WO2002079247A2

WO2002079247A2 - Intimins in the prevention or treatment of infections:ii

Info

Publication number: WO2002079247A2
Application number: PCT/GB2002/001462
Authority: WO
Inventors: Gadi Frankel; Gordon Dougan; Steve Matthews
Original assignee: Imperial College Innovations Limited
Priority date: 2001-03-29
Filing date: 2002-03-26
Publication date: 2002-10-10
Also published as: EP1372709A2; AU2002251202A1; AU2002241162A1; WO2002079247A3; WO2002079240A2; WO2002079240A3

Abstract

Mutated intimin polypeptides, for example at the residue equivalent to V162/252/911, T165/255/914 of Int190alpha/Int280alpha/full length intimin-alpha, or at a residues that form part of a solvent-exposed loop bordering the Tir binding site, for example residues equivalent to residue 230, 231, 232 and/ or 233 of Int280 alpha. A food product, pharmaceutical composition or vaccine comprising such an intimin polypeptide or polynucleotide.

Description

BIOLOGICAL MATERIALS AND METHODS FOR USE IN THE PREVENTION OR TREATMENT OF INFECTIONS :II

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.

Induction of an 'attaching and effacing' (A/E) lesion on the intestinal mucosa is a pathogenic mechanism shared by a number of enteric human and animal pathogens. The A/E lesion is characterised by localised destruction (effacement) of brush border microvilli, intimate attachment of the bacillus to the host cell plasma membrane and the formation of an underlying pedestal-like structure in the host cell consisting of polymerised actin, ezrin, talin and myosin (reviewed in Frankel et al., 1998b; Kaper et al., 1998), as well as WASP (Wiskott-Aldrich syndrome family of proteins) and Arp2/3 complex (Kalman et al., 1999). A/E lesions were first described for strains of enteropathogenic E. coli (EPEC) (Moon et al., 1983; Ulshen and Rollo, 1980), an established etiological agent of human infantile diarrhoea in the developing world (Nataro and Kaper, 1998). Similar intestinal lesions have later been associated with other enteric mucosal pathogens including enterohaemorrhagic E. coli (EHEC) (Phillips et al., 2000; Tzipori et al., 1986), an emerging microbial pathogen associated with food poisoning that is often associated with life threatening complications (for example acute gastro-enteritis, bloody diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (HUS)) due to the production of Verocytotoxins (Nataro and Kaper, 1998), rabbit diarrhoeagenic E. coli (PvEPEC) (Robins-Browne et al., 199 ) and the mouse pathogen Citrobacter rodentium (Schauer and Falkow, 1993a). Using animal and ex vivo infection models, A/E lesion formation was shown, in most cases, to be essential for colonisation of the intestinal mucosa and in all case for pathogenicity (Dean-Nystrom et al., 1998; Donnenberg et al., 1993b; Hicks et al, 1998; Marches et al., 2000; McKee et al., 1995; Schauer and Falkow, 1993b).

The genes encoding the A/E phenotype are encoded on a pathogenicity island termed the Locus of Enterocyte Effacement (LEE) (McDaniel et al., 1995; Perna et al., 1998). The majority ofthe LEE genes are organised in 5- polycistronic operons (LEE1, LEE2, LEES, tir and LEE4) (Mellies et al., 1999). LEE1, LEE2 and LEE3 encode components of a type III secretion system, LEE4 encodes proteins secreted by the TTSS termed E. coli secreted proteins (Esps) (reviewed in Elliott et al., 1998; Frankel et al., 1998b) and the tir operon encodes for the outer membrane adhesion molecule, intimin (Jerse et al., 1990), the translocated intimin receptor (Tir) (Deibel et ah, 1998; Kenny et al, 1997), and CesT (the Tir chaperon) (Abe et al, 1999; Elliot et al, 1999).

Although the genetic basis of A/E lesion formation is well documented (Frankel et al., 1998b; Kaper et al., 1998), relatively little is known about the initial intestinal stage of the infection involving colonisation of the gut, a necessary first step in the infection process. The only adherence factor that has been demonstrated to play a role in intestinal colonisation in vivo in animal models is the 94 to 97 kDa adhesion molecule - intimin (Jerse and Kaper, 1991; Jerse et al., 1990). Intimin, which is encoded by the eae gene (Jerse et al., 1990; Yu and Kaper, 1992), mediates intimate bacterial attachment to epithelial cells through binding to a second bacterial protein, the translocated intimin receptor, Tir/EspE (Deibel et al., 1998; Kenny et al., 1997), which is delivered to the host cell membrane by a type III secretion system. Intimin-mediated intimate attachment was demonstrated in a number of infection models including EHEC in calves (Dean-Nystrom et al, 1998) and pigs (Donnenberg et al, 1993b; McKee et al, 1995), REPEC (Marches et al, 2000) and Citrobacter rodentium in mice (Schauer and Falkow, 1993b). For human disease, the importance of intimin has been shown for EPEC infection by volunteer studies in which an eae mutant was significantly attenuated compared to the wildtype parent stain (Donnenberg et al, 1993a). The importance of intimin in human disease is also supported by the presence of a high titre of serum intimin antibodies in individuals infected with EHEC (Jenkins et al, 2000) and in colostrum of mothers in Brazil where EPEC infection is endemic (Loureiro et al, 1998).

At least five distinct intimin types, designated α, β, γ, δ, ε, have been identified thus far (Adu-Bobie et al, 1998; Oswald et al, 2000). Importantly, intimin α is associated with one evolutionary branch of EPEC known as EPEC 1. Intimin β is associated both with EPEC and EHEC belonging to their respective clone 2 and with C. rodentium, whereas intimin γ is specifically associated with EHEC O157:H7 and the related strain EPEC O55:H7 (Adu-Bobie et al, 1998). Recent results, including intimin-type exchange studies in piglets (Tzipori et al, 1995) and our work on human intestinal explants (Phillips and Frankel, 2000), suggested that different intimin types might play a role in determining the pattern of colonisation and tissue tropism in the host. Moreover, a C. rodentium strain engineered to express the EHEC intimin γ was avirulent in the mouse model (Hartland et al, 2000). This, together with the fact the intimin can bind to uninfected cells (Tir-independent binding activity) (An et al, 1997; Deibel et al, 2001; Frankel et al, 1994; Frankel et al, 1995) suggest that intimin also bind to a receptor encoded by the host. The receptor/s-binding activity of intimin was localised to its C-terminal 280 amino acids (Int280) (Frankel et al, 1994). The global fold of Int280α, determined by NMR and crystallography (Kelly et al, 1999; Luo et al, 2000), shows that it is built from three globular domains. The first two domains (residues 1-91 and 93-181) each comprise β-sheet sandwiches that resemble the immunoglobulin super family (IgSF). Despite no significant sequence homology, the topology of the C-terminal domain (residues 183- 280) is reminiscent of the C-type lectin domains (CTLD), a family of proteins responsible for cell-surface carbohydrate recognition. Based on structural alignment, W240 (within Int280) was found to be highly conserved in intimins and CTLDs (including invasin from Yersinia ((Isberg et al, 1987))). Although W240 is buried, it is located just below surface residues implicated in Tir binding and may therefore provide a structural scaffold for the binding site. Indeed, mutating this residue in Int280α and the intact intimin (W899) disrupted intimin-Tir interaction and prevented EPEC from forming A/E lesions (Batchelor et al, 2000).

Significant progress has been made defining the molecular basis of EPEC- host cell interactions and defining the role of EPEC's virulence determinants in the regulation of host cell cytoskeletal rearrangement. However, very little is known about the host response to infection in either humans or animals. Indeed, it remains unclear whether humans or animals infected with these pathogens develop protective immunity. A better understanding of this neglected aspect of EPEC and EHEC infection is important for the design of new vaccines and novel approaches which prevent infection- driven diarrhoea. The absence of small animal models to study EPEC or EHEC directly has made the study of host response to infection problematic. In this case, conclusions about EPEC and EHEC need to be drawn from studies of other pathogens which colonise via A/E lesion formation. In this respect, C. rodentium infection of mice offers an advantage because of the wide availability of gene knockout strains and immunological reagents for this species. C. rodentium infection of mice represents the best small animal model in which to study lumenal microbial pathogens relying on A/E lesion formation for colonisation of the host.

