Title:  Vaccines against Escherichia coli O157 infection

United States Patent:  6,858,211

Issued:  February 22, 2005

Inventors:  Szu; Shousun Chen (Bethesda, MD); Robbins; John B. (Chevy Chase, MD); Konadu; Edward (late of Ashanti Region, GH); Konadu; Yvonne Ageyman (Bronx, NY)

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC)

Appl. No.:  744289

Filed:  August 1, 2001

PCT Filed:  July 20, 1998

PCT NO:  PCT/US98/14976

371 Date:  August 1, 2001

102(e) Date:  August 1, 2001

PCT PUB.NO.:  WO00/04922

PCT PUB. Date:  February 3, 2000

Abstract

This invention relates to conjugates of the O-specific polysaccharide of E. coli O157 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: (.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)- .alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and conjugates thereof, to induce serum antibodies having bactericidal (killing) activity against hemolytic-uremic syndrome (HUS) causing E. coli, in particular E. coli O157. 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 O157, and are useful to prevent and/or treat illnesses caused by E. coli O157. The invention further relates to the antibodies which immunoreact with the O-specific polysaccharide of E. coli O157 and/or the carrier, that are induced by these conjugates and/or compositions thereof. The invention also relates to methods and kits using one or more of the polysaccharides, conjugates or antibodies described above.

Description of the Invention

FIELD OF THE INVENTION

This invention relates to conjugates of the O-specific polysaccharide of Shiga toxin-producing bacteria, such as E. coli O517, 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: (.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)- .alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and conjugates thereof, to induce serum antibodies having bactericidal (killing) activity against E. coli, in particular E. coli O517. 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 O517, and are useful to prevent and/or treat illnesses caused by E. coli O517.

The invention further relates to the antibodies which immunoreact with the O-specific polysaccharide of E. coli O517 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 O517, using one or more of the polysaccharides, conjugates or antibodies described above.

BACKGROUND

The most successful of all carbohydrate pharmaceuticals so far have been the carbohydrate-based, antibacterial vaccines [1]. 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. The inability of most polysaccharides to elicit protective levels of anti-carbohydrate antibodies in infants and adults with weakened immune systems could be overcome by their covalent attachment to proteins that conferred T-cell dependent properties [2]. This principle led to the construction of vaccines against Haemophilus influenzae b (Hib) [3] and in countries where these vaccines are routinely used, meningitis and other diseases caused by Hib have been virtually eliminated [4]. Extension of the conjugate technology to the O-specific polysaccharides of Gram-negative bacteria has provided a new generation of glycoconjugate vaccines that are undergoing various phases of clinical trials [5].

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 O517 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 O517 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 (Stx1). This toxin has been variously referred to in the literature as Vero cytotoxin 1 (VT1), Shiga-like toxin 1 (SLT-I), and Shiga toxin 1(Stx-I or Stx1). In some cases a second toxin (variously referred to as VT2, SLT-II, Stx-II, or Stx2), structurally and functionally related to Stx1 and having a cross-reactive A subunit, is also produced. Infection with Stx-producing organisms has been correlated with HUS, and E. coli O517:H7 is a common serotype that produces these toxins. However, strains of E. coli O517 without Stx have been isolated from patients with hemorrhagic colitis.

The pathogenicity of E. coli O517 has been compared to that of Shigella dysenteriae type 1 [9, 10]. Both E. coli O517 and S. dysenteriae type 1 secrete almost identical exotoxins (Stx1 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 O517, 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 O517 infection in humans, through contamination of drinking water and meat.

Most adults have low or nondetectable levels of serum antibodies to E. coli O517 O-SP and to Shiga toxins. High levels of O-SP antibodies and low or nondetectable levels of antitoxin are regularly found following infection with E. coli O517 and the subsequent complication HUS. It is not known whether immunity follows infection with this pathogen.

