EP1372709A2 - Substances biologiques et procedes pour leur utilisation dans la prevention ou le traitement d'infections - Google Patents

Substances biologiques et procedes pour leur utilisation dans la prevention ou le traitement d'infections

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Publication number
EP1372709A2
EP1372709A2 EP02720132A EP02720132A EP1372709A2 EP 1372709 A2 EP1372709 A2 EP 1372709A2 EP 02720132 A EP02720132 A EP 02720132A EP 02720132 A EP02720132 A EP 02720132A EP 1372709 A2 EP1372709 A2 EP 1372709A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
intimin
fragment
polypeptides
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02720132A
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German (de)
English (en)
Inventor
Gadi Frankel
Gordon Dougan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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Filing date
Publication date
Priority claimed from GB0107840A external-priority patent/GB0107840D0/en
Priority claimed from GB0107837A external-priority patent/GB0107837D0/en
Application filed by Imperial College Innovations Ltd filed Critical Imperial College Innovations Ltd
Publication of EP1372709A2 publication Critical patent/EP1372709A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This invention relates to intimin polypeptides and polynucleotides encoding them, products comprising them and their use in the treatment of bacterial infections, particularly those which cause food borne diseases.
  • Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli are important causes of severe infantile diarrhoeal disease in many parts of the world.
  • EPEC and EHEC colonise the gastrointestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the "attaching and effacing" (A/E) lesion.
  • the A/E lesion is characterised by localised destruction (effacement) of brush border microvilli, intimate attachment of the bacterium to the host cell membrane and the formation of an underlying pedestal-like structure in the host cell.
  • EPEC and EHEC are members of a family of enteric bacterial pathogens which use A/E lesion formation to colonise the host.
  • E. coli capable of forming A/E lesions have also been recovered from diseased cattle [China, 1999], dog and cats [Beutin, 1999], rabbits [Jerse, 1991 ] and pigs [An, 2000].
  • Citrobacter rodentium colonises gut enterocytes via A/E lesion formation and, like EHEC in humans, causes disease in the large bowel.
  • EPEC Enteropathogenic
  • EHEC enterohaemorrhagic
  • HUS haemolytic uraemic syndrome
  • the LEE pathogenicity island which is present in EPEC, EHEC and C. rodentium, is necessary and sufficient for bacteria to promote the induction of A/E lesions on epithelial cells.
  • the LEE region encodes a type III secretion system, three secreted proteins EspA, EspB and EspD, an outer membrane adhesin, intimin, and a translocated intimin receptor, Tir.
  • the first gene to be associated with A/E activity was eae encoding the intimate EPEC and EHEC adhesin, intimin (Jerse et al; (1990) Proc Natl Acad Sci U S A SI, 7839- 7843.).
  • Intimin exists as at least five antigenically distinct subtypes that have been named intimin , ⁇ , ⁇ , ⁇ and ⁇ (Adu-Bobie et al (1998) J Clin Microbiol 36, 662-668; Oswald et al (2000) Infection and Immunity 68, 64-71).
  • EPEC/EHEC intimins exhibit homology at their amino-termini to the invasin polypeptides of Yersinia (Isberg (1987) Cell 50, 769-778) and like Yersinia invasin ( Leong et al (1990) EMBO J 9, 1979-1989), intimin harbours receptor binding activity at the C-terminus of the polypeptide (Frankel et al (1994) Infect Immun 62, 1835-1842; Frankel et al (1995) Infect Immun 63, 4323-4328), localised to the C-terminal 280 amino acids (Int280).
  • a 76-amino acid motif enclosed by a disulphide bridge between two cysteines lies within the C-terminal domain of intimin. This is absolutely required for intimin binding to the host cell, A/E lesion formation and colonisation of mucosal surfaces (Frankel et al (1995) Infect Immun 63, 4323-4328; Frankel et al (1996) J Biol Chem 271, 20359-20364; Frankel et al (1998b) Mol Microbiol 29, 559-570; Hicks et al (1998) Infect Immun 66, 1570-1578; Higgins et al (1999a) Science 285, 588-591 ; Higgins et al (1999b) Infect Immun 67, 3031-3139).
  • the C-terminal domain of invasin also harbours two Cys residues, in similar locations to those found in intimin ( Leong et al (1993) J Biol Chem 268, 20524-20532).
  • Intimin ⁇ is specifically expressed by a group of EPEC clone 1 strains. Intimin ⁇ is mainly associated with clone 2 EPEC and EHEC strains, C. rodentium and rabbit diarrhoeagenic E. coli (REPEC), while intimin ⁇ is associated with EHEC 0157:H7.
  • the first two domains each comprise ⁇ -sheet sandwiches that resemble the immunoglobulin super family (IgSF).
  • IgSF immunoglobulin super family
  • the topology of the C-terminal domain is reminiscent of the C-type lectins, a family of proteins responsible for cell-surface carbohydrate recognition.
  • a domain between residues 558-650 within the extracellular portion of intimin aligns with 35% identity (44% conservations) with the first domain of Int280, identifying a further Ig- like domain (Kelly et al 1999). This produces at least four domains that protrude from the bacterial membrane for interaction with the host cell.
  • the domains in Int280 are therefore renamed as D2, D3 and D4 respectively.
  • the presence of a disulphide bridge in the C-type lectin-like module of Int280 ⁇ is essential for correct folding of this domain and for carbohydrate binding by other C-type lectins (Weis & Drickamer (1996) Annu Rev Biochem 65, 441-473)
  • the extracellular portion of intimin comprises an articulated rod of immunoglobulin domains that extend from the bacterium surface conveying a highly accessible 'adhesive tip' to the target cell.
  • the interpretation of NMR-titration and mutagenesis data enabled us to identify the binding site for Tir, which is located at the extremity of the Intl90 moiety.
  • the intimin-binding region of Tir has been localised to a central region encompassing a 55 amino acid motif between two putative membrane-spanning helices (de Grado et al, 1999; Hartland et al, 1999; Kenny, 1997; 1999).
  • D4 comprises four helices that surround two anti-parallel ⁇ -sheets.
  • the C-terminal strand is disulphide bonded to helix I, and together with the N-terminus of D4 forms the two principal strands of the first sheet.
  • Helix III protrudes from the main structure into the solvent and therefore was not observed in our low-resolution structure of Int280 (Kelly et al, 1999). Interestingly, this helix contains an unusual kink at residue 139, which is replaced by proline in other intimin types.
  • Intimin is also the target of host immune responses in infected animals and humans, as described in the Examples, although little is known about the host immune response to other LEE encoded antigens. Intimin has also been promoted as a potential candidate vaccine antigen based on the ability of antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) 0157:H7 to inhibit adherence of this strain to HEp-2 cells.
  • EHEC enterohemorrhagic Escherichia coli
  • C. rodentium infection of mice offers an advantage because of the wide availability of gene knockout strains and immunological reagents for this species. Whilst an imperfect model of EPEC and EHEC infection, C. rodentium infection of mice nevertheless represents the best small animal model in which to study lumenal microbial pathogens relying on A/E lesion formation for colonisation of the host.
  • C. rodentium harbours a LEE pathogenicity island. This genetic locus contains eae and espB homologs that are essential for A/E lesion formation and colonisation of mice [Schauer, 1993; Newman, 1999].
  • the A/E lesion induced by C. rodentium is ultrastructurally identical to those formed by EHEC and EPEC in animals and human intestinal in vitro organ culture (IVOC).
  • IVOC human intestinal in vitro organ culture
  • large numbers of C. rodentium can be recovered from the colon and infection is associated with crypt hyperplasia, mucosal erosion and focal crypt abscesses. Oral infection of mice with live wild-type C.
  • rodentium or intra-colonic inoculation of dead bacteria induces a CD3 + and CD4 + T- cell infiltrate into the colonic lamina intestinal and a strong T helper type 1 immune response. This response is not observed in mice inoculated with an eae mutant of C. rodentium, but is seen in mice inoculated with C. rodentium complemented with intimin ⁇ from EPEC E2348/69.
  • a first aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprising or encoding a polypeptide or polypeptides in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ - intimin or a fragment thereof, together with a pharmaceutically acceptable diluent or carrier, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • a pharmaceutical composition of the invention may comprise a polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprising or encoding a polypeptide or polypeptides in combination comprising at least two of (1) at least eight contiguous amino acids derived from Int280 ⁇ (2) at least eight contiguous amino acids derived from Int280 ⁇ (3) at least eight contiguous amino acids derived from Int280 ⁇ (4) at least eight contiguous amino acids derived from Int280 ⁇ and (5) at least eight contiguous amino acids derived from Int280 ⁇ .
  • a pharmaceutical composition of the invention may comprise a polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprising or encoding a polypeptide or polypeptides in combination comprising at least two of (1) an epitope derived from Int280 ⁇ , (2) an epitope derived from Int280 ⁇ , (3) an epitope derived from Int280 ⁇ , (4) an epitope derived from Int280 ⁇ , (5) an epitope derived from Int280 ⁇ .