Like EPEC and EHEC, 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 & Falkow, 1993b; Newman et al 1999 infect Immun 67, 6019-6025). 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). In experimentally or naturally infected mice, 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 propria 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 α from EPEC E2348/69.

Here we report modification of intimin and modulation of intimin activity, as shown by in vitro (HEp-2 cells), ex vivo (human intestinal explants) and in vivo (mice) infection models as well as biochemical and genetic analysis. We report uses for the modified intimins in science and medicine.

A first aspect of the invention provides a method of treating a human or animal with or at risk of bacterial infection, comprising the step of administering to the human or animal an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide, wherein the intimin polypeptide has the following properties:

(a) it is capable of inducing in a human or animal an immune response which is protective against infection by a bacterial strain expressing a wild- type intimin polypeptide; and

(b) it is either (1) a polypeptide (test polypeptide) which has the structural domains present in a wild-type intimin but does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to induce colonic hypeφlasia in a mouse, wherein the polypeptide is not a polypeptide comprising the cell-binding domain of wild-type intimin-γ, or (2) a fragment of such a polypeptide, or fusion of said fragment which does not confer on a strain of Citrobacter rodentium lacking a functional eae gene (for example by expression in the C. rodentium cell from a recombinant polynucleotide encoding the fusion polypeptide) the ability to induce colonic hypeφlasia in a mouse, wherein the fusion of said fragment is not a polypeptide comprising the cell-binding domain of wild-type intimin-γ.

A second aspect of the invention provides a food product comprising a foodstuff and an intimin polypeptide or recombinant polynucleotide as defined in relation to the first aspect ofthe invention.

The food product may be adapted for consumption by animals or by humans. In relation to all aspects of the invention, it is preferred that the human is a child or infant. It is preferred that the animal is livestock, poultry or a domestic animal, still more preferably a young such animal, as discussed further below. Thus, the food product may preferably be adapted for consumption by children or infants or by young animals. A third aspect of the invention provides a vaccine effective against bacterial infection, for example EHEC and/or EPEC infection, comprising an effective amount of the intimin polypeptide or recombinant polynucleotide as defined in relation to the first aspect of the invention. Additional possible components ofthe vaccine are discussed further below.

A fourth aspect of the invention provides a pharmaceutical composition comprising an intimin polypeptide or recombinant polynucleotide as defined in relation to the first aspect of the invention together with a pharmaceutically acceptable diluent or carrier. It is preferred that the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein-fusion with the C-terminal 181 residues of intimin-γ in which Nal906 is replaced by an alanine residue.

A fifth aspect of the invention provides an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide as defined in relation to the first aspect of the invention, wherein the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein- usion with the C-terminal 181 residues of intimin-γ in which Nal906 is replaced by an alanine residue. Liu et al (1999) Mol Microbiol 34, 67-81 and Frankel et al (1998) Mol Microbiol 29, 559-570 describe such intimin mutants (which are excluded from this aspect of the invention) but do not suggest any use for them.

A sixth aspect ofthe invention provides the use of an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection, wherein the intimin polypeptide or recombinant polynucleotide is as defined in relation to any of the preceding aspects ofthe invention, preferably the first aspect ofthe invention.

A seventh aspect of the invention provides an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide for use in medicine, wherein the intimin polypeptide is as defined in relation to any of the preceding aspects of the invention, preferably the first aspect of the invention.

The intimin polypeptide as defined in relation to the first (and subsequent) aspect of the invention may be considered to be "detoxified" relative to a wild-type intimin. The intimin polypeptide is considered to be able to generate a protective immune response, whilst being less likely than a wild- type intimin to provoke a harmful response (for example, an inflammatory response) in the treated human or animal. Colonic hypeφlasia in the mouse is an example of such an inflammatory response which may conveniently be assessed, and which is provoked by wild-type intimins (other than intimin- γ). It is considered that the intimin polypeptide is less likely to provoke a harmful inflammatory response in a human or animal, for example livestock (such as cattle), poultry or domestic animals than a wild-type intimin polypeptide.

By "polypeptide which has the structural domains present in a wild-type intimin" is included the meaning that the polypeptide has the same _. global structure as a full-length wild-type intimin, for example intimin-α. Thus, the polypeptide which is tested (for example by its expression from a recombinant polynucleotide) for the ability to confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to induce colonic hypeφlasia in a mouse, has N-terminal domains equivalent to the N-terminal domains of full-length wild-type intimin, ie an intracellular domain and a transmembrane domain. The intracellular domain may be formed by residues equivalent to approximately residues 1 to 439 of intimin-γ (as discussed in Liu et al (1999). The transmembrane domain lies C-terminal to this region, but N-terminal to the cell binding domain. An intracellular domain and transmembrane domain may be necessary for the intimin polypeptide to be directed to the correct location in the cell; thus, the requirement for the intracellular and transmembrane domains is satisfied if the polypeptide reaches the correct location in the cell ie in the outer membrane ofthe cell.

The test polypeptide has C-terminal domains equivalent to the C-terminal domains of full-length wild-type intimin, for example intimin-α. Intimin has four domains that protrude from the bacterial membrane for interaction with the host cell, as discussed in Batchelor et al (2000) and Luo et al (2000). Thus, the tested polypeptide has domains corresponding to each of these four domains. The presence of such domains may be demonstrated by structural studies, for example using methods as described in Batchelor et al (2000) and references cited therein (such as Kelly et al (1999)) or Luo et al (2000). It may also be inferred from the ability of the polypeptide or a fragment of the polypeptide comprising all or part of the extracellular portion, preferably the fragments corresponding to or comprising domains D3 and D4 (notation of Batchelor et al (2000)), for example the C-terminal 190 (Intl90) or 188 (Intl88) or 280 (Int280) amino acid residues of intimin, to bind a Tir polypeptide. However, it is not essential that the tested polypeptide or fragment thereof binds to a Tir polypeptide, though this is preferred. The required domain structure may be present even if the polypeptide does not bind a Tir polypeptide. Binding to a Tir polypeptide may be assessed in a yeast two hybrid screen and/or a gel overlay assay, as discussed further below.

It is not necessary for all structural features of the intimin domains to be present in the tested polypeptide so long as the overall domain structure is maintained. For example, a loop located near the Tir binding site (discussed further below) may be deleted.

It is preferred that the test polypeptide does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to mediate intestinal colonisation in a mouse.

The test polypeptide may be a polypeptide which is capable of conferring on a strain of EPEC or EHEC lacking a functional eae gene the ability to colonise and/or form A/E (attachment/effacement lesions) on an intestinal in vitro organ culture, for example from a human. The intimin polypeptide may be such a polypeptide, or a fragment of such a polypeptide.

It is preferred that the intimin polypeptide is capable of binding to a Tir polypeptide in a yeast two hybrid screen and/or a gel overlay assay.

The properties of an intimin polypeptide which does not have all of the domains present in a full-length intimin may be assessed by (1) determining the sequence of the intimin polypeptide, for example using techniques of protein chemistry or from information concerning its production, (2) obtaining (for example using techniques of molecular biology, as well known to those skilled in the art) a polynucleotide encoding the intimin polypeptide fused with one or more portions of a wild-type intimin (for example intimin-α; preferably the wild-type intimin from which the intimin polypeptide under consideration is most closely related) so as to form a polypeptide having all of the domains present in a full-length intimin, (3) determining whether expression of the polypeptide having all of the domains present in a full-length intimin does or does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to induce colonic hypeφlasia in a mouse. Techniques by which this may be done are described in the Examples. Colonic hypeφlasia is present if, for example, the weight of the distal 6cm of the colon 12 days after oral challenge is significantly greater for mice challenged with Citrobacter rodentium lacking a functional eae gene but expressing the test polypeptide than for mice challenged with otherwise equivalent Citrobacter rodentium lacking a functional eae gene, as described in the Examples. The C. rodentium lacking a functional eae gene may be strain DBS255, as discussed in the Examples.

Of course, the intimin polypeptide may also be assessed by comparison of its sequence with that of detoxified intimin polypeptides as described herein, as will be apparent to those skilled in the art.

The intimin polypeptide may differ from a wild-type intimin polypeptide (or fragment thereof) in that it is mutated at one or a limited number of residues (for example up to 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 residues) relative to the wild-type polypeptide. The wild-type polypeptide may be intimin-α, intimin-β, intimin-γ, intimin-δ or intimin-ε, preferably intimin-α or intimin- β or intimin-γ.