Although there is no consensus on the host factors that might confer immunity to E. coil O157, the O-specific polysaccharide portion of the lipopolysaccharides of the similar genus Shigella have emerged as possible protective antigens [13,14]. These polysaccharides were shown to be essential for the virulence of Shigella, and it is now well-established that the protection is serotype specific. Since each serotype is characterized by a distinct O-specific polysaccharide, it is fair to say that protection against E. coli O517 is also O-specific polysaccharide specific. The safety and immunogenicity of a protein conjugate of the O-specific polysaccharides of S. sonnei, S. flexneri 2a, and S. dysenteriae type 1 has been demonstrated in human volunteers, and preliminary clinical trials have established the efficacy of these vaccines [9, 15, 16, 17].

The immunogenicity of saccharides, alone or as protein conjugates, is related to several variables: 1) species and the age of the recipient; 2) molecular weight of the saccharide; 3) density of the saccharide on the protein; 4) configuration of the conjugate (single vs. multiple point attachment); and 5) the immunologic properties of the protein.

Because high molecular weight 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 (.gtoreq.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 (.gtoreq.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.

Synthesis of conjugates for use as vaccines in humans has special considerations. LPS is not suitable for parenteral administration to humans because of toxicity mediated by the lipid A domain. Usually, 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 of the core region.

In the case of E. coli O517, vaccine development has been hindered because there is little information about mechanisms of immunity [9], and there are no valid animal models for diseases caused by E. coli O517[10].

There have been some efforts to date to attempt to obtain effective vaccine compositions against E. coli. See, e.g., Cryz et al. (U.S. Pat. No. 5,370,872), which describes the isolation of O-SP derived from LPS of 12 serotypes of E. coli and their covalent linkage to P. aeruginosa toxin A as a carrier protein [18]. The twelve monovalent conjugates were combined to form a polyvalent vaccine, which was described as being safe and immunogenic in both rabbits and humans when administered by injection. An antibody response to both the O-SP and toxin A moieties was reported, and protection of rabbits against E. coli sepsis was demonstrated upon passive immunization with the resulting IgG antibodies. However, neither bactericidal activity of the antibodies nor protection after vaccination with the conjugates was shown, and antibodies against E. coli strain O517 and protection against E. coli O517 infection are not mentioned.

Because anti-LPS or anti-O-SP antibody-mediated protection is likely to be serotype-specific, it is unlikely that the polyvalent vaccine described in U.S. Pat. No. 5,370,872 would induce a significant level of antibodies against E. coli O517 O-SP or LPS. There remains a need, therefore, for compositions and methods of inducing a significant level of antibodies against E. coli O517. There also remains a need compositions and methods for inducing antibodies which have bactericidal activity against E. coli O517, and which also prevent or ameliorate HUS.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the invention to produce antigens based on the O-specific polysaccharide of Shiga toxin-producing bacteria, particularly E. coli O517, conjugated 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 is an object of the invention to provide conjugates having polysaccharides containing the tetrasaccharide repeat unit: (.fwdarw.3)-.alpha.-D-GalpNAc-(1.fwdarw.2)-.alpha.-D-PerpNAc-(1.fwdarw.3)- .alpha.-L-Fucp-(1.fwdarw.4)-.beta.-D-Glcp-(1.fwdarw.), and compositions thereof, to induce serum antibodies having bactericidal (killing) activity against E. coli, in particular E. coli O517. 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 O157, and are useful to prevent and/or treat illnesses caused by E. coli O517.

It is yet another object of the present invention to provide conjugates of E. coli O517 O-SP bound to the non-toxic B-subunit of Shiga toxin 1 (StxB1), or mutated non-toxic holotoxin of Shiga toxin 1 or Shiga toxin 2. 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 (Stx1 or Stx2)[19,20,21].

It is also an object of the invention to provide antibodies which immunoreact with the O-specific polysaccharide of E. coli O517 and/or the carrier, that are induced by these conjugates and/or compositions thereof. Such antibodies may be isolated, or may be provided in the form of serum containing these antibodies.