  • a second aspect of the invention provides a vaccine effective against bacterial infection, for example EHEC and/or EPEC infection, comprising an effective amount of the polypeptide or polypeptides and/or polynucleotide or polynucleotides as defined in relation to the preceding aspect of the invention.
  • a third aspect of the invention provides a food product comprising a foodstuff and a polypeptide or polypeptides and/or polynucleotide or polynucleotides as defined in relation to the preceding aspects of the invention.
  • a fourth aspect of the invention provides a kit of parts comprising a polypeptide and polynucleotide, polypeptides and/or polynucleotides as defined in relation to the preceding aspects of the invention and optionally a pharmaceutically acceptable diluent or carrier.
  • the polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprise or encode a polypeptide or polypeptides which comprise or consist in combination of at least two of (1) Int280 ⁇ or Intl90 ⁇ or Intl50 ⁇ or a fragment any thereof, (2) Int280 ⁇ or Intl90 ⁇ or Intl50 ⁇ or a fragment any thereof, (3) Int280 ⁇ or Intl90 ⁇ or Intl 50 ⁇ or a fragment any thereof, (4) Int280 ⁇ or Intl90 ⁇ or Intl 50 ⁇ or a fragment any thereof, and (5) Int280 ⁇ or Intl90 ⁇ or Intl 0 ⁇ or a fragment any thereof.
  • polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprise or encode a polypeptide or polypeptides in combination comprising or consisting of at least ⁇ -intimin or Int280 ⁇ (or Intl90 ⁇ or Intl50 ⁇ ) or a fragment thereof and ⁇ -intimin or Int280 ⁇ (or Intl90 ⁇ or Intl 50 ⁇ ) or a fragment thereof.
  • composition, vaccine, kit of parts or food product may comprise a polypeptide or polypeptides which in combination comprise (for example) at least two of (1) ⁇ - intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • composition, vaccine, kit of parts or food product may comprise a polynucleotide or polynucleotides in combination encoding a polypeptide or polypeptides in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ - intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • the composition, vaccine, kit of parts or food product may comprise a polynucleotide (or polynucleotides) and a polypeptide (or polypeptides).
  • the polynucleotide may encode an intimin polypeptide of one type (for example an intimin- ⁇ -derived polypeptide), and the polypeptide may be an intimin polypeptide of a different type (for example an intimin- ⁇ derived . polypeptide); between them, the polynucleotide and polypeptide are capable of providing intimin polypeptides of at least two types.
  • Intimin- ⁇ , ⁇ and ⁇ are considered to be the intimins of greatest prevalence and medical relevance; hence the preference for inclusion of sequences derived from these intimins.
  • Intimin- ⁇ is expressed by some EHEC, including the pathogenic E. coli 0157.
  • Different individual intimin polypeptides or combinations of intimin-derived sequences may be preferable, depending upon the nature of the individual (human or animal) to be treated.
  • inclusion of sequences derived from intimin- ⁇ and/or intimin- ⁇ and/or intimin- ⁇ and possibly also intimin- ⁇ (but not necessarily intimin- ⁇ ) may be appropriate when the medicament is for vaccination of cattle or other livestock (for example pigs or pigeons), whereas inclusion of sequences derived from intimin- ⁇ , intimin- ⁇ , intimin- ⁇ , intimin- ⁇ and/or intimin- ⁇ may be appropriate when the medicament is for vaccination of human patients prior to foreign travel.
  • preferred combinations of intimin-derived sequences correspond to combinations of intimins from which the target recipient/population (human or animal) are most likely to be at risk (for example because the bacteria expressing a particular intimin are, for example, particularly harmful, or less harmful but more prevalent.
  • a particularly useful combination may be of intimins derived from intimin- ⁇ , intimin- ⁇ , intimin- ⁇ and probably also intimin- ⁇ .
  • the pharmaceutical composition, vaccine, food product or kit of parts may further comprise a further polypeptide sequence (or polynucleotide encoding such a sequence) from a further type of intimin (ie from an intimin which can be distinguished (by sequence or by antigenicity, preferably antigenicity) from intimin types ⁇ , ⁇ , ⁇ , ⁇ or ⁇ ).
  • the recipient may be human, for example a human baby or infant or child or other human with or at risk of bacterial infection.
  • the recipient may be an animal, for example a domesticated animal or animal important in agriculture (ie livestock), for example cattle, sheep, goats, or poultry, for example chickens and turkeys.
  • the recipient may preferably be a young animal, for example a calf.
  • the bacterial infection causes an histopathologic effect on intestinal epithelial cells, the effect being known as attachment and effacement (A/E).
  • the bacterial infection comprises infection by enteropathogenic E. coli (EPEC) and/or enterohemorrhagic E. coli (EHEC), and particularly E. coli 0157:H7.
  • EHEC enterohemorrhagic E. coli
  • the infection may be selected from one or more of the infections which cause diseases affecting humans or domestic farm animals such as cows, sheep and goats, particularly food borne diseases, notably diarrhoea, haemorrhagic colitis, acute gastroenteritis or haemolytic uraemic syndrome (HUS).
  • the polypeptide or polypeptides comprise at least part of a Tir binding site of an intimin, preferably of at least two intimins, preferably of each intimin represented in the polypeptide or polypeptides. It is preferred that the epitope is part of a Tir binding site of the intimin.
  • the Tir binding site is described in GB patent application No 0013115.1 filed on 31 May 2000 (incorporated herein by reference) and in Batchelor et al (2000).
  • the epitope comprises at least one residue identified in GB patent application No 0013115.1 as part of the Tir binding site (for example as defined in claim 2 of that application).
  • polypeptide may comprise regions N-terminal to the Tir binding site, identified as containing immunodominant epitopes in Adu-Bobie et al (1998) infect Immun 66, 5643-5649.
  • the epitope may be part of a region identified in Adu-Bobie et al (1998).
  • mimotopes By epitopes is included mimotopes, as well known to those skilled in the art.
  • the medicament may further comprise a sequence (or epitope (including a mimotope)) derived from any other naturally occurring intimin (other than solely from the Gly387 to Lys666 region), for example from the Int280 region of any such intimin.
  • the medicament may further comprise a polypeptide with a sequence (or epitope (including a mimotope) or peptidomimetic equivalent) derived from one or more other "LEE" polypeptides, for example from TirM, EspB or EspA, preferably EspA, as discussed in the Examples and well known to those skilled in the art.
  • These other "LEE"-derived sequences may be included in the same or a different polypeptide (or polynucleotide, as appropriate) as the intimin-derived sequences.
  • intimin- ⁇ , ⁇ , ⁇ , ⁇ or ⁇ is included variants, fragments and fusions that have interactions or activities which are substantially the same as those of the intimin sequences described herein and/or those disclosed in Frankel et al (1994) Infect Immun 62, 1835-1842; Adu-Bobie et al (1998) J Clin Microbiol 36, 662-668; Oswald et al (2000) Infection and Immunity 68, 64-71 and/or public databases.
  • the intimin or fragment thereof is a naturally occurring intimin or fragment thereof, or a fusion of such an intimin or fragment with a non-intimin-derived polypeptide.
  • the intimin-derived sequence may be fused with a moiety that aids expression, stability and/or purification, for example a maltose binding protein (MBP) moiety or His tag, as well known to those skilled in the art.
  • MBP maltose binding protein
  • a "variant” will have a region which has at least 50% (preferably 60,70, 80,90, 95 or 99%) sequence identity with an intimin polypeptide as described herein or in the references indicated above, as measured by the Bestfit Program of the Wisconsin Sequence Analysis Package, version 8 for Unix.
  • the percentage identity may be calculated by reference to a region of at least 50 amino acids (preferably at least 60, 75, or 100) of the candidate variant molecule, and the most similar region of equivalent length in the intimin sequence, allowing gaps of up to 5%.
  • the percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids res.
  • the GAP program utilizes the alignment method of Neddleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2.482. 1981).
  • the preferred default parameters for the GAP program include : (1) a comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Bribskov and Burgess, Nucl. Acids Res.
  • the intimin fragment consists of the C-terminal 280 amino acids of the intimin, or fragments, variants, or fusions thereof which exhibit substantially the same or higher Tir binding activity than the intimin or C-terminal 280 amino acids of the intimin.
  • a preferred intimin "fragment” comprises all or for example 50, preferably 60, 75, or
  • ie for intimin- ⁇ taken from the following Int280 ⁇ sequence (using the single letter code for amino acid designations): ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTEKTDTNG YAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNIEIVGTGVKGKLP TVWLQYGQVNLKASGGNGKYTWRSANPAIASVDASSGQVTLKEKGTTTISVISSDNQ TATYTIATPNSLIVPNMSKRVTYNDAVNTCKNFGGKLPSSQNELENVFKA GAANKY EYYKSSQTIISWVQQTAQDAKSGVASTYDLVKQNPLNNIKASESNAYATCVK
  • Substitutions, deletions, insertions or any subcombination may be used to arrive at a final construct. Since there are 64 possible codon sequences but only twenty known amino acids, the genetic code is degenerate in the sense that different codons may yield the same amino acid. Thus there is at least one codon for each amino acid, ie each codon yields a single amino acid and no other. It will be apparent that during translation, the proper reading frame must be maintained in order to obtain the proper amino acid sequence in the polypeptide ultimately produced.