Thus, one (or more) amino acid residues of a wild-type intimin polypeptide may be replaced with an alternative amino acid residue. Alternatively, one or more amino acid residues (for example one, two, three, four, five or six residues) may be inserted or deleted. It is strongly preferred that the mutation is within the C-terminal 280 amino acids of full-length intimin (termed Int280), still more preferably within the C-terminal 190 or 188 amino acids of full-length intimin (termed Intl90 or Intl88), still more preferably within the amino acids forming the C-type lectin domain (CTLD) ie the C-terminal domain within Int280, formed by residues 183-280 of Int280.

The mutation may be a mutation (preferably a substitution (as opposed to deletion or insertion)) of a residue that is considered to form part of the Tir binding site, most preferably a solvent-exposed residue. Suφrisingly, we have found that mutation of a residue that is considered to form part of the Tir binding site may usefully modify the properties of the intimin, without preventing the mutated intimin polypeptide from being useful in raising a protective immune response, or binding to Tir.

The ability of a mutated intimin to raise a protective immune response may be checked by methods that will be apparent to those skilled in the art. Suitable methods are described in the Examples.

As described in Batchelor et al (2000) and GB 0013115.1 filed on 31 May 2000, residues of the Tir binding site are identifiable by comparing the 1H- ¹⁵N HSQC spectrum of an intimin molecule with and without saturating amounts of the Tir55 peptide; amide resonances which move and/or broaden in the presence of the Tir55 peptide are indicative of intimin residues involved in binding Tir.

Recently, 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). Analysis of line-widths and chemical shifts for Intl88 amide resonances in the presence of the Tir55 peptide facilitated mapping of the Tir-binding surface. Amides, which exhibit large chemical shift changes and/or line broadening in the presence of Tir55, reveal residues likely to be directly involved in binding. A number of amide resonances move or broaden whilst the majority of the spectrum remains unchanged, which is indicative of a specific complex between Intl88 and Tir55. The affected residues are concentrated within a region located in the C-terminal domain, D4. Residues involved include Y140, K142, 1147, 1148, S149, W150, T154, Q156, D157, A158, V162, A163, S164, T165, K170, Q171, N176, 1177, S180, E181, N183, A184, Y185, T187, and V189. For clarity these positions are numbered according to Intl90 and marked by asterisks in the sequence alignment figure (Figure 8). Principally, this region is located at the tip of the structure and forms a long thin surface that is centred on the upper, solvent exposed surface of the second β-sheet (strands C, D and E). This putative binding site is highly localised, covering an area on intimin that measures approximately 20 A by 8A. The highly localised nature of this region with the three-dimensional structure suggests that the portion of Tir in contact with intimin is likely to be shorter than the 55 amino acids currently proposed. Based on the structure, we hypothesise that the number of amino acid residues of Tir in contact with intimin may be as short as 5, if the conformation is completely extended, or as many as 14 if helical.

Thus, the Tir binding site is considered to comprise, or consist of, one or more, or all, of the following residues of Intl90; Y140, Y142, 1147, 1148, S149, W150, T154, Q156, D157, A158, N162, A163, S164, T165, K170, Q171, W176, 1177, S180, E181, Ν183, A184, Y185, T187, and N189; or the corresponding residues of another intimin molecule. Addition of 90 to each of these numbers gives the corresponding position in Int280. Addition of a further 659 gives the corresponding position in full length intimin-α.

It is preferred that the intimin is mutated at the residue equivalent to Y140, K142, 1147, 1148, S149, W150, T154, Q156, D157, A158, V162, A163, S164, T165, K170, Q171, N176, 1177, S180, E181, N183, A184, Y185, T187, and/or V189 (numbering of Intl90).

It is preferred that the mutated residue is not the residue equivalent to W899 of full-length intimin-α (W150 of Intl90; W240 of Int 280). W150 is not a solvent-exposed residue; it is buried. As discussed in Batchelor et al (2000) and GB 0013115.1 filed on 31 May 2000, it is located just below surface residues involved in binding and may therefore provide a structural scaffold for the Tir binding site. Mutation of this residue may decrease the ability of the intimin polypeptide to provoke a protective immune response in an individual. It may disrupt the Tir binding site and/or surrounding region of an intimin polypeptide to too great an extent. Replacement of W899 with alanine did not affect surface expression of intimin-α but resulted in adherent bacteria that are unable to initiate host cytoskeletal rearrangements. In contrast to the fusion protein MBP-Intl90, which showed a clear interaction with Tir-M in gel overlay experiments, no interaction could be detected with MBP-Intl90 or MBP-Int280 containing the modification (MBP-Intl90W150A and MBP-Int280W240A). For the yeast two-hybrid system DNA encoding Intl90W150A and Int280W240A did not support growth on the selective media when the bait was Tir. Measuring the β-galactosidase activities in these strains also revealed background levels. These experiments indicate that the mutated intimin polypeptides do not bind Tir. A further aspect of the invention provides an intimin polypeptide and polynucleotide encoding an intimin polypeptide wherein the residue equivalent to residue N252/911, T255/914 and/or 1237/897 of Int280α/full length intimin-α is mutated, preferably substituted, still more preferably substituted by an alanine residue.

The mutation may alternatively be a mutation (which may be a substitution, deletion or insertion) of a residue (or residues) that is considered to form part of a region bordering the Tir binding site, for example a solvent- exposed loop bordering the Tir binding site. Such a loop may be formed by residues 230 to 233 (numbering of Int280α) or equivalent residues of other intimins. This loop has the sequence YEYY in Intimin-α. In other intimins the residue equivalent to the "E" residue (for example) may be a different type of residue. Suφrisingly, we have found that mutation of a residue or residues that is considered to form part of a region bordering the Tir binding site, for example a solvent-exposed loop bordering the Tir binding site may usefully modify the properties of the intimin, without preventing the mutated intimin polypeptide from being useful in raising a protective immune response or binding to Tir.

It is particularly preferred that one or more, preferably all of the residues equivalent to residues 230 to 233 of Int280α (which have the sequence YEYY) are substituted by one or more alanine residues A or deleted. The properties of such mutants are indicated in Table 3. An intimin polypeptide in which residues 230 to 233 (Int280 numbering) are deleted is considered to be particularly useful as a vaccine component.

A further aspect of the invention provides an intimin polypeptide and polynucleotide encoding an intimin polypeptide wherein the residue equivalent to one or more, preferably all of the residues equivalent to residues 230 to 233 of Int280α are substituted, preferably by one or more alanine residues A or deleted.

The mutation may be a mutation of the C-terminal amino acid of Intimin (L939; numbering of full-length intiminα). The residue may be deleted or substituted. It is preferred that the C-terminus is not deleted by 3 amino acids or more, because this would delete one of the cysteine residues that is involved in formation of a disulphide bridge.

The mutation may be within the amino acids forming an Ig-like domain of intimin (residues 1 to 91 or 93 to 181 of Int280), as discussed in, for example, Batchelor et al (2000). It is preferred that the mutation is in the C- terminal Ig domain. It is particularly preferred that the mutation is of a residue equivalent to residue 120 of Int280α. This residue is a Tyr in Int280α. It may be mutated to, for example, an alanine residue.

It will be appreciated that the intimin polypeptide mutated at a position discussed above may also be mutated in other ways, for example by insertion, deletion, truncation or fusion, as known to those skilled in the art. As noted above, it may be preferred (for example when an isolated polypeptide, or polynucleotide encoding it, is to be administered to an individual) that the intimin polypeptide is a fragment of full-length intimin (or a fusion of such a fragment), for example Int280, Intl 90, Intl 88 or Intl 50. It is preferred that the intimin is not further mutated (in particular by amino acid substituion) in a way that may materially further affect the intimin polypeptide 's biological behaviour, for example its Tir binding activity (as judged by gel overlay or yeast-two hybrid assay) or cell binding activity, or its immunogenicity. It will be appreciated that the polynucleotide encoding the intimin polypeptide may therefore be mutated (compared with a sequence encoding wild-type intimin) at a position discussed above and may also be mutated in other ways in order to encode a variant intimin polypeptide, for example by insertion, deletion, substitution, truncation or fusion, as known to those skilled in the art. Preferred polynucleotides may thus be polypeptides derived from naturally occurring polynucleotides encoding intimin polypeptides. Examples of preferred polynucleotides are described in Example 1.

