GalpNAc-(1→2)-α-D-PerpNAc-(1→3)-α-L-Fucp-(1→4)-β-D-Glcp-(1→) et de conjugués de ladite unité pour la production d'anticorps sériques ayant une activité bactéricide (tueuse) contre E. coli provoquant le syndrome de Gasser, en particulier E. coli O157. Lesdits conjugués et les compositions les contenant sont utiles en tant que vaccins destinés à produire des anticorps sériques qui ont une activité bactéricide ou bactériostatique contre E. coli, en particulier contre E. coli O157, et sont utiles pour prévenir et/ou traiter des maladies provoquées par E. coli O157. La présente invention concerne en outre des anticorps qui présentent une réaction immunitaire contre le polysaccharide à spécificité O de E. coli O157 et/ou le véhicule et qui sont induits par ces conjugués et/ou compositions. Elle concerne encore des procédés et des kits reposant sur l'utilisation d'un ou plusieurs des polysaccharides, conjugués ou anticorps décrits ci-dessus.
PCT/US1998/014976 1998-07-20 1998-07-20 Vaccins contre l'infection due a l'escherichia coli o157 WO2000004922A1 (fr)

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BR9815953-4A BR9815953A (pt) 1998-07-20 1998-07-20 Vacinas contra infecção provocada por escherichia coli o157
PCT/US1998/014976 WO2000004922A1 (fr) 1998-07-20 1998-07-20 Vaccins contre l'infection due a l'escherichia coli o157
US09/744,289 US6858211B1 (en) 1998-07-20 1998-07-20 Vaccines against Escherichia coli O157 infection
CA2714833A CA2714833A1 (fr) 1998-07-20 1998-07-20 Vaccins contre l'infection due a l'escherichia coli o157
AU85758/98A AU767047B2 (en) 1998-07-20 1998-07-20 Vaccines against (escherichia coli) O157 infection
CA2338093A CA2338093C (fr) 1998-07-20 1998-07-20 Vaccins contre l'infection due a l'escherichia coli o157
US10/987,428 US7553490B2 (en) 1998-07-20 2004-11-12 Vaccines against Escherichia coli O157 infection
US11/015,436 US7247307B2 (en) 1998-07-20 2004-12-16 Vaccines against Escherichia coli O157 infection
US12/471,049 US8168195B2 (en) 1998-07-20 2009-05-22 Vaccines against Escherichia coli O157 infection

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7048927B2 (en) 1999-08-10 2006-05-23 Allergan, Inc. Botulinum neurotoxin eluting stent
US8173135B2 (en) 2006-03-17 2012-05-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for preparing complex multivalent immunogenic conjugates
US11260119B2 (en) 2018-08-24 2022-03-01 Pfizer Inc. Escherichia coli compositions and methods thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356170A (en) * 1981-05-27 1982-10-26 Canadian Patents & Development Ltd. Immunogenic polysaccharide-protein conjugates
US4711779A (en) * 1985-07-05 1987-12-08 Sclavo S.P.A. Glycoproteinic conjugates having trivalent immunogenic activity
US5371197A (en) * 1991-09-24 1994-12-06 Merck & Co., Inc. Protein-dimeric polysaccharide conjugate vaccine
US5585100A (en) * 1992-02-11 1996-12-17 Henry Jackson Foundation Dual carrier immunogenic construct
US5693326A (en) * 1993-09-22 1997-12-02 Henry M. Jackson Foundation For The Advancement Of Military Medicine Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents
US5773007A (en) * 1990-09-17 1998-06-30 National Research Council Of Canada Vaccine compositions
US5785973A (en) * 1988-02-01 1998-07-28 Praxis Biologics, Inc. Synthetic peptides representing a T-cell epitope as a carrier molecule for conjugate vaccines