  • exemplary techniques include oligonucleotide-mediated site-directed mutagenesis and the polymerase chain reaction.
  • Oligonucleotide site-directed mutagenesis in essence involves hybridizing an oligonucleotide coding for a desired mutation with a single strand of DNA containing the region to be mutated and using the single strand as a template for extension of the oligonucleotide to produce a strand containing the mutation. This technique, in various forms, is described in Zoller and Smith (1982) Nucl. Acids Res. 10, 6487.
  • intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminal domain are described in Frankel et al (1998) Mol Microbiol 29(2), 559-540, the disclosure of which is incorporated herein by reference.
  • Preferred variants may be variants which may be less toxic to the treated human or animal than a naturally occurring intimin or fragment thereof. Such variants are discussed in the copending patent application filed by the applicant on the same date as this application, entitled "BIOLOGICAL MATERIALS AND METHODS FOR USE IN THE PREVENTION OR TREATMENT OF INFECTIONS:II".
  • the polypeptide or polypeptides may preferably comprise 1) a first epitope selected from epitopes present in ⁇ -intimin, ⁇ -intimin, ⁇ -intimin, ⁇ -intimin or ⁇ -intimin and 2) a second epitope (and optionally further epitopes) selected from said epitopes, wherein the first and second epitope are (i) not common to all said intimins and (ii) are present in different said intimins.
  • the pharmaceutical composition, vaccine, food product or kit of parts comprises epitopes from more than one intimin polypeptide.
  • polypeptides may comprise more than one copy of an epitope. This may be useful in promoting an immune response, as well known to those skilled in the art.
  • the pharmaceutical composition, vaccine, food product, medicament or kit of parts may comprise further polypeptides or polynucleotides, as will be apparent to those skilled in the art.
  • the polypeptide(s) or polynucleotide(s) may, for example, be included in the pharmaceutical composition, vaccine, food product or kit of parts in the form of a recombinant organism or part thereof, preferably microorganism, preferably capable of expressing the polypeptides(s) ie capable of expressing the two or more intimin amino acid sequences, or alternatively capable of delivering nucleic acid encoding the polypeptide(s) to a host cell for expression therein.
  • the recombinant microorganism is preferably a non-virulent microorganism, as well known to those skilled in the art.
  • the recombinant microorganism may be, for example, a Bifidobacterium or a lactobacillus, or an attenuated Salmonella or BCG or attenuated E. coli.
  • the recombinant organism may alternatively be a plant, for example making use of the teaching of WO97/40177.
  • the pharmaceutical composition may be useful as a vaccine, for example as a prophylactic or therapeutic vaccine, as well known to those skilled in the art.
  • the said polypeptide or polypeptides are intended as target antigens, which are intended to promote a protective or therapeutic immune response in the treated human or animal.
  • a further aspect of the invention provides a chimaeric polypeptide comprising or consisting of one or more copies of at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, wherein the polypeptide does not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • chimaeric polypeptide Preferences for the components of the chimaeric polypeptide are as indicated above in relation to the polypeptide or polypeptides useful in the manufacture of a medicament.
  • the chimaeric polypeptide may comprise sequences derived from a further intimin type or types.
  • the chimaeric polypeptide may comprise at least two of (1) at least eight contiguous amino acids derived from Int280 ⁇ (2) at least eight contiguous amino acids derived from Int280 ⁇ (3) at least eight contiguous amino acids derived from Int280 ⁇ (4) at least eight contiguous amino acids derived from Int280 ⁇ and (5) at least eight contiguous amino acids derived from Int280 ⁇ .
  • the chimaeric polypeptide may comprise at least two of (1) an epitope derived from Int280 ⁇ , (2) an epitope derived from Int280 ⁇ , (3) an epitope derived from Int280 ⁇ , (4) an epitope derived from Int280 ⁇ , (5) an epitope derived from Int280 ⁇ .
  • the at least two epitopes are not common to all said intimins.
  • a further aspect of the invention provides a polynucleotide encoding a chimaeric polypeptide of the invention.
  • the polynucleotide may be in the form of a vector molecule, for example a replicable vector molecule, as well known to those skilled in the art.
  • a further aspect of the invention provides a recombinant microorganism, preferably bacterium, comprising a polynucleotide (for example a replicable vector) of the invention.
  • a further aspect of the invention provides a peptidomimetic compound corresponding to the chimaeric polypeptide of the invention.
  • a further aspect of the invention provides a food product comprising a foodstuff and a chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention.
  • a still further aspect of the invention provides a pharmaceutical composition comprising a chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention and a pharmaceutically acceptable diluent or carrier.
  • a further aspect of the invention provides a vaccine effective against bacterial infection, for example EHEC and/or EPEC infection, comprising an effective amount of a chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention.
  • a f rther aspect of the invention provides a pharmaceutical composition, vaccine, food product, kit of parts, chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism according to any preceding aspect of the invention for use in medicine.
  • a still further aspect of the invention provides the use of a pharmaceutical composition, vaccine, food product, kit of parts, chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection.
  • a further aspect of the invention provides the use of a pharmaceutical composition, vaccine, food product, kit of parts, chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention in the manufacture of a composition for use as a food supplement or a food additive.
  • a further aspect of the invention provides the use of a polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprising or encoding a polypeptide or polypeptides in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • a further aspect of the invention provides a method for treating a human or animal with or at risk of bacterial infection, wherein the human or animal is administered a pharmaceutical composition, vaccine, food product, kit of parts, chimaeric polypeptide, peptidomimetic compound, polynucleotide or recombinant microorganism of the invention.
  • a further aspect of the invention provides the use of (1) a peptidomimetic compound or compounds corresponding to the polypeptide or polypeptides (therapeutic polypeptide(s)) as defined in relation to any of the preceding aspects of the invention, and/or (2) antibodies or an antibody preparation reactive with the said polypeptide or polypeptides, preferably reactive with two or more epitopes (derived from two or more intimins) as defined in relation to a preceding aspect of the invention, in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection.
  • the antibody preparation or antibodies are reactive with intimin epitopes other than epitopes found in the conserved region Gly387 to Lys666.
  • the peptidomimetic compound, polypeptide, polynucleotide or antibodies/antibody preparation may also be useful in the manufacture of a diagnostic reagent for use in diagnosis of a human or with or at risk of bacterial infection.
  • a further aspect of the invention provides a method of treating a human or animal with or at risk of bacterial infection, wherein the human or animal is administered (1) a polypeptide or polypeptides and/or polynucleotide or polynucleotides as defined in relation to any of the preceding aspects of the invention and/or (2) a peptidomimetic compound or compounds corresponding to the said encoded or comprised polypeptide or polypeptides (therapeutic polypeptide(s)), as defined in relation to any of the preceding aspects of the invention, and/or (3) an antibody preparation or antibodies reactive with an epitope as defined in relation to a preceding aspect of the invention.
  • peptidomimetic refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent, but that avoids the undesirable features.
  • morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin.
  • Therapeutic applications involving peptides are limited, due to lack of oral bioavailability and to proteolytic degradation.
  • peptides are rapidly degraded in vivo by exo- and endopeptidases, resulting in generally very short biological half-lives.
  • Another deficiency of peptides as potential therapeutic agents is their lack of bioavailability via oral administration.
  • Degradation of the peptides by proteolytic enzymes in the gastrointestinal tract is likely to be an important contributing factor. The problem is, however, more complicated because it has been recognised that even small, cyclic peptides which are not subject to rapid metabolite inactivation nevertheless exhibit poor oral bioavailability.
  • Retro-inverso peptidomimetics in which the peptide bonds are reversed, can be synthesised by methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159 3230-3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • a common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework.
  • the cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased affinity of the peptide for a particular biological receptor.
  • An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.
  • RCM ring closing metathesis
  • an antibody is included an antibody or other immunoglobulin, or a fragment or derivative thereof, as discussed further below.
  • variable heavy (VJJJ and variable light (VjJ domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains that antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the VJI and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
  • ScFv molecules we mean molecules wherein the V ⁇ -[ and VL partner domains are linked via a flexible oligopeptide.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • Whole antibodies, and F(ab')2 fragments are "bivalent". By “bivalent” we mean that the said antibodies and F(ab')2 fragments have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.
  • the antibody has an affinity for the epitope of between about lO ⁇ .M"! to about l ⁇ l2.M _ l, more preferably at least lO ⁇ .M 1 .