A "variant" will have a region which has at least 50% (preferably 60,70, 80,90, 95 or 99%) sequence identity with a wild-type 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. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). 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. 14:6745, 1986 as described by Schwarts and Dayhoff, eds, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

A preferred intimin "fragment" comprises all or for example 50, preferably 60, 75, or 100 amino acids ofthe intimin Tir binding domain sequence (as discussed in GB 0013115.1 and above), mutated as discussed above. Thus for Int280-α using the single letter code for amino acid designations, a preferred sequence is:

ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI EIVGTGVKGKLPTVWLQYGQVNLKASGGNGKYTWRSANPAIASVDASSGQV TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF GGKLPSSQNELENVFKAWGAANKYEYYKSSQTIISWVQQTAQDAKSGVAST YDLVKQNPLNNIKASESNAYATCVK

A preferred detoxified intimin polypeptide is

ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI EIVGTGVKGKLPTV LQAGQVNLKASGGNGKYTWRSANPAIASVDASSGQV TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF GGKLPSSQNELENVFKAWGAANKYEYYKSSQTIIS VQQTAQDAKSGVAST YDLVKQNPLNNIKASESNAYATCVK (residue 120 mutated to Ala) or

ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI EIVGTGVKGKLPTVWLQYGQVNLKASGGNGKYT RSANPAIASVDASSGQV TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF GGKLPSSQNELENVFKA GAANKYEYAKSSQTIISW VQQTAQDAKS GVASTYDLVK QNPLNNIKAS ESNAYATCVK (Y233 mutated to Ala) or

ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI EIVGTGVKGKLPTVWLQYGQVNLKASGGNGKYTWRSANPAIASVDASSGQV TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF GGKLPSSQNELENVFKAWGAANKYEYYKSSQTIISWVQQTAQDAKSGAAST YDLVKQNPLNNIKASESNAYATCVK (N252 mutated to Ala) or ITEIKADKTTAVAΝGQDAITYTVKVMKGDKPVSΝQEVTFTTTLGKLSΝSTE KTDTΝGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGΝI EIVGTGVKGKLPTVWLQYGQVΝLKASGGΝGKYTWRSAΝPAIASVDASSGQV TLKEKGTTTISVISSDΝQTATYTIATPΝSLIVPΝMSKRVTYΝDAVΝTCKΝF GGKLPSSQΝELEΝVFKAWGAAΝKYEYYKSSQTIISWVQQTAQDAKSGVASA YDLVKQNPLNNIKASESNAYATCVK (T255 mutated to Ala) or

ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI EIVGTGVKGKLPTVWLQYGQVNLKASGGNGKYTWRSANPAIASVDASSGQV TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF GGKLPSSQNELENVFKAWGAANKKSSQTIISWVQQTAQDAKSGVASTYDLV KQNPLNNIKASESNAYATCVK (deletion of"YEYY" loop) or ITEIKADKTTAVANGQDAITYTVKVMKGDKPVSNQEVTFTTTLGKLSNSTE

KTDTNGYAKVTLTSTTPGKSLVSARVSDVAVDVKAPEVEFFTTLTIDDGNI

EIVGTGVKGKLPTVWLQYGQVNLKASGGNGKYTWRSANPAIASVDASSGQV

TLKEKGTTTISVISSDNQTATYTIATPNSLIVPNMSKRVTYNDAVNTCKNF

GGKLPSSQNELENVFKAWGAANKYEYYKSSQTIISWVQQTAQDAKSGVAST

YDLVKQNPLNNIKASESNAYATCV

(deletion of K280)

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.

Techniques for additions, deletions or substitutions at predetermined amino acid sites having a known sequence are well known. Exemplary techniques include oligonucleotide-mediated site-directed mutagenesis and the polymerase chain reaction, for example as described in Example 1.

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. Techniques for the generation of intimin derivatives with differing biological activities using site-directed mutagenesis ofthe intimin C-terminal domain are described in Frankel et al (1998) Mol Microbiol 29(2), 559-540, the disclosure of which is incoφorated herein by reference.

As noted above, the intimin polypeptide may be a fusion polypeptide. For example, 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.

By "equivalent of W899 in full-length intiminα" (for example) is meant the amino acid residue that occupies a position in the native three dimensional structure of an intimin polypeptide corresponding to the position occupied by W899 in the native two or three dimensional structure of intiminα, for example as described in Batchelor et al (2000), Kelly et al (1999) or Luo et al (2000), and structures referred to in those papers. W899 is considered to lie beneath the Tir binding region of intiminα.

The comparison of amino acid sequences or three dimension structure (for example from crystallography or computer modelling based on a known structure) may be carried out using methods well known to the skilled man, as detailed below.

Structural features of intimin are discussed above and in the references cited above concerning the structure of intimin polypeptides, for example Batchelor et al (2000), Kelly et al (1999) or Luo et al (2000). The residue equivalent to W899 in full-length intiminα may be identified by alignment of the sequence of the intimin polypeptide with that of known intimins in such a way as to maximise the match between the sequences. The alignment may be carried out by visual inspection and/or by the use of suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, which will also allow the percent identity of the polypeptides to be calculated. The Align program (Pearson (1994) in: Methods in Molecular Biology, Computer Analysis of Sequence Data, Part II (Griffin, AM and Griffin, HG eds) pp 365-389, Humana Press, Clifton).

It will be appreciated that in the case of truncated forms of intimin or in forms where simple replacements of amino acids have occurred it is facile to identify the "equivalent residue".

By wild-type intimin-α, β, γ, δ or ε is included 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.

It may be useful to use a combination (including a concatemer or fusion polypeptide) of "detoxified" intimin polypeptides (or polynucleotide or polynucleotides encoding such a combination (including a concatemer or fusion polypeptide) in treating a human or animal with or at risk of bacterial infection, for example as described in the copending application entitled "BIOLOGICAL MATERIALS AND METHODS FOR USE IN THE PREVENTION OR TREATMENT OF INFECTIONS: I" filed on the same day as this application (incoφorated herein by reference). Intimin-α, β and γ are considered to be the intimins of greatest prevalence and medical relevance; hence the preference for inclusion of sequences derived from these intimins. Intimin-γ 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. For example, inclusion of sequences derived from intimin-γ and/or intimin- β and/or intimin-ε and possibly also intimin-α (but not necessarily intimin-δ) 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-α, intimin- β, intimin- γ, intimin-δ and/or intimin-ε may be appropriate when the medicament is for vaccination of human patients prior to foreign travel. Thus, preferred combinations of intimin-derived sequences correspond to combinations of intimins from which the target individual/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-α, intimin-β, intimin-γ and probably also intimin-ε.

The individual to be treated may be human, for example a human baby or infant or child or other human with or at risk of bacterial infection. Alternatively, the individual 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 animal may preferably be a young animal, for example a calf. If used to assess an intimin polypeptide or test polypeptide, it is preferred that intestinal in vitro organ culture is performed on intestine from the same species as the individual to be treated, for example a human. The intimin polypeptide (or test polypeptide) may (preferably) or may not (less preferably) confer on a strain of EPEC or EHEC lacking a functional eae gene the ability to colonise and/or form A/E (attachment/effacement lesions) on an intestinal in vitro organ culture, preferably a human intestinal in vitro organ culture. This may be relevant in determining whether the intimin polypeptide is likely to provoke a protective response even if the intimin polypeptide is to be used for treating animals, for example cattle.

It is preferred that the bacterial infection causes an histopathologic effect on intestinal epithelial cells, the effect being known as attachment and effacement (A/E).

Advantageously, the bacterial infection comprises infection by enteropathogenic E. coli (EPEC) and/or enterohemorrhagic E. coli (EHEC), and particularly E. coli O157:H7. Infection by other EHEC serotypes and shiga toxigenic E. coli (including human and bovine strains), Hafnia alvei and Citrobacter rodentium, as indicated above, are also included. 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 human or animal may further be administered (or 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 sequence or sequences.

The polypeptide may comprise more than one copy of an intimin sequence, for example an epitope-forming sequence. This may be useful in promoting an immune response, as well known to those skilled in the art.