It is also an object of the invention to provide a method for the treatment or prevention of E. coli O517 infection in a mammal, by administration of compositions containing the antibodies of the invention, or serum containing the antibodies of the invention.

The invention also provides methods and kits for identifying, detecting, and/or diagnosing E. coli O517 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.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides conjugates of an E. coli O517 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. In a preferred embodiment, the carrier is the non-toxic B subunit of Shiga toxin 1 or 2 (StxB1, StxB2), or a non-toxic mutant of Stx1 or Stx2 holotoxin. In yet another preferred embodiment, the particular E. coli O157-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 O157. In the preferred embodiment, 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 O517 are useful for the identification, detection, and/or diagnosis of E. coli O517 colonization and/or infection. Antibodies which have bactericidal or bacteriostatic activity against E. coli O517 are useful to prevent and/or treat illnesses caused by E. coli O157. 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.

Pharmaceutical compositions of this invention are capable, upon injection into a human of an amount containing 25 .mu.g of E. coli O517 O-specific polysaccharide, of inducing in the serum bactericidal activity against E. coli O517, such that the serum kills, in the presence of complement, 50% or more of E. coli O517 at a serum dilution of 1300:1 or more. Preferred compositions can induce serum bactericidal activity against E. coli O517 such that the serum kills 50% or more of E. coli O517 at a serum dilution of 32,000:1 or more, and the most preferred compositions can induce serum bactericidal activity against E. coli O517 such that the serum kills 50% or more of E. coli O517 at a serum dilution of 64,000:1 or more. The O-SP conjugate vaccines of this invention are designed to induce serum IgG antibodies that will inactivate an inoculum of E. coli O517 at the entrance of the 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 O517 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 O517, and to induce antibodies specific to E. coli O517 O-SP and LPS. The E. coli O517 vaccine is composed of non-toxic bacterial components, suitable for infants, children of all ages, and adults.

The conjugates of this invention, and/or compositions thereof, as well as the antibodies thereto, will be useful in increasing resistance to, preventing, ameliorating, and/or treating E. coli O517 infection in humans, and in reducing or preventing E. coli O517 colonization in humans.

This invention also provides compositions, including but not limited to mammalian serum, plasma, and immunoglobulin fractions, which contain antibodies which are immunoreactive with E. coli O517 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 O517. 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-O517 sera, or antibodies isolated therefrom, could be used for therapeutic treatment for patients with E. coli O157 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 O517 infections, and are also useful in providing passive protection to an individual exposed to E. coli O517.

The present invention also provides diagnostic tests and/or kits for E. coli O517 infection and/or colonization, using the conjugates and/or antibodies of the present invention, or compositions thereof.

The present invention also provides an improved method for synthesizing an O-SP peptide conjugate, particularly the E. coli O517 O-SP conjugated to the B subunit of Shiga toxin 1 or 2 (Stx1 or Stx2), or to a mutant, non-toxic Stx1 or Stx2 holotoxin.

A number of primary uses for the conjugates of this invention are envisioned. The E. coli LPS-protein conjugates of this invention, and the antibodies they induce, are expected to be useful for several purposes, including but not limited to:

1) a vaccine for high-risk groups (children under 5 and senior citizens);

2) high-titered globulin for plasmapheresis, for prophylaxis and treatment of E. coli O517-infected patients; and

3) diagnostic reagents for detecting and/or identifying E. coli O517.

The invention is intended to be included in the routine immunization schedule of infants and children, and in individuals at risk for E. coli O517 infection. It is also planned to be used for intervention in epidemics caused by E. coli O517. Additionally, it is may be used as a component of a multivalent vaccine for E. coli O517 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 O517, for therapy of established infection. The invention is also intended to provide a diagnostic test for E. coli O517 infection and/or colonization.