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in Monoclonal Antibodies: A manual of techniques, H Zola (CRC Press, 1988) and in Monoclonal Hybridoma Antibodies: Techniques and Applications, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799). Suitably prepared non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • a further aspect of the invention provides the use of a polypeptide or polypeptides and/or polynucleotide or polynucleotides in combination comprising or encoding a polypeptide or polypeptides in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, in the manufacture of a composition for use as a food supplement or a food additive. It is preferred that the polypeptide or polypeptides (including encoded polypeptide or polypeptides) do not consist of the Gly387 to Lys666 region region, or a fragment thereof, of two or more intimins.
  • the food product may be adapted for consumption by animals or adapted for consumption by humans.
  • the food is preferably a milk substitute.
  • the food is suitable for administration to a human baby or infant or a young animal.
  • it may be suitable for any human or animal which is susceptible to a bacterial infection, including older humans or animals.
  • Exemplary animals include domestic cattle, especially calves; and poultry such as chickens and turkeys
  • the invention also relates to a food product comprising a foodstuff and an agent as defined above.
  • the chimaeric polypeptide or polynucleotide of the invention may also be useful in diagnosis of a bacterial infection, for example as a control for a diagnostic test, for example an immunodiagnostic test or a nucleic acid detection/characterisation test, for example involving PCR, as well known to those skilled in the art.
  • a diagnostic test for example an immunodiagnostic test or a nucleic acid detection/characterisation test, for example involving PCR, as well known to those skilled in the art.
  • Polypeptides in which one or more of the amino acid residues are chemically modified, before or after the polypeptide is synthesised, may be used as antigen providing that the function of the polypeptide, namely the production of a specific immune response in vivo, remains substantially unchanged.
  • modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the polypeptide from in vivo metabolism.
  • the polypeptide may be mannosylated or otherwise modified to increase its antigenicity, or combined with a compound for increasing its antigenicity and/or immunogenicity.
  • the polypeptide may comprise a viral polypeptide, for example a HBV polypeptide, as known to those skilled in the art.
  • the epitope(s) may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the polypeptide to be formed as a loop, with the N-terminal and C-terminal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the epitope, for example epitope-forming amino acid sequence, is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the epitope-forming amino acid sequence forms a loop.
  • a carrier preferably a polypeptide
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system.
  • the epitope(s) as defined above in relation to the preceding aspects of the invention may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • Intimin may itself act as a carrier or adjuvant.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly- Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).
  • Any of the conventional ways of cross-linking polypeptides may be used, such as those generally described in O'Sullivan et al Anal. Biochem.
  • the first portion may be enriched with thiol groups and the second portion reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N- hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), a heterobifunctional cross-linking agent which incorporates a disulphide bridge between the conjugated species.
  • NHS iodoacetic acid
  • SPDP N-succinimidyl-3-(2- pyridyldithio)propionate
  • Amide and thioether bonds for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.
  • cross-linking agents include S-acetylthioglycolic acid N- hydroxysuccinimide ester (SATA) which is a thiolating reagent for primary amines which allows deprotection of the sulphydryl group under mild conditions (Julian et al (1983) Anal Biochem. 132, 68), dimethylsuberimidate dihydrochloride and N,N'-o- phenylenedimaleimide.
  • SATA S-acetylthioglycolic acid N- hydroxysuccinimide ester
  • polypeptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the polypeptide as a fusion product with a peptide sequence which acts as a carrier.
  • Kabigen's "Ecosec" system is an example of such an arrangement.
  • Suitable vectors or constructs which may be used to prepare a suitable recombinant polypeptide or polynucleotide will be known to those skilled in the art.
  • a polynucleotide capable of expressing the required polypeptide or polypeptides may be prepared using techniques well known to those skilled in the art.
  • the polynucleotide may be capable of expressing the polypeptide(s) in the recipient, so that the human or animal may be administered the polynucleotide, leading to expression of the antigenic polypeptides (ie sequences derived from two or more intimins) in the human or animal.
  • the polypeptide(s) for example Int280 ⁇ and Int280 ⁇ , as appropriate, may be expressed from any suitable polynucleotide (genetic construct) as is described below and delivered to the recipient.
  • the genetic construct which expresses the polypeptide comprises the said polypeptide coding sequence operatively linked to a promoter which can express the transcribed polynucleotide (eg mRNA) molecule in a cell of the recipient, which may be translated to synthesise the said polypeptide.
  • a promoter which can express the transcribed polynucleotide (eg mRNA) molecule in a cell of the recipient, which may be translated to synthesise the said polypeptide.
  • Suitable promoters will be known to those skilled in the art, and may include promoters for ubiquitously expressed genes, for example housekeeping genes or for tissue-selective genes, depending upon where it is desired to express the said polypeptide (for example, in dendritic cells or other antigen presenting cells or precursors thereof, or in mucosal cells).
  • a dendritic cell or dendritic precursor cell-selective promoter is used, but this is not essential, particularly if delivery or uptake of the polynucleotide is targeted to the selected cells, eg dendritic cells or precursors.
  • Dendritic cell-selective promoters may include the CD83 or CD36 promoters.
  • the nucleic acid sequence capable of expressing the polypeptide(s) is preferably operatively linked to regulatory elements necessary for expression of said sequence.
  • a coding sequence "operatively linked" to regulatory elements refers to a configuration wherein the nucleic acid sequence encoding the antigen can be expressed under the control of the regulatory sequences.
  • regulatory sequences refers to nucleic acid sequences necessary for the expression of an operatively linked coding sequence in a particular host organism.
  • the regulatory sequences which are suitable for eukaryotic cells are promotors, polyadenylation signals, and enhancers.
  • Vectors means a DNA molecule comprising a single strand, double strand, circular or supercoiled DNA. Suitable vectors include refroviruses, adenoviruses, adeno- associated viruses, pox viruses and bacterial plasmids. Retroviral vectors are refroviruses that replicate by randomly integrating their genome into that of the host. Suitable retroviral vectors are described in WO 92/07573. Adenovirus is a linear double-standard DNA Virus. Suitable adenoviral vectors are described in Rosenfeld et al, Science, 1991, Vol. 252, page 432.
  • Adeno-associated viruses belong to the parvo virus family and consist of a single strand DNA of about 4-6 KB.
  • Pox viral vectors are large viruses and have several sites in which genes can be inserted. They are thermostable and can be stored at room temperature. Safety studies indicate that pox viral vectors are replication-defective and cannot be transmitted from host to host or to the environment.
  • Targeting the vaccine to specific cell populations may be achieved, for example, either by the site of injection, use of targeting vectors and delivery systems, or selective purification of such a cell population from the recipient and ex vivo administration of the peptide or nucleic acid (for example dendritic cells may be sorted as described in Zhou et al (1995) Blood 86, 3295-3301 ; Roth et al (1996) Scand. J. Immunology 43, 646-651).
  • targeting vectors may comprise a tissue- or tumour-selective promoter which directs expression of the antigen at a suitable place.
  • the genetic construct can be DNA or RNA it is preferred if it is DNA.
  • the genetic construct is adapted for delivery to a human cell.
  • the constructs of the invention may be introduced into the cells by any convenient method, for example methods involving refroviruses, so that the construct is inserted into the genome of the (dividing) cell.
  • Targeted refroviruses are available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into preexisting viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • Preferred retroviral vectors may be lentiviral vectors such as those described in Verma & Somia (1997) Nature 389, 239-242.
  • a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody.
  • the polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone.
  • the adenovirus because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.
  • Bacterial delivery methods which may be suitable are described in Dietrich (2000) Antisense Nucleic Acid Drug Delivery 10, 391-399.
  • attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces.
  • attenuated bacteria are generally engineered to express heterologous antigens
  • a further approach employs intracellular bacteria for the delivery of eukaryotic antigen expression vectors (DNA vaccines).
  • DNA vaccines eukaryotic antigen expression vectors
  • APC professional antigen-presenting cells
  • DC dendritic cells
  • the bacteria used for DNA vaccine delivery either enter the host cell cytosol after phagocytosis by the APC, for example, Shigella and Listeria, or they remain in the phagosomal compartment, such as Salmonella. Both intracellular localizations of the bacterial carriers may be suitable for successful delivery of DNA vaccine vectors of the present invention.
  • Expression of the intimin polypeptide may be under the control of inducible bacterial promoters, for example promoters that are induced when the bacterium encounters or enters a host organism environment (for example the host's gut) or binds to or enters a host cell.
  • inducible bacterial promoters for example promoters that are induced when the bacterium encounters or enters a host organism environment (for example the host's gut) or binds to or enters a host cell.
  • Bacterial delivery is a preferred method of delivery in relation to the present invention.
  • Oral bacterial delivery of expressed intimin antigens may be a useful delivery route.
  • injection of purified intimin polypeptide(s) is considered also to be effective.
  • the DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.
  • a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids.
  • Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell.
  • the transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).
  • complexes of transferrin- polycation and the DNA constructs or other genetic constructs of the invention are supplied to the target cells, a high level of expression from the construct in the cells is expected.
  • High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used.
  • This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.
  • This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.
  • Non- viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.
  • Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein.
  • a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention.
  • suitable viruses or virus-like particles include HSV, AAV, vaccinia, lentivirus and parvovirus.