The pharmaceutical composition, vaccine, food product or medicament may comprise further polypeptides or polynucleotides, as will be apparent to those skilled in the art. The intimin polypeptide or polynucleotide encoding the intimin polypeptide may, for example, be included in the medicament in the form of a recombinant organism or part thereof, preferably microorganism, preferably capable of expressing the intimin polypeptide (which in this case may preferably be a full-length intimin polypeptide or intimin polypeptide comprising a transmembrane domain), or alternatively capable of delivering nucleic acid encoding the polypeptide 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 organism. The recombinant organism may alternatively be a plant, for example making use ofthe teaching of WO97/40177.

The medicament 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 intimin polypeptide is intended as a target antigen, which is 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) a detoxified α-intimin derived polypeptide as defined above, (2) a detoxified β-intimin derived polypeptide as defined above, (3) a detoxified γ-intimin derived polypeptide as defined above, (4) a detoxified δ-intimin derived polypeptide as defined above, and (5) a detoxified ε-intimin derived polypeptide as defined above.

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.

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) ofthe invention.

A further aspect of the invention provides a chimaeric polypeptide, polynucleotide or recombinant microorganism, preferably bacterium, of the invention for use in medicine. A still further aspect of the invention provides the use of a chimaeric polypeptide, polynucleotide or recombinant bacterium 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 a recombinant microorganism, preferably bacterium, comprising a polynucleotide (for example a replicable vector) encoding an intimin polypeptide in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection, wherein the intimin polypeptide is as defined in relation to earlier aspects of the invention, for example the first aspect of the invention. Preferences for the intimin polypeptide are as indicated in relation to earlier aspects of the invention, but it may further be preferred that the intimin polypeptide is exposed on the surface of the microorganism, for example is located in or on an outer membrane of the microorganism. Preferences for the microorganism for use in a medicament, for example a vaccine, will be apparent to those skilled in the art and are discussed above.

A further aspect of the invention provides a recombinant microorganism, preferably bacterium, comprising a polynucleotide (for example a replicable vector) encoding an intimin polypeptide for use in medicine, wherein the intimin polypeptide is as defined in relation to preceding aspects of the invention.

It is preferred that the recombinant bacterium is not capable of expressing any other intimin polypeptide, for example is not capable of expressing a wild-type intimin polypeptide. A further aspect of the invention provides use of an intimin polypeptide, or recombinant polynucleotide encoding an intimin polypeptide, or a recombinant microorganism, preferably bacterium, comprising a polynucleotide (for example a replicable vector) encoding an intimin polypeptide, in the manufacture of a composition for use as a food supplement or a food additive, wherein the intimin polypeptide is as defined in relation to preceding aspects ofthe invention, for example the first aspect ofthe invention.

A further aspect of the invention provides the use of a chimaeric polypeptide, polynucleotide or recombinant bacterium of the invention in the manufacture of a composition for use as a food supplement or a food additive.

The food is preferably a milk substitute. Preferably, the food is suitable for administration to a human baby or infant or a young animal. However, it may be suitable for any human or animal which is susceptible to a bacterial infection, including older individuals. 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.

A further aspect of the invention provides the use of a peptidomimetic compound or compounds corresponding to the intimin polypeptide (detoxified intimin) as defined in relation to any of the preceding aspects 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 a method for treating a human or animal with or at risk of bacterial infection, the method comprising the step of administering to the human or animal a peptidomimetic compound or compounds corresponding to the intimin polypeptide (detoxified intimin) as defined in relation to any ofthe preceding aspects ofthe invention.

The term "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. For example, moφhine is a compound which can be orally administered, and which is a peptidomimetic ofthe peptide endoφhin.

Therapeutic applications involving peptides are limited, due to lack of oral bioavailability and to proteolytic degradation. Typically, for example, 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. This is likely to be due to poor transport across the intestinal membrane and rapid clearance from the blood by hepatic extraction and subsequent excretion into the intestine. These observations suggest that multiple amide bonds may interfere with oral bioavailability. It is thought that the peptide bonds linking the amino acid residues in the peptide chain may break apart when the peptide drug is orally administered. There are a number of different approaches to the design and synthesis of peptidomimetics. In one approach, such as disclosed by Sherman and Spatόla, J. Am. Chem. Soc, 112: 433 (1990), one or more amide bonds have been replaced in an essentially isoteric manner by a variety of chemical functional groups. This stepwise approach has met with some success in that active analogues have been obtained. In some instances, these analogues have been shown to possess longer biological half-lives than their naturally-occurring counteφarts. Nevertheless, this approach has limitations. Successful replacement of more than one amide bond has been rare. Consequently, the resulting analogues have remained susceptible to enzymatic inactivation elsewhere in the molecule. When replacing the peptide bond it is preferred that the new linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond.

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.

In another approach, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilised by a covalent modification, such as cyclisation or by incoφoration of γ-lactam or other types of bridges. See, eg. Veber et al, Proc. Natl. Acad. Sci. USA, 75:2636 (1978) and Thursell et al, Biochem. Biophys. Res. Comm., 111 :166 (1983).

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.

One approach to the synthesis of cyclic stabilised peptidomimetics is ring closing metathesis (RCM). This method involves steps of synthesising a peptide precursor and contacting it with a RCM catalyst to yield a conformationally restricted peptide. Suitable peptide precursors may contain two or more unsaturated C-C bonds. The method may be carried out using solid-phase-peptide-synthesis techniques. In this embodiment, the precursor, which is anchored to a solid support, is contacted with a RCM catalyst and the product is then cleaved from the solid support to yield a conformationally restricted peptide.

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. Such 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-butoxy carbonyl. 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 intimin polypeptide, for example a detoxified intimin fragment comprising regions contributing to the Tir binding site) 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 intimin polypeptide is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the intimin polypeptide, particularly an epitope-forming amino acid sequence (for example the Tir binding site or part thereof) forms a loop.

According to current immunological theories, a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. The polypeptide 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. 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.

Alternatively, several copies ofthe same or different epitope (for example two or more different detoxified intimin polypeptides) may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking. Suitable 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. (1979) 100, 100-108. For example, 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 incoφorates a disulphide bridge between the conjugated species. Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.

Further useful 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.

If the 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.

It may be desirable for the polynucleotide to 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 polypeptide (ie detoxified intimin polypeptide) in the human or animal. The polypeptide (or polypeptides, for example detoxified Int280α or Int280γ and Int280β), as appropriate, may be expressed from any suitable polynucleotide (genetic construct) as is described below and delivered to the patient. Typically, 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. Suitable promoters will be known to those skilled in the art, and may include promoters for ubiquitously expressed, 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). Preferably, 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.

"Operatively linked" refers to juxtaposition such that the normal function of the components can be performed. Thus, 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. For example, 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 retroviruses, adenoviruses, adeno-associated viruses, pox viruses and bacterial plasmids. Retroviral vectors are retroviruses 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-stranded DNA Virus. Suitable adenoviral vectors are described in Rosenfeld et al, Science, 1991, Vol. 252, page 432. Adeno-associated viruses (AAV) belong to the parvo virus family and consist of a single strand DNA or 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, for example antigen presenting cells, 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 ofthe 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). In addition, targeting vectors may comprise a tissue- or tumour-selective promoter which directs expression ofthe antigen at a suitable place.

Although the genetic construct can be DNA or RNA it is preferred if it is DNA.

Preferably, the genetic construct is adapted for delivery to a human cell.

Means and methods of introducing a genetic construct into a cell in or removed from an animal body are known in the art. For example, the constructs of the invention may be introduced into the cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the (dividing) cell. Targeted retroviruses are available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing 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.

Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653). Other methods of delivery include adenoviruses carrying external DNA via an antibody- polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transferrin- polycation conjugates as carriers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methods 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 poly lysine.

Bacterial delivery methods which may be suitable are described in Dietrich (2000) Antisense Nucleic Acid Drug Delivery 10, 391-399. For example, attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces. Whereas 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). This strategy allows a direct delivery of DNA to professional antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), through bacterial infection. 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 localisations of the bacterial carriers may be suitable for successful delivery of DNA vaccine vectors ofthe 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.

Bacterial delivery is a preferred method of delivery in relation to the present invention.

The DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.

In the second of these methods, 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 poly cations 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). When 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.

"Naked DNA" and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the patient to be treated. 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. Mutant adenoviruses which replicate selectively in p53-deficient human tumour cells, such as those described in Bischoff et al (1996) Science 21 A, 373-376 are also useful for delivering the genetic construct of the invention to a cell. Thus, it will be appreciated that a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention. Other suitable viruses or virus-like particles include HSV, AAV, vaccinia, lentivirus and parvo virus.