Polymeric Carriers

Carriers are chosen to increase the immunogenicity of the 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.

Examples of water insoluble carriers include, but are not limited to, aminoalkyl SEPHRAROSE, e. g., aminopropyl or aminohexyl SEPHAROSE (Pharmacia Inc., Piscataway, N.J.), 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.

Methods for Attaching Polymeric Carriers

Methods for binding a polysaccharide to a protein are well known in the art. For example, a polysaccharide containing at least one carboxyl group, through carbodiimide condensation, 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 of the present invention better preserve the native structure of the 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. To conjugate without a linker, for example, 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].

To conjugate with a linker or crosslinking agent, either or both of the polysaccharide and the carrier may be covalently bound to a linker first. The 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)-.beta.-alaninate-propionate (SIAP), succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC), 3,3'-dithiodipropionic acid, and the like. Also among the class of heterobifunctional linkers area omega-hydroxy and omega-amino alkanoic acids.

More specifically, attachment of the E. coli O517 O-specific polysaccharide to a protein carrier can be accomplished by methods known to the art. In a preferred embodiment, 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, 1-cyano-4-(N,N-dimethylamino)pyridine tetrafluoroborate, or the like. Several such cyanylation reagents are known to those skilled in the art [27]. The resulting cyanated E. coli O517 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. 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 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the like.

More preferably, the cyanated E. coli O157 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 of the customary linker provides a more effective immunogen in the case of the E. coli O517 O-specific polysaccharide.

Regardless of the precise method used to prepare the conjugate, after the coupling reactions have been carried out 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 of the polysaccharide and the carrier bound directly or through a linker.

Dosage for Vaccination

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 O517 depends, among other things, on the species of mammal inoculated, the body weight of the 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.

The term "unit dose" as it pertains to the inocula 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. 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 O517. In order to increase the antibody level, 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.

Antibodies

An antibody of the present invention in one embodiment is characterized as comprising antibody molecules that immunoreact with E. coli O517 O-SP or LPS.

An antibody of the present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing an E. coli O517 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 of the 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 of the 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 O157 at 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 O517 at a dilution of 64,000:1 or more.

Alternatively, the antibodies of the 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, IgG₁, IgG₂, IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferred for purposes of passive protection. The antibodies of the 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 O517 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. In one such assay, for example, the biological sample is contacted with first antibodies of the present invention, and a labeled second antibody is used to detect the presence of E. coli O517 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 of the present invention are also useful in prevention and treatment of infections and diseases caused by E. coli O517.

In providing the antibodies of the present invention to a recipient mammal, preferably a human, 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.

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 of the mammal, although a lower or higher dose may be administered. The antibodies of the 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 of the infection by E. coli O517. 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 of the infection by Shigatoxin producing organisms, such as E. coli strains O517, O111, O26, and O17.

The administration of the agents of the invention may be for either "prophylactic" or "therapeutic" purpose. When provided prophylactically, the agents are provided in advance of any symptom. The prophylactic administration of the agent serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the agent is provided at (or shortly after) the onset of a symptom of infection. The agent of the present invention may, thus, be provided prior to the anticipated exposure to E. coli O517 (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.

For all therapeutic, prophylactic and diagnostic uses, the 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.

Claim 1 of 17 Claims

We claim:

1. A pharmaceutical composition, comprising:

about 25 .mu.g of E. coli O157 O-specific polysaccharide covalently bound to a carrier, wherein the carrier is a B subunit of Shiga toxin 1, a B subunit of Shiga toxin 2, a non-toxic mutant Shiga toxin 1 holotoxin, or a non-toxic mutant Shiga toxin 2 holotoxin, and

a pharmaceutically acceptable agent,

wherein injection into a human of a therapeutically effective amount of said composition produces in serum of said human bactericidal activity against E. coli O157 such that the bactericidal activity in the serum kills 50% or more of E. coli O157 at a serum dilution of 1,300:1 or more.

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