  • Immunoliposomes are especially useful in targeting to cell types which over-express a cell surface protein for which antibodies are available, as is possible with dendritic cells or precursors, for example using antibodies to CD1 , CD 14 or CD83 (or other dendritic cell or precursor cell surface molecule, as indicated above).
  • MPB-PE N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine
  • MPB-PE N-[4-(p- maleimidophenyl)butyryl]-phosphatidylethanolamine
  • MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface.
  • the liposome is conveniently loaded with the DNA or other genetic construct of the invention for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min.
  • Freshly prepared MPB-PE- liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight.
  • the immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min.
  • Immunoliposomes may be injected, for example intraperitoneally or directly into a site where the target cells are present, for example subcutaneously.
  • polypeptide(s) for example antigenic polypeptides
  • expression of the polypeptide(s) is directly or indirectly (see below) under the control of a promoter that may be regulated, for example by the concentration of a small molecule that may be administered to the recipient when it is desired to activate or repress (depending upon whether the small molecule effects activation or repression of the said promoter) expression of the polypeptide.
  • the expression construct is stable ie capable of expressing the polypeptide (in the presence of any necessary regulatory molecules) in the said cell for a period of at least one week, one, two, three, four, five, six, eight months or one or more years. It is preferred that the expression construct is capable of expressing the polypeptide in the said cell for a period of less than one month.
  • a preferred construct of the invention may comprise a regulatable promoter.
  • regulatable promoters include those referred to in the following papers: Rivera et al (1999) Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orally bioavailable drug, using two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor); Magari et al (1997) J Clin Invest 100(11), 2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22 (review of adeno-associated viral vectors); Bohl et al (1998) Blood 92(5), 1512-7 (control by doxycycline in adeno-associated vector); Abruzzese et al (1996) J Mol Med 74(7), 379-92 (reviews induction factors e.g., hormones, growth factors, 379
  • Tetracycline - inducible vectors may also be used. These are activated by a relatively -non toxic antibiotic that has been shown to be useful for regulating expression in mammalian cell cultures. Also, steroid-based inducers may be useful especially since the steroid receptor complex enters the nucleus where the DNA vector must be segregated prior to transcription.
  • This system may be further improved by regulating the expression at two levels, for example by using a tissue-selective promoter and a promoter controlled by an exogenous inducer/repressor, for example a small molecule inducer, as discussed above and known to those skilled in the art.
  • one level of regulation may involve linking the appropriate polypeptide-encoding gene to an inducible promoter whilst a further level of regulation entails using a tissue-selective promoter to drive the gene encoding the requisite inducible transcription factor (which controls expression of the polypeptide (for example the antigenic polypeptide)-encoding gene from the inducible promoter).
  • Control may further be improved by cell-type-specific targeting of the genetic construct.
  • the genetic constructs of the invention can be prepared using methods well known in the art.
  • the aforementioned therapeutic molecules may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • Preferred routes include oral, intranasal or intramuscular injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • an inducer for example small molecule inducer as discussed above may preferably be administered orally.
  • Methods of delivering genetic constructs for example adeno viral vector constructs to cells of a recipient will be well known to those skilled in the art.
  • an adoptive therapy protocol may be used or, more preferably, a gene gun may be used to deliver the construct to dendritic cells, for example in the skin.
  • Adoptive therapy protocols are described in Nestle et al (1998) Nature Med. 4, 328- 332 and De Bruijn et al (1998) Cancer Res. 58, 724-731.
  • the therapeutic agent may be given to a subject who is being treated for the disease by some other method.
  • the method of treatment may be used alone it is desirable to use it as an adjuvant therapy, for example alongside conventional preventative or therapeutic methods.
  • a therapeutic molecule as described herein for example an antigenic molecule, construct or chimaeric polypeptide
  • the carrier(s) must be "acceptable” in the sense of being compatible with the therapeutic molecule (which may be a nucleic acid or polypeptide) and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the pharmaceutical composition may further comprise a component for increasing the antigenicity and/or immieuxicity of the composition, for example an adjuvant and/or a cytokine.
  • a component for increasing the antigenicity and/or immavouricity of the composition for example an adjuvant and/or a cytokine.
  • a polyvalent antigen may be useful.
  • Nasal sprays may be useful formulations.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (for an antigenic molecule, construct or chimaeric polypeptide of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the therapeutic molecule can be delivered to the locus by any means appropriate for localised administration of a drug.
  • a solution of the therapeutic molecule can be injected directly to the site or can be delivered by infusion using an infusion pump.
  • the construct for example, also can be incorporated into an implantable device which when placed at the desired site, permits the construct to be released into the surrounding locus.
  • the therapeutic molecule may be administered via a hydrogel material.
  • the hydrogel is non-inflammatory and biodegradable. Many such materials now are known, including those made from natural and synthetic polymers.
  • the method exploits a hydrogel which is liquid below body temperature but gels to form a shape-retaining semisolid hydrogel at or near body temperature.
  • Preferred hydrogel are polymers of ethylene oxide-propylene oxide repeating units. The properties of the polymer are dependent on the molecular weight of the polymer and the relative percentage of polyethylene oxide and polypropylene oxide in the polymer.
  • Preferred hydrogels contain from about 10% to about 80% by weight ethylene oxide and from about 20% to about 90% by weight propylene oxide.
  • a particularly preferred hydrogel contains about 70% polyethylene oxide and 30% polypropylene oxide. Hydrogels which can be used are available, for example, from BASF Corp., Parsippany, NJ, under the tradename Pluronic ⁇ .
  • a further aspect of the invention provides a vaccine effective against bacterial infection, for example EHEC and/or EPEC, comprising an effective amount of the polypeptide(s) or polynucleotide(s) as defined in relation to the first and second aspects of the invention.
  • the nucleic acid vaccine may comprise any suitable nucleic acid delivery means, as noted above.
  • the nucleic acid preferably DNA, may be naked (ie with substantially no other components to be administered) or it may be delivered in a liposome or as part of a viral vector delivery system.
  • the nucleic acid vaccine may be administered without adjuvant.
  • the nucleic acid vaccine may also be administered with an adjuvant such as BCG or alum.
  • suitable adjuvants include Aquila's QS21 stimulon (Aquila Biotech, Worcester, MA, USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and proprietary adjuvants such as Ribi's Detox.
  • Quil A another saponin-derived adjuvant, may also be used (Superfos, Denmark).
  • Other adjuvants such as Freund's may also be useful. It is preferred if the nucleic acid vaccine is administered without adjuvant.
  • an antibody preparation reactive against two or more intimins comprising administering said two or more intimins or fragments thereof, as discussed above, to an animal and collecting and purifying the directly or indirectly resulting antibody, wherein the said intimin fragments do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • the antibody may preferably be polyclonal.
  • the preparation may be made by combination of two or more antibodies (for example monoclonal antibodies) or antibody preparations reactive with regions of different intimins other than the Gly387 to Lys666 region.
  • antibody in accordance with the invention we include molecules which comprise or consist of antigen binding fragments of an antibody including Fab, Fv, ScFc and dAb. We also include agents which incorporate such fragments as portions for targeting antigens and/or cells or viruses which display such antigens.
  • an antibody preparation preferably a polyclonal antibody preparation reactive against two or more intimins for use in medicine.
  • the invention also provides the use of the antibody preparation in the manufacture of a medicament (or food supplement composition) for use in the prevention or treatment of a bacterial disease.
  • the invention also provides a method of treatment of a human or animal with or at risk of a bacterial disease, wherein the human or animal is administered the said antibody preparation.
  • the antibody preparation is capable of binding to two or more intimins, still more preferably capable of binding to portions of the intimins outside the conserved regions, ie Gly387 to Lys666 region, (though the antibody preparation may also bind to the conserved regions).
  • the antibody preparation binds to the tir-binding region of two or more intimins.
  • a further aspect of the invention provides a method of preventing and./or treating a bacterial disease comprising administering to a subject an effective amount of a polypeptide or polypeptides (or corresponding peptidomimetic compounds, as discused above) in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ - intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • a further aspect of the invention provides a method of preventing and./or treating a bacterial disease comprising administering to a human or animal an effective amount of a polynucleotide encoding, or polynucleotides encoding in combination, a polypeptide or polypeptides in combination comprising at least two of (1) ⁇ -intimin or a fragment thereof, (2) ⁇ -intimin or a fragment thereof, (3) ⁇ -intimin or a fragment thereof, (4) ⁇ -intimin or a fragment thereof, and (5) ⁇ -intimin or a fragment thereof, wherein the polypeptide or polypeptides do not consist of the Gly387 to Lys666 region, or a fragment thereof, of two or more intimins.
  • Preferences in relation to the polypeptide(s) and polynucleotide(s) are as indicated in relation to preceding aspects of the invention.
  • the subject may be administered a combination of polypeptides and polynucleotides
  • the subject may be administered two or more intimin polypeptides.