Immunoliposomes (antibody-directed liposomes) 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). For the preparation of immuno-liposomes MPB-PE (N-[4-(p-maleimidophenyl)butyryl]- phosphatidylethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288. MPB-PE is incoφorated 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 μm and 0.2 μm 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.

It will be appreciated that it may be desirable to be able to regulate temporally expression of the polypeptide(s) (for example antigenic polypeptides) in the cell. Thus, it may be desirable that 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 patient 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. It will be appreciated that this may be of particular benefit if 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. Examples of 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, cytokines, cytostatics, irradiation, heat shock and associated responsive elements). 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. Thus, 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 ofthe genetic construct.

The genetic constructs of the invention can be prepared using methods well known in the art.

The aforementioned therapeutic molecules, for example antigenic molecule, for example a chimaeric molecule or construct of the invention or a formulation thereof, 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. It will be appreciated that an inducer, for example small molecule inducer as discussed above may preferably be administered orally.

Methods of delivering genetic constructs, for example adenoviral vector constructs to cells of a recipient will be well known to those skilled in the art. In particular, 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 (vaccine) may be given to a recipient who is being treated for the disease by some other method. Thus, although 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.

Whilst it is possible for a therapeutic molecule as described herein, for example an antigenic molecule, construct or chimaeric polypeptide, to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. 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. Typically, 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 immungenicity of the composition, for example an adjuvant and/or a cytokine.

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 ofthe 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 the 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 ofthe 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.

It should be understood that in addition to the ingredients particularly mentioned above the 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.

It will be appreciated that the therapeutic molecule can be delivered to the locus by any means appropriate for localised administration of a drug. For example, 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 incoφorated 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. In a preferred embodiment, 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 Coφ., Parsippany, NJ, under the tradename Pluronic^R.

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 or polynucleotide or recombinant microorganism (or chimaeric polypeptide or peptidomimetic compound) as defined in relation to previous aspects ofthe invention.

Conveniently, 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. Other 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 proprietory 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.

A further aspect of the invention provides a pharmaceutical composition comprising a polypeptide, polynucleotide or recombinant microorganism as defined in relation to previous aspect of the invention (for example the first aspect of the invention), vaccine or chimaeric polypeptide or peptidomimetic compound ofthe invention, and a pharmaceutically acceptable carrier.

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 an intimin polypeptide as defined in relation to previous aspects of the invention, for example the first aspect ofthe invention.

Preferences in relation to the polypeptide and polynucleotide are as indicated in relation to preceding aspects ofthe invention.

The subject may be administered two or more detoxified intimin polypeptides. Preferably the detoxified intimin polypeptides are from at least two of the groups of α, β, γ, δ and ε intimins. Preferably the subject is administered an γ intimin, a β intimin and optionally further an α intimin (including fragments thereof). It is preferred that the polypeptide(s) comprise 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 subject an effective amount of a chimaeric polypeptide, peptidomimetic compound, recombinant microorganism or polynucleotide ofthe invention.

By "effective amount" we include the meaning that sufficient quantities of the agent are provided to produce a desired pharmaceutical effect beneficial to the health ofthe recipient.

All documents referred to herein are, for the avoidance of doubt, hereby incoφorated by reference. The invention is now described by reference to the following, non-limiting, figures and examples.

Figure 1: Schematic representation of the structure of D2 and D3 of Int280 comprising the Intl 90 super-domain (Batchelor et al (2000)). The residues selected for mutagenesis V252 (911) and 1237 (897) lie at opposite ends of the Tir-binding site and T255 (914) is located centrally within Tir-binding region. Numbers by the amino acid residue represent positions in Int280 and numbers in brackets are in the full-length intimin.

Figure 2: Detection of Int280-Tir interactions using the yeast two-hybrid sustem. β-galactosidase assays showing a 6.4 to 10 fold increase in enzymatic activity in strains co-expressing the whole Tir polypeptide and the intact Int280 or Int280 mutants compared with yeast strains expressing either Int280 or Tir only.

Figure 3: Detection of Int280-Tir interactions using gel overlays. Western blots of Tir-M overlayed with the different MBP-Int280 derviatives were developed with a rabbit MBP antiserum. Similar levels of MBP-Ibt280 (wild type) (lane 1), MBP-Int280ι₂₃₇/89_7A (lane 2) and MBP-Int280v25₂/9-i_A (lane 3) did not react with the immobilised Tir-M.

Figure 4: Intimin fluorescence (A, C, E, G, I) and FAS test (B, D, F, H, J) of CVD206(pCVD438) derivatives. Infected Hep-2 cells were stained 3 h post-infection. Similar intensity of staining was seen for all the CVD206(pCVD438) derivatives: A CVD206(pCVD438); E CVD206(pICC83); G CVD206(pICC84) and I CVD206(pICC85). No staining was observed with CVD206, C. All the recombinant strains, but not CVD206 (D), produced FAS positive reaction (arrows): B CVD206(pCVD438); F CVD206(pICC83); H CVD206(pICC84) and J (CVD206 (pICC85).

Figure 5: Western blot of CVD206(ρCVD438)-derivative whole cell lysates. The nitrocellulose membrane was probed with the Int280α antiserum. Full-length intimin was detected only in strains harbouring the pCVD438 derivatives. Lane 1, CVD206(pCVD438); lane 2, CVD206; lane 3, CVD206 (pICC83); lane4, CVD206(pICC84); lane 5, CVD206(pICC85).

Figure 6: Scanning electron microscopy (SEM) showing CVD206(pCVD438) (A), CVD206(pICC83) (B) and CVD206(pICC85) (C) adhering to small intestinal explants with typical SEM appearance of A/E lesion; CVD206(pICC84) did not colonise the small intestinal tissue (D). Distinctive appearance of dome-shaped follicle associated epithelium of the Peyer's patch (uninfected) is presented in E and CVD206(pICC84) colonising and inducing A/E lesions on the follicle-associated epithelium is shown in F.

Figure 7: A. Virulence of C. rodentium strains expressing mutated intimin molecules. The data depicts the number of C. rodentium recovered from colonic tissue of individual mice orally infected 12 days previously. Mice infected with DBS255(pCVD438) and DBS255(pICC83) had equivalent pathogen burdens. In contrast, mice infected with DBS255(pICC84) and DBS255(pICC85) had significantly lower bacterial loads (P<0.05). B. The distal 6 cm of the colon was weighed 12 days post-challenge. The weights of colons from mice infected with DBS255(pCVD438) and DBS255(pICC83) were significantly greater than colons from mice infected with DBS255, DBS255(pICC84) and DBS255(pICC85) (PO.05). Figure 8: Sequence alignment and topology for Intl 88 and invasin. The approximate location of secondary structure elements is also indicated; helices are delineated as open tubes and β-strands as black arrows. The amino acid positions are shown for Intl 90 from EPEC O127:H6 and correspond to residues 750-939 in full-length intimin. The residues highlighted represent identity and conservation with the EPEC O127:H6 sequence. Intimins from E. coli O127:H6, E. coli O26.Η and E. coli O157:H7 have been immunologically categorised into types α, β and γ, respectively. The two invasin sequences, Yersinia pseudotuberculosis and Yersinia enterocolitica, are aligned on the basis of the structural superimposition provided by DALI. Asterisks represent amide resonances that are significantly perturbed upon the addition of Tir55 peptide.

Example 1: Site directed mutagenesis of intimin α modulates intimin- mediated tissue tropism and host specificity

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching & effacing" lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor binding activity of intimin is localised to the C-terminal 280 amino acids and at least five distinct intimin types (α, β, γ, δ and ε) have been identified thus far. In addition to binding to Tir, intimin can also bind to a receptor encoded by the host. The consequence of this interaction may determine tissue tropism and host specificity. In this study we targeted three amino acids in intimin, implicated in Tir binding, for site directed mutagenesis. We have used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and mouse models). We report that while an I897A substitution in intimin α did not have any effect on its biological activity, T914A substitution attenuated intimin activity in vivo. In contrast, the mutation V911A affected tissue targeting in the human intestinal explants model and attenuated the biological activity of intimin in the mouse model. This study provides first clues as to the molecular basis of intimin-mediated tissue tropism and host specificity.