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4356170A (en) * 1981-05-27 1982-10-26 Canadian Patents & Development Ltd. Immunogenic polysaccharide-protein conjugates
US4711779A (en) * 1985-07-05 1987-12-08 Sclavo S.P.A. Glycoproteinic conjugates having trivalent immunogenic activity
US5785973A (en) * 1988-02-01 1998-07-28 Praxis Biologics, Inc. Synthetic peptides representing a T-cell epitope as a carrier molecule for conjugate vaccines
US5773007A (en) * 1990-09-17 1998-06-30 National Research Council Of Canada Vaccine compositions
US5371197A (en) * 1991-09-24 1994-12-06 Merck & Co., Inc. Protein-dimeric polysaccharide conjugate vaccine
US5585100A (en) * 1992-02-11 1996-12-17 Henry Jackson Foundation Dual carrier immunogenic construct
US5693326A (en) * 1993-09-22 1997-12-02 Henry M. Jackson Foundation For The Advancement Of Military Medicine Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
CHU C. ET AL: "Preparation, Characterization, and Immunogenicity of Conjugates Composed of the O-Specific Polysaccharide of Shigella Dysenteriae Type 1 (Shiga's Bacillus) Bound to Tetanus Toxoid", INFECTION AND IMMUNITY, vol. 59, no. 12, December 1991 (1991-12-01), USA, pages 4450 - 4458, XP002912811 *
COLI E. S. ET AL: "Influence of Shiga-Like Toxin Production in Enteric Infection with an Enteropathogenic", GASTROENTEROLOGY, vol. 95, no. 5, May 1987 (1987-05-01), USA, pages 1643, XP002912816 *
CRYZ S.J. ET AL: "Synthesis and Characterization of Escherichia Coli O18 O-Polysaccharide Conjugate Vaccines", INFECTION AND IMMUNITY, vol. 58, no. 2, February 1990 (1990-02-01), USA, pages 373 - 377, XP002912814 *
DICK W.E. ET AL: "Glycoconjugates of Bacterial Carbohydrate Antigens", CONJUGATE VACCINES, vol. 10, 1989, pages 48 - 114, XP002912808 *
GUPTA R.K. ET AL: "Comparative Immunogenicity of Conjugates Composed of Escherichia Coli O111 O-Specific Polysaccharide, Prepared by Treatment with Acetic Acid or Hydrazine, Bound to Tetanus Toxoid by Two Synthetic Schemes", INFECTION AND IMMUNITY, vol. 63, no. 8, August 1995 (1995-08-01), USA, pages 2805 - 2810, XP002912810 *
KONADU E. ET AL: "Preparation, Characterization, and Immunological Properties in Mice of Escherichia Coli O157 O-Specific Polysaccharide-Protein Conjugate Vaccines", INFECTION AND IMMUNITY, vol. 62, no. 11, November 1994 (1994-11-01), USA, pages 5048 - 5054, XP002912809 *
KONADU E.Y. ET AL: "Investigational Vaccine for Escherichia Coli O157: Phase 1 O157 O- Specific Polysaccharide- Pseudomonas Aeruginosa Recombinant Exoprotein A Conjugates in Adults", THE JOURNAL OF INFECTIONS DISEASES, vol. 177, February 1998 (1998-02-01), USA, pages 383 - 387, XP002912815 *
ROBBINS J.B. ET AL: "O-Specific Side-Chain Toxin-Protein Conjugates as Parenteral Vaccines for the Prevention of Shigellosis and Related Diseases", REVIEWS OF INFECTIOUS DISEASES, vol. 13, no. 4, 1991, USA, pages S362 - S365, XP002912812 *
TAYLOR D.N. ET AL: "Synthesis, Characterization, and Clinical Evaluation of Conjugate Vaccines Composed of the O-Specific Polysaccharides of Shigella Dysenteriae Type 1, Shigella Flexneri Type 2a, and Shigella Sonnei (Plesiomonas Shigelloides) Bound to Bacterial Toxoids", INFECTION AND IMMUNITY, vol. 61, no. 9, September 1993 (1993-09-01), USA, pages 3678 - 3687, XP002912813 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7048927B2 (en) 1999-08-10 2006-05-23 Allergan, Inc. Botulinum neurotoxin eluting stent
US7223399B2 (en) 1999-08-10 2007-05-29 Allergan, Inc. Methods for treating restenosis with a botulinum neurotoxin
US8173135B2 (en) 2006-03-17 2012-05-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for preparing complex multivalent immunogenic conjugates
US8557250B2 (en) 2006-03-17 2013-10-15 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for preparing complex multivalent immunogenic conjugates
US9175033B2 (en) 2006-03-17 2015-11-03 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for preparing complex multivalent immunogenic conjugates
EP3006041A1 (fr) 2006-03-17 2016-04-13 The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services Procédé de préparation de conjugués immunogéniques multivalents complexes
US11260119B2 (en) 2018-08-24 2022-03-01 Pfizer Inc. Escherichia coli compositions and methods thereof

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