  • the intimin polypeptides are from at least two of the groups of ⁇ , ⁇ , ⁇ , ⁇ and ⁇ intimins.
  • the subject is administered an ⁇ intimin, a ⁇ intimin and optionally further an ⁇ intimin (including fragments thereof other than merely the conserved (Gly387 to Lys666) region).
  • the polypeptide(s) comprises a tir binding site.
  • a further aspect of the invention provides a method of preventing and./or treating a bacterial disease comprising administering to a human or animal an effective amount of a chimaeric polypeptide, polynucleotide, antibody preparation or combination of the invention.
  • FIG. 1 Mice infected with C. rodentium mount IgG and IgA antibody responses to LEE encoded virulence determinants.
  • the data depicts the mean serum (A) and IgG and (B) IgA antibody titers 14, 28, 42 and 56 day post infection with C. rodentium.
  • FIG. 2 Mice infected with C. rodentium develop robust acquired immunity.
  • A Mean number of C. rodentium bacteria of DBS255(pCVD438) recovered from colons or
  • B the MLN's of convalescent mice or age matched na ⁇ ve mice 1 1 days after oral challenge showing significant fewer bacteria recovered.
  • C The mean colon weights of convalescent or naive mice 11 days post challenge showing significant differences.
  • Fig. 3 Humoral immune response to Int280 ⁇ in mice immunised with Int280 ⁇ with or without enterotoxin-based adjuvant.
  • A. Mean IgA, IgGl and IgG2a serum antibody responses in mice s.c. immunised with 10 ⁇ g of Int280 ⁇ with or without 1 ⁇ g of adjuvant.
  • Fig. 4 Splenic T cell responses in mice immunised with Int280 ⁇ with or without enterotoxin adjuvant.
  • Vaccination using Int280 ⁇ alone protects mice from C. rodentium colonisation.
  • Mice were vaccinated s.c. x3 with 10 ⁇ g of the irrelevant antigen OVA, 10 ⁇ g of Int280 ⁇ or PBS. Mice were infected 14 days after the last immunisation. Bacterial levels were measured 14 days post challenge in (A) colon, (B) mesenteric lymph nodes and (C) spleen showing significant fewer bacteria in colons and MLN's.
  • Vaccination using Int280 ⁇ alone does not prevent colonic colonisation by C. rodentium expressing Int280 ⁇ .
  • Mice were immunised with 10 ⁇ g Int280 ⁇ and infected with either WT or BDS255(pCVD438) strains. Number of bacteria, measured 15 days post challenge, reveals significantly fewer DBS255(pCVD438) bacteria compared with naive and WT infected mice.
  • Antibodies may be produced in a number of ways.
  • Antibodies can also be produced by molecular biology techniques, with expression in bacterial or other heterologous host cells (Chiswell, D.J. & McCafferty, J. (1992) Phage antibodies: will new "coli-clonal” antibodies replace monoclonal antibodies?" Trends Biotechnol. 10, 80-84).
  • the purification method to be adopted will depend on the source material (serum, cell culture, bacterial expression culture, etc.) and the purpose of the purification (research, diagnostic investigation, commercial production). The major methods are as follows:
  • Ammonium sulphate precipitation The ⁇ -globulins precipitate at a lower concentration than most other proteins, and a concentration of 33% saturation is sufficient. Either dissolve in 200g ammonium sulphate per litre of serum, or add 0.5 vol of saturated ammonium sulphate. Stir for 30 minutes, then collect the ⁇ - globulin fraction by centrifugation, redissolve in an appropriate buffer, and remove excess ammonium sulphate by dialysis or gel filtration.
  • Staphylococcus aureus Outer coat protein known as Protein A
  • F c invariant region
  • IgG2a > an d I ⁇ Gr3 all of which behave differently on elution from Protein A.
  • Protein G from G from a Streptococcus sp. can be used. This is more satisfactory with immunoglobulins from farm animals such as sheep, goats and cattle, as well as with certain subclasses of mouse and rabbit IgGs. The most specific affinity adsorbent is the antigen itself.
  • the process of purifying an antibody on an antigen adsorbent is essentially the same as purifying the antigen on an antibody adsorbent.
  • the antigen is coupled to the activated matrix, and the antibody-containing sample applied. Elution requires a process for weakening the antibody-antigen complex. This is particularly useful for purifying a specific antibody from a polyclonal mixture.
  • Monoclonal antibodies can be prepared to most antigens.
  • the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982). Chimaeric antibodies are discussed by Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).
  • Non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • one or more polypeptides comprising in combination polypeptide sequences of two or more of intimin types ⁇ , ⁇ , ⁇ , ⁇ and ⁇ (or further intimin types) are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the subject.
  • the subject is administered polypeptides corresponding to two or more intimin types.
  • the polypeptides comprise Tir binding sites from two or more intimin polypeptides.
  • the subject is administered two or more of Int280 ⁇ , Int280 ⁇ , Int280 ⁇ , Int280 ⁇ and Int280 ⁇ .
  • polypeptides By polypeptides is included peptidomimetic molecules, containing intimin peptides or full length intimin or chimaeric polypeptides of the invention.
  • Suitable adjuvants include Freund's complete or incomplete adjuvant, detoxified cholera toxin or heat labile E. coli toxin, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic” is a Registered Trade Mark. It may be advantageous not to include such an adjuvant, as discussed in Example 1.
  • a DNA vaccine may be administered.
  • Preferred formulations include those suitable for oral administration, including topical oral administration, intranasal (mucosal) administration and parenteral administration, including intramuscular or subcutaneous injection.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of an active ingredient. Examples of formulations that may be useful with the present invention are described below. Other formulations may also be used.
  • Example A Injectable Formulation Active ingredient 0.200 g
  • Example B Intramuscular injection
  • the active ingredient is dissolved in the glycofurol.
  • the benzyl alcohol is then added and dissolved, and water added to 3 ml.
  • the mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).
  • the sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added.
  • the active ingredient is added and dispersed.
  • the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.
  • Example D Suppository mg/suppository Active ingredient (63 :m)* 250 Hard Fat, BP (Witepsol H 15 - Dynamit Nobel) 1770
  • the active ingredient is used as a powder wherein at least 90% of the particles are of 63 ⁇ m diameter or less.
  • One fifth of the Witepsol HI 5 is melted in a steam-jacketed pan at 45°C maximum.
  • the active ingredient is sifted through a 200 ⁇ m sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol HI 5 is added to the suspension and sti ⁇ ed to ensure a homogenous mix.
  • the entire suspension is passed through a 250 ⁇ m stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.
  • the aforementioned active agents or a formulation thereof may be administered in a variety of ways, for non-limiting example, by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time, depending on the characteristics (for example, age, weight and condition) of the subject (which may be an animal, and which may have no symptoms of disease) and/or the state of the particular bacterial disease against which the treatment (which may be prophylactic treatment) is directed.
  • agents and other compounds of the invention may also find utility as diagnostic agents. Skilled persons will appreciate that the agents and other compounds of the invention can readily be provided for use in ELISA techniques. They may also be useful in isolating or identifying bacteria (for example E. coli) expressing any type of intimin from a sample (for example a biological or food sample), for example using immuno-rnagnetic separation techniques. They may also be useful in diagnosis to determine if a subject has been exposed to any intimin.
  • the agents of the invention may find particular utility in the prevention of bacterial infections.
  • the agents can be administered to humans or animals at particular risk of exposure to bacterial infections. Such risks may arise when a human or animal is likely to, or has already, come into contact with an affected human or animal.
  • Intimin-specific immune responses prevent bacterial colonisation and disease caused by Citrobacter rodentium (a model for EHEC and EPEC colonisation)
  • EHEC Enterohaemo ⁇ hagic
  • EPEC enteropathogenic Escherichia coli
  • A/E attaching and effacing
  • rodentium developed robust acquired immunity and were resistant to reinfection with w.t. C. rodentium or a recombinant C. rodentium strain (DBS255(pCVD438) which expressed intimin from EPEC strain E2348/69.
  • the eukaryotic cell-binding domain of intimin polypeptides is located within the carboxy- terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimes using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280 ⁇ from EPEC strain E2348/69.
  • This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int3gg_ 7) as a vaccine.
  • mice Female, specific pathogen free C3H/Hej mice (6-8 weeks old) were purchased from Harlan Olac (Bichester, United Kingdom). All mice were housed in individual ventilated cages with free access to food and water.
  • DBS255 is an eae mutant of C. rodentium and is avirulent in mice.
  • Plasmid pCVD438 is a recombinant plasmid containing the eae gene from EPEC strain E2348/69 (intimin ⁇ ). Therefore DBS255(pCVD438) is a C. rodentium eae mutant complemented with the eae gene from EPEC strain E2348/69 (intimin ⁇ ). This strain expresses biologically active intimin and is virulent in mice.
  • mice For intranasal (i.n.) immunisations, groups of mice were lightly anaesthetised with gaseous halothane and 30 ⁇ l of Ag in PBS applied to the nasal nares. Mice were i.n. immunised on day 0, 14 and 28 and orally challenged between days 42-44.