Material and Methods Bacterial strains and plasmids

Bacterial strains used in this study include wild-type EPEC strain E2348/69 (O127:H6) (Levine et al, 1985), eae deletion mutants of E2348/69, strain CVD206 (Donnenberg and Kaper, 1991) and C. rodentium, strain DBS255 (Schauer and Falkow, 1993b) and E. coli XLl-Blue and BL21. Bacterial strains were grown in L-broth. Media were supplemented with 50 μg/ml kanamycin, 30 μg/ml chloramphenicol or 100 μg/ml ampicillin where appropriate. The plasmids are listed in Table 1. Site directed mutagenesis

Site-directed mutagenesis was performed using the QuickChange Site- directed mutagenesis kit (Stratagene) following manufacturer's instructions, using the pCVD438-encoding intimin α (Donnenberg and Kaper, 1991) vector as template. Complimentary mutagenesis oligonucleotide pairs incoφorating single amino acid substitutions are as follows; Sense oligonucleotides:

5'-CTAGTCAGACTGCTATTTCATGGGTAC-3* (pICC83), 5'-GAAGAGTGGTGCTGCAAGTACATACG-3* (pICC84), 5'-GTGTTGCAAGTGCTTACGATTTAG-3' (pICC85) Antisense oligonucleotides;

5'-GTACCCATGAAATAGCAGTCTGACTAG-3* (pICC83), 5'-CGTATGTACTTGCAGCACCACTCTTC-3* (pICC84), 5'-CTAAATCGTAAGCACTTGCAACAC-3' (pICC85). Mutated plasmid containing staggered nicks was generated by extension of primers annealed to opposite strands of the denatured plasmid by temperature cycling (1 cycle of 95°C, 30 sec, then 16 cycles of 95°C, 30 sec, 55°C, 1 min, 68°C, 18 min) in the presence of the high fidelity Pfu DNA polymerase. Synthesised DNA containing the desired mutation was selected from the original DNA template by incubation with Dpnlat 37°C for 1 h, on the basis that dam methylated parental DNA template would be susceptible to digestion whereas the newly synthesised unmethylated mutated plasmid would not. Nicks in the plasmid were repaired following transformation of 1 μl of the synthesised products into competent E. coli XLl-Blue cells. Chloramphenicol-resistant transformants were randomly selected and inoculated to overnight L-broths for preparation of plasmid mini-preps (Qiagen). Correct incoφoration of each mutation was monitored by DNA sequencing using an automated DNA sequencer. The mutated plasmids were then transformed into an eae deletion mutant of EPEC, strain CVD206 (Donnenberg & Kaper (1991)).

The mutagenised pCVD438-derivative plasmids were then used as templates to amplify by PCR the DNA fragment encoding the mutated Int280 regions using one set of primers (forward primer 5'- GGAATTCATTACTGAGATTAAGGCT-3'; reverse primer 5'- CGGGATCCTTATTTTACACAAGTGGC-3'). Following digestion with EcoRllBamHi the DNA fragments were sub-cloned in either the yeast two hybrid vector pGAD424 or into pMAL-c2. Yeast two hybrid system pGAD424-encoding Int280α-derivative plasmids (carrying the GAL4 activation domain, AD) were transformed, individually or with pICCIO (pGBT9-encoding Tit and carrying the GAL4 binding domain, BD), into the yeast two-hybrid host PJ69-4A (MATa trpl-901 leu2-3,112 urα3-52 his 3- 200 gαl4D gαl80D LYS2::GAL1-HIS3 GAL2-ADE1 met2::GAL7-lαcZ), which confers the advantage of three independent reporter genes under the control of three different GAL promoters (James et αl, 1996). They were initially selected for the plasmid encoded TRP1 and LEU2 genes. The resulting transformants were then replica plated onto 3-aminotriazole containing medium to select for the HIS3 reporter, and onto SC minus Tφ, Leu, Ade to select for the ADE2 reporter. The function of LacZ reporter was quantified in cell extracts by assaying for β-galactosidase activity using o-nitrophenyl β-D-galactopyranoside as a substrate (Hartland et αl, 1999; Miller, 1972). Gel overlays

Purified His-Tir-M, expressed from pICC26 in E. coli BL21, was purified as described (Hartland et αl., 1999). MBP-Int280 derivatives were expressed from the pMal-c2 derivatives and purified from E. coli XLl-Blue as described (Frankel et α , 1994). His-Tir-M was separated by SDS- PAGE, blotted onto a nitrocellulose membrane which was blocked with 10% skim-milk in PBS, 0.1% Tween-20 overnight. The nitrocellulose membranes were reacted with 5 μg/ml of the purified MBP-Int280 fusions or MBP in PBS, 0.1% Tween-20 for 2 h and washed twice for 5 min in PBS, 0.1% Tween-20. MBP-Int-280 fusion proteins binding to Tir were detected with anti- MBP antiserum (1:2,000 for 1 h) and then anti-rabbit antibodies conjugated to alkaline phosphatase (1:2,000 for 1 h) as described (Hartland et αl., 1999). FAS assays, detection of surface intimin expression and Western blots

The ability of intimin derivative strains to induce A/E lesion formation was assessed using the fluorescent actin stain (FAS) test developed by Knutton et al. (Knutton et al, 1989). Briefly, HEp2 cells were grown to approximately 80% confluency on coverslips in 24-well plates. Cells were infected with 10 μl of static overnight L-broth cultures in medium lacking antibiotic for 3 h. The monolayers were then washed with PBS, fixed by the addition of 300 μl 10% formalin for 20 min, washed again and then permeabilised with 1% Triton-X 100 for 4 minutes. Filamentous actin was subsequently stained with 5 μl fiuorescein isothiocyanate labelled phalloidin in PBS (0.1 μg/ml) for 20 min (FAS test), and viewed by fluorescence microscopy following extensive washes with PBS.

Infected cultures were similarly fixed then probed for detection of EPEC associated intimin with anti-Int280 polyclonal antiserum (1:50 dilution) for 45 min in DMEM. Coverslips were mounted on slides following incubation with anti-rabbit FITC labelled secondary antibody (1:250 dilution) for 30 min, and labelled intimin visualised by fluorescence microscopy as described (Adu -Bobie et al, 1998; Frankel et al, 1998a).

Expression of the intimin derivatives was also determined by Western blotting. Briefly, stationary L-broth cultures were diluted 1 :100 in DMEM and incubated for 3 h at 37°C. An equivalent of an optical density (600) (OD600) of 0.5 was loaded onto 7.5 % SDS-polyacrylamide gel electrophoresis as described. The electrophoresed polypeptides were transferred to a nitrocellulose membrane and immunodetection of intimin was performed using anti-Int280, diluted 1 :500 as described (Adu -Bobie et al, 1998; Frankel et al, 1998a). IVOC adhesion assay

Tissue was obtained with fully informed parental consent and ethical approval using grasp biopsy forceps during routine endoscopic (Olympus PCF paediatric endoscope) investigation of intestinal disorders. Duodenal, ileal and terminal ileal Peyer's patch tissue was taken from patients from areas showing no endoscopic abnormality. Light microscopy subsequently showed no histological abnormality. IVOC infection was performed as described previously (Hicks et al, 1998; Phillips and Frankel, 2000). The assay was tenninated at 8 h. Each bacterial strain was examined in IVOC on several occasions using tissue from different children. Challenge of mice with C. rodentium Challenge of mice with C. rodentium Female specific pathogen free C3H/Hej mice (6-8 week old) were purchased from Harlan Olac (Bichester, United Kingdom). All mice were housed in individual ventilated cages with free access to food and water. Bacterial inoculums were prepared by culturing bacteria overnight at 37°C in 10 ml of L-broth containing nalidixic acid (100 μg/ml) plus chloramphenicol (50 μg/ml). 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 unanaesthetised mice orally inoculated with 200 μl of the bacterial suspension using a gavage needle. The viable count of the inoculum was determined by retrospective plating on L-agar containing appropriate antibiotics.

Measurement of pathogen burden

Mice were killed twelve days post-infection by cervical dislocation. The distal 6 cm of colon was removed and weighed after removal of faecal pellets. Colons were then homogenised mechanically using a Seward 80 stomacher (London, England) and the number of viable bacteria in colonic homogenates determined by viable count on L-agar containing appropriate antibiotics.