  • mice were s.c. immunised on 0, 14 and 28 and orally challenged between day 42-44.
  • Bacterial inoculums were prepared by culturing bacteria overnight at 37°C in L-broth containing nalidixic acid (lOO ⁇ g/ml) (C. rodentium) or L-broth containing nalidixic acid (lOO ⁇ g/ml) plus chloramphenicol (50 ⁇ g/ml) (DBS255(pCVD438)). After incubation, bacteria were harvested by centrifugation and resuspended in an equal volume of PBS. A 1/10 dilution of bacteria in PBS was then prepared and mice orally inoculated, without anaesthetic, using a gavage needle with 200 ⁇ l of the bacterial suspension. The viable count of the inoculum was determined by retrospective plating on L-agar containing appropriate antibiotics.
  • Recombinant porcine LT, LTK63 and LTR72 were kindly provided by M. Pizza and R. Rappuoli (Chiron Vaccines, Siena, Italy), and were prepared as described previously [Magagnoli, 1996].
  • Recombinant Int280 ⁇ which represents the C-terminal 280 amino acids of intimin (Int660-939) fr° m EPEC strain E2348/69, was purified as described previously [Kelly, 1998].
  • Int388-667 > which co ⁇ esponds to two putative Ig- like domains upstream of Int280, and Tir-M, the intimin-binding domain of Tir, were purified as poly-histidine tagged polypeptides as described, [Batchelor, 1999].
  • EspA was similarly purified as a poly-histidine tagged polypeptide [Batchelor, 1999].
  • EspB was cloned from EPEC strain E2348/69, expressed as maltose-binding protein fusion proteins in E. coli and purified by nickel affinity chromotagraphy as previously described [Knutton, 1998; Frankel, 1996].
  • a preparation of soluble proteins from C. rodentium was generated by repeated sonication of a concentrated suspension of bacteria cultured overnight in L broth. Insoluble proteins were removed by centrifugation at 13,000 RPM for 5 minutes and the supernatant removed and stored at -20°C. The concentration of protein solutions was determined using a BCA protein assay kit (Pierce, Rockford, USA).
  • mice were killed by cardiac exsanguination under terminal anaesthesia or by cervical dislocation. Spleens, livers, mediastinal and caudal lymph nodes were then aseptically removed. The distal 6cm of the colon was also removed and the colon weighed after removal of faecal pellets. In some experiments, the distal 1cm of colon was removed for histological analysis. Spleens, livers, lymph nodes and colons were then homogenised mechanically using a Seward 80 stomacher (London, England) and the number of viable bacteria in organ homogenates determined by viable count.
  • Seward 80 stomacher London, England
  • Spleens from immunised mice were aseptically removed and single cell suspensions prepared by passing organs through 100 ⁇ m nylon sieves (Marathon Laboratories, London, UK). After lysis of splenic erythrocytes with Tris-ammonium chloride, a total of 10 ⁇ leukocytes were cultured in the presence of 1 ⁇ g/ml Int280 ⁇ in RPMI 1640 (Sigma, St Louis, MO) containing 10% FCS (Sigma), 5x10-5 M 2-ME, 2mM L-glutamine (Sigma), 100 units penicillin/ml (Sigma) and 100 ⁇ g/ml streptomycin (cRPMI) in triplicate for 24 h in wells of a 24 well plate (Costar).
  • ELISPOT plates were prepared as follows: nitrocellulose-based 96-well microtitre plates (Multiscreen-HA, Millipore, Hertfordshire, UK) were coated overnight at 4 ⁇ C with 50 ⁇ l/well of either anti-IFN- ⁇ (4 ⁇ g/ml) (R46A2) or anti-IL-4 (4 ⁇ g/ml)(l IBl 1) mAb diluted in carbonate buffer pH 9.6.
  • the distal 1cm of colon was removed, cut longitudinally and rolled, then snap frozen in liquid nitrogen. From frozen colonic tissue, 5 ⁇ m thick, cryostat cut sections were mounted on poly-L-lysine coated glass microscope slides. Staining for bacterially expressed intimin ⁇ in frozen tissue was performed as previously described [Higgins, 1999].
  • mice infected with wild-type C. rodentium were analysed to determine if LEE encoded proteins were recognised by the host immune system.
  • Mice infected orally with C. rodentium mounted serum IgG (Fig. 1 A) and IgA (Fig. IB) antibody responses which recognised antigens in a whole cell lysate of C. rodentium.
  • Infected mice also mounted serum IgG and IgA responses which cross- reacted with EspA, EspB, TirM and Int388-667 from EPEC 2348/69 (Fig.
  • IgA responses developed more rapidly and were strongest to Int280 ⁇ . IgA responses to all antigens remained detectable 8 weeks post-infection. These data imply that several LEE encoded antigens are expressed in vivo during an infection with C. rodentium and are targets of the host immune response.
  • mice infected with C. rodentium develop acquired immunity
  • the development of acquired immunity to enteric bacterial pathogens which colonise via the formation of A/E lesions has been implied [Donnenberg, 1998], but never formally shown in animals or humans.
  • two groups of C3H/Hej mice were orally infected with 7x10 ⁇ cfu of C. rodentium.
  • three months later, one group of convalescent mice were re-challenged with 8x10$ cfu of wild- type C. rodentium and the second group with 2x10 ⁇ cfu of a C. rodentium strain expressing ⁇ intimin (DBS255(pCVD438)).
  • mice Age and sex-matched naive mice were orally challenged in parallel with convalescent mice. Fourteen days after oral challenge the pathogen burden in mouse tissues was determined in all groups. Compared with naive animals, convalescent mice harboured significantly fewer challenge bacteria in colons (Fig. 2 A) and draining lymph nodes (Fig. 2B). Further, the colon weights of challenged mice, a good indicator of the degree of infection-driven pathology in the mucosa [Higgins, 1999], were substantially lower in convalescent mice compared to the naive animals (Fig 2C). These data clearly show that mice infected with C. rodentium develop acquired immunity to re-infection with C. rodentium strains expressing either homologous or heterologous intimin types.
  • Intimin plays an essential role in the formation of A/E lesions and an important role in the pathogenesis of EPEC, EHEC, and C. rodentium [Donnenberg, 1993; Donnenberg, 1993; Frankel, 1996]).
  • the demonstrated importance of intimin in facilitating bacterial colonisation in vivo led to the hypothesis that an intimin-based vaccine may prevent infections caused by bacteria which colonise the host via A/E lesion formation.
  • a highly purified preparation of recombinant Int280 ⁇ from EPEC E2348/69 was used as an immunogen in mucosal and parenteral vaccination regimes. Mice were vaccinated intranasally or subcutaneously with or without the use of Escherichia coli heat-labile toxin (LT) or mutant derivatives as adjuvants.
  • LT heat-labile toxin
  • mice were s.c. immunised three times, on day 0, 14 and 28, with lOmg of Int280 ⁇ with or without adjuvant.
  • the co-administration of LT or LTR72 with Int280 ⁇ prompted a more rapid Ig response to Int280 ⁇ (data not shown), but did not, however, increase the magnitude of the final Int280 ⁇ -specific IgGl or IgG2a titre compared to that obtained in mice s.c. immunised with Int280 ⁇ alone (Fig 3 A).
  • Int280 ⁇ -specific IgGl was the predominant IgG subclass elicited by parenteral vaccination, although the ratio of IgGl :IgG2a was reduced when Int280 ⁇ was co-administered with the adjuvants LT or LTR72 (Fig. 3 A).
  • mice were immunised three times, on day 0, 14 and 28, with lOmg of Int280 ⁇ with or without an enterotoxin-based adjuvant.
  • Mice i.n. administered lOmg of Int280 ⁇ mounted serum IgGl and IgG2a, but not IgA, antibody responses to Int280 ⁇ .
  • Co-delivery of lmg of LT, LTR72 or LTK63 with Int280 ⁇ significantly increased the serum IgGl and IgG2a antibody response to Int280 ⁇ .
  • the addition of a mucosal adjuvant resulted in the induction of Int280 ⁇ - specific serum IgA responses (Fig. 3B).
  • Int280 ⁇ -specific T cells elicited by selected immunisation strategies were evaluated using cytokine-specific ELISPOT.
  • Int280 ⁇ -specific T cell responses were compared in mice i.n. immunised with PBS, lmg of LTR72, lmg of LTR72 plus lOmg of Int280 ⁇ or lOmg of Int280 ⁇ alone.
  • Int280 ⁇ -specific T cell responses were also assessed in mice s.c. immunised with lmg of LTR72 plus lOmg of Int280 ⁇ or lOmg of Int280 ⁇ alone. Mice were immunised on day 0, 14 and 28 and killed on day 42.
  • Splenocytes from immunised mice were stimulated with media or 1 ug/ml of recombinant Int280 ⁇ for 18 hrs before being washed and cultured in the absence of antigen for another 18 hrs on IL-4 or IFN- ⁇ ELISPOT plates. ELISPOT plates were then developed and counted.
  • mice immunised with Int280 ⁇ i ⁇ espective of the vaccination route, there was a predominance of IFN- ⁇ SFC's over IL-4 SFC's (Fig. 4).
  • Mice vaccinated i.n. three times with lOmg of Int280 ⁇ alone had the highest number of IFN- ⁇ SFC's.
  • mice immunised s.c. with lmg of LTR72 plus lOmg of Int280 ⁇ followed by mice immunised i.n. with lOmg of Int280 ⁇ alone (Fig. 4).
  • DBS255(pCVD438) a recombinant C. rodentium strain which only expresses intimin ⁇ , is virulent in mice and induces similar mucosal pathology in the distal colon as wild-type C. rodentium [Higgins, 1999].
  • mice were i.n. or s.c. immunised three times, on day 0, 14 and 28, with lOmg of Int280 ⁇ with or without adjuvant.
  • mice were orally challenged with between 2-3xl0 7 cfu of DBS255(pCVD438) 13 or 16 days after the last immunisation.
  • mice were killed 14 days post-challenge, the colon was weighed, homogenised and the pathogen burden determined by viable count.
  • Mice immunised s.c. with PBS or adjuvant alone had uniformly high C. rodentium counts in the colon (Fig 5A).
  • the colons of mice immunised s.c. (Fig. 5A) with Int280 ⁇ alone harboured significantly fewer challenge bacteria than the colons of naive or control animals.
  • mice immunised with Int280 ⁇ together with a mucosal adjuvant were more susceptible to colonic infection than mice which received Int280 ⁇ alone (Fig. 5A). Similar results were obtained in i.n. immunised mice. Mice immunised i.n.
  • intimin subtypes ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ) have been described based on sequence variation within the C-terminal 280 amino acids. These intimin subtypes are also antigenically different [Adu-Bobie, 1998]. Therefore, a vaccine based on Int280 ⁇ may not necessarily protect against infections caused by E. coli strains expressing a heterologous intimin type. To address this hypothesis in the context of C. rodentium infection, C3H/Hej mice were vaccinated s.c.
  • mice were vaccinated s.c. with Int280 ⁇ plus or minus Al(OH)3.
  • Measurement of serum IgG antibody responses to the three different antigens demonstrated the induction of robust humoral immune responses to each antigen (Fig 8A).
  • the co-administration of Alum with Int280 ⁇ or OVA did not, however, increase the final magnitude of the IgG response (Fig 8A).
  • mice including naive controls, were orally challenged with 6xl0 7 cfu of DBS255(pCVD438) 12 days after the last immunisation.
  • mice immunised s.c. with Int280 ⁇ were resistant to DBS255(pCVD438) challenge. Resistance to challenge in mice vaccinated with Int280 ⁇ was independent of the use of Al(OH)3 as an adjuvant.
  • mice immunised with Int388-667 were as susceptible to DBS255(pCVD438) colonisation as naive mice or mice immunised with OVA (Fig. 8B).
  • Immunohistological examination of colons from susceptible mice showed that pathogen colonisation was focal and characterised by intimate bacterial attachment to the epithelial surface (Fig. 8C).
  • bacterial staining was substantially reduced or absent in mice immunised with Int280 ⁇ (Fig. 8C) and absent in uninfected mice (data not shown).
  • rodentium infection of mice can also be employed as a model system in which to test LEE encoded proteins as candidate vaccine antigens.
  • Int280 ⁇ was used as a vaccine to ameliorate the severity of an infection caused by a recombinant C. rodentium strain expressing intimin ⁇ . This represents the first in vivo evidence to support the use of defined intimin domains as candidate EPEC/EHEC vaccine antigens.
  • the intimin binding domain of Tir, TirM is also recognised by serum IgG and colostrum IgA antibodies from Brazilian mothers [Sanches, 2000]. Children infected with EHEC also mount serum Ig responses to Intimin, Tir, EspA and EspB [Li, 2000]. These data from humans matches the spectrum of antibody responses detected in sera of mice infected with C. rodentium. Infected mice develop serum IgG and IgA antibody responses to Int280 ⁇ , TirM, EspB and EspA. These studies complement existing data demonstrating induction of mucosal IgA responses to intimin and EspB in C. rodentium infected mice [Frankel, 1996].
  • Intimin is an essential virulence determinant of C. rodentium in mice [Schauer, 1993] and EHEC in gnotobiotic pigs [Donnenberg, 1993]. Intimin also contributes markedly to the virulence of EPEC in humans [Donnenberg, 1993]. In these pathogens, intimin most likely contributes to virulence by facilitating tight binding of the bacterium to the epithelial cell membrane via Intimin-Tir interactions. The aim of the studies described here was to determine whether vaccine-induced immune responses to Int280 ⁇ could modulate or prevent in vivo bacterial colonisation by C. rodentium strains expressing either homologous or heterologous intimin types.
  • Vaccination using Int280 ⁇ clearly imparted a degree of type-specific protective immunity to mice.
  • the anatomical location and immunological mechanisms through which vaccination confers resistance to DBS255(pCVD438) colonisation remains unknown. Indeed, few clues are provided by comparing immune responses elicited by s.c. or intranasally administered Int280 ⁇ with those of other, less efficacious vaccination methods.
  • Vaccination by s.c. or i.n. administration of Int280 ⁇ elicited strong Int280 ⁇ -specific serum IgG responses, with a bias towards IgGl over
  • IgG2a was typically less pronounced in these animals. Additionally, the use of a mucosal adjuvant with Int280 ⁇ evoked serum IgA responses in mucosally immunised mice. Despite the absence of an immunological co ⁇ elate of protection in appropriately immunised animals, the concept of efficacious vaccination against mucosal pathogens by parenteral immunisation is not new. Numerous parenteral vaccines used in humans, including the Salk polio vaccine and the pneumococcal, Haemophilus influenzae type b and Shigella O-specific polysaccharide conjugates have proven efficacious [Cohen, 1997; Robbins, 1997; Kaul, 1998].
  • Int280 ⁇ -specific immune responses mediate their affect on C. rodentium
  • IgG IgG
  • Int280 ⁇ specific antibodies which have translocated from the serum to the gut lumen via transhepatic delivery mechanisms [Bouvet, 1999] may interact with luminal C. rodentium and exhibit anti-adhesin properties.
  • One aspect of the humoral immune response to Int280 ⁇ which was not examined in this study is the avidity of the antigen-specific antibody response. Potentially, administration of Int280 ⁇ evokes specific antibody responses which are of higher avidity than those elicited by adminisfration of Int280 ⁇ with an enterotoxin-based mucosal adjuvant. Biologically, relatively high avidity Int280 ⁇ -specific antibody may have reduced opsonic activity or blocking capacity and may thereby have a reduced ability to inhibit or limit colonisation of DBS255(pCVD438) on the colonic epithelium.
  • Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB. Infect Immun. 64(12):5315-25.

Abstract

L'invention concerne une composition pharmaceutique, un vaccin, un produit alimentaire ou un nécessaire, comprenant un polypeptide ou des polypeptides ou un polynucléotide ou des polynucléotides combinés comprenant un polypeptide ou des polypeptides combinés ou codant pour eux comprenant au moins deux parmi (1) l'intimine α ou un fragment de celle-ci, (2) l'intimine β ou un fragment de celle-ci, (3) l'intimine η ou un fragment de celle-ci, (4) l'intimine δ ou un fragment de celle-ci et (5) l'intimine ε ou un fragment de celle-ci, avec un support ou un vecteur pharmaceutiquement acceptable. Selon l'invention, le ou les polypeptides ne correspondent pas à la région Gly387 à Lys666, ou à un fragment de celle-ci, de deux ou plusieurs intimines. La vaccination avec la région Gly387 à Lys666, qui se retrouve entre différents types d'intimine, n'est en effet pas considérée comme induisant une immunité protectrice. En revanche, un vaccin combiné peut fournir une telle immunité protectrice et peut être utile pour fournir une protection contre les infections bactériennes.
EP02720132A 2001-03-29 2002-03-26 Substances biologiques et procedes pour leur utilisation dans la prevention ou le traitement d'infections Withdrawn EP1372709A2 (fr)

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GB2473523A (en) * 2009-09-10 2011-03-16 Solus Scient Solutions Ltd Nalidixic acid and lithium in a base medium for the culture of Listeria
CN103212060B (zh) * 2013-04-23 2014-11-05 中国人民解放军军事医学科学院微生物流行病研究所 蛋白及其组合物与在制备预防、诊断或治疗ehec感染产品中的应用
CN105821065A (zh) * 2016-04-29 2016-08-03 南方医科大学 一种双抗原重组蛋白及其制备方法和应用

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WO2002079240A2 (fr) 2002-10-10
WO2002079240A3 (fr) 2003-06-26
WO2002079247A2 (fr) 2002-10-10
AU2002251202A1 (en) 2002-10-15
WO2002079247A3 (fr) 2003-10-30
AU2002241162A1 (en) 2002-10-15

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