Results

Site directed mutagenesis of intimin residues

The intimin-binding site of Tir has been localised to a central region (Tir-M) located between two putative membrane spanning helices (de Grado et al, 1999; Hartland et al, 1999; Kenny, 1999). Analysis of intimin-Tir complexes revealed intimin residues likely to be directly involved in binding (Batchelor et al, 2000; Luo et al, 2000). These residues are concentrated within a solvent exposed region located in the CTLD. In this study we selected three residues in this region, 1237/897, V252/911 and T255/914 (positions numbered according to Int280α/whole intimin α) (Fig. 1), for site directed mutagenesis to alanine. The effect of the mutagenesis on the biological activity of intimin was investigated using protein binding assays as well as in vitro, ex vivo and in vivo infection models. Int280-Tir protein interaction: the yeast two hybrid system and gel overlays In a previous report we have demonstrated Int280α-Tir interaction using both the yeast two-hybrid system, designed to identify protein-protein interactions through the functional restoration of the yeast GAL4 transcriptional activator in vivo (James et al, 1996), and gel overlays (Hartland et al, 1999). In this study we expressed the mutations, individually, from a recombinant pGAD424 yeast two-hybrid system vector encoding Int280, plasmids pICC77 (Int280τ_237A), pICC78 (Int280v_252A) and pICC79 (Int280_T255A) (Table 1). We previously reported that in the yeast two-hybrid system, the interaction of Int280 (pICC19) with the whole Tir polypeptide is more efficient than the interaction of Int280 with Tir-M (Hartland et al, 1999). Accordingly, the DNA fragment encoding the whole Tir polypeptide, expressed from the second yeast two-hybrid system vector, pGBT9 (pICCIO) (Hartland et al, 1999), was used to determine the effect of the mutagenesis on Int280-Tir protein interaction.

Plasmid pICCIO was co-transformed with each of the different pGAD424- based plasmids into a derivative of the yeast stain PJ69-4A selected previously as a reporter for intimin-Tir interaction (Hartland et al, 1999). Replica plating these colonies onto selective media yielded vigorously growing colonies, and hence a positive two-hybrid phenotype, in yeast strains expressing both Tir and Int280 (pICCIO and pICC19), Tir and Int280_I237A (pICCIO and pICC77), Tir and Int280_V252A (pICCIO and pICC78) and Tir and Int280_T255A (pICCIO and pICC79). No yeast colonies were observed using single plasmid transformants (data not shown and Table 2).

The function of the non-selective reporter, lacZ, was also assessed in these strains by measuring β-galactosidase activities (Fig. 2). The host (data not shown) or single plasmid-bearing strains exhibited low levels of β- galactosidase activity, whereas the strains expressing Int280 or Int280 mutants and Tir showed 6.4 to 10 fold induction of β-galactosidase units.

In order to investigate the effect ofthe mutagenesis on Int280-Tir interaction using gel overlays, the single amino acid substitutions were also expressed from a recombinant pMal-c2 vector encoding maltose binding protein (MBP)-Int280 fusion, plasmids pICC80 (Int280_I237A), pICC81 (Int280_{V25 A}) and ρICC82 (Int280_T255A) (Table 1). The MBP-Int280 fusion proteins were purified by affinity chromatography (Frankel et al, 1994) and used together with purified Tir-M in a gel overlay binding assay. This revealed that while MBP-Int280, MBP-Int280_I237A and MBP-Int280_T255A bound to the immobilised Tir-M, no binding was observed with MBP-Int280_V252A or MBP alone (Fig. 3; Table 2). These findings are consistent with the results of the yeast two-hybrid system for Int280, Int280_{I23 A} and Int280τ₂₅₅v₅ but are inconsistent for Int280_V252A- As the gel overlay binding assay probed for interactions between Int280 and denatured Tir-M, this suggests that binding of Int280_V25_2A to Tir is conformational dependent.

The effect of the mutagenesis on intimin-mediated A/E lesion formation in vitro In order to determine the effect of the mutagenesis on intimin-mediated A/E lesion formation on HEp-2 cells, the mutations were introduced, individually, into pCVD438, a recombinant pACYC184 vector harbouring the eae gene encoding intimin α of EPEC (Donnenberg and Kaper, 1991), generating plasmids pICC83 (intimin_I897A), ρICC84 (intimin_V9iι_A) and pICC85 (intimin_{T91 A}) (positions numbered according to intimin α) (Table 1). The mutant pCVD438-derivatives were introduced into the EPEC strain CVD206, which harbours a null deletion in eae (Donnenberg and Kaper, 1991). The mutant strains were then subjected to a number of biological assays designed to determine the influence of the mutations on intimin function in vitro.

Experiments were conducted to confirm that the mutations introduced into the C-terminus of intimin did not affect surface expression of the intimin polypeptide. Indirect immuno-fluorescence staining, using polyclonal antiserum raised in rabbits against Int280α, showed that after a 3 h incubation with HEp-2 cells all the adhering CVD206(pCVD438)- derivatives, but not CVD206 (Fig. 4), expressed intimin. Intimin expression was also determined by Western blot analysis of whole cell lysates prepared from the different CVD206(pCVD438)-derivatives. Again, lysates from all the CVD206(ρCVD438)-derivatives, but not from CVD206, reacted similarly with the antiserum (Fig. 5).

Since expression of intimin was not detectably affected by introduction of the site-directed mutations, the ability of the different CVD206(pCVD438) derivatives to induce actin polymerisation in infected HEp-2 cells (FAS test; (Knutton et al, 1989)), a marker for A/E lesion formation, was determined. All the recombinant strains, but not CVD206, produced a FAS positive reaction, although CVD206(pICC84) consistently produced a weaker FAS staining (Fig. 4, Table 2). However, as FAS is not a quantitative measure of the interaction and is usually considered to provide a yes or no answer, these results show that the mutations, when expressed on the surface of an EPEC bacterium, did not affect the ability ofthe strain to cause A/E lesions in vitro.

Interaction of CVD206(pCVD438)-derivatives with human intestinal in vitro organ cultures (IVOC)

Human IVOC has been used previously to study EPEC pathogenesis. In particular Knutton et al. (Knutton et al, 1987) used this system to show that a human EPEC isolate induces A/E lesions on adult duodenal mucosa which were indistinguishable from those seen in vivo. Hicks et al. (Hicks et al, 1998) showed that A/E formation by EPEC in IVOC is an intimin-dependent event. More recently, we have shown that different intimin types targeted adhesion of EPEC to different regions of the human gut; i. e. while strains expressing intimin α efficiently colonised any part of the small intestinal mucosa and inefficiently colonic explants, strains expressing intimin γ showed restricted tropism to the follicle associated epithelium of the Peyer's patches (Phillips and Frankel, 2000). Normal tissue obtained from the distal duodenum and terminal ileum of children was examined after infection with the different CVD206(pCVD438)-derivatives to investigate the effect of the site directed mutagenesis on intimin-mediated mucosal attachment and A/E lesion formation using an ex vivo infection model. Examination of the infected biopsies by scanning electron microscopy revealed that CVD206(pCVD438), CVD206(pICC83) and CVD206(pICC85) colonised and formed A/E lesions on the small intestinal mucosal explants (Fig. 6; Table 2). In contrast, CVD206(pICC84) did not colonise the small intestinal biopsies (Fig. 6; Table 2). Subsequently, the ability of CVD206(pICC84) to colonise mucosal surfaces of the follicle-associated epithelium of the Peyer's patch was investigated. We found that like the CVD206(pCVD438) control, CVD206(pICC84) colonised the tissue efficiently and induced A/E lesions (Fig. 6, Table 2). These results show that substitution of Val911 with alanine in intimin α was coupled with conversion of the function to an intimin γ-like activity.

Colonic colonisation of mice challenged with DBS255(pCVD438)- derivatives The absence of convenient small animal models to study EPEC directly has made the study of pathogen/host interactions problematic. In this case, conclusions about EPEC (and EHEC) need to be drawn from studies of other pathogens which colonise via A/E lesion formation. Citrobacter rodentium causes transmissible colonic hypeφlasia in mice (Barthold et al, 1976), an infection associated with the formation of A/E lesions similar to those described for human EPEC (Schauer and Falkow, 1993a). C. rodentium has been shown to encode an eae homologue that directs the expression of intimin β (Adu-Bobie et al, 1998), a protein that is essential for A E lesion formation and infection of mice (Schauer and Falkow, 1993b). Moreover, expression of intimin α from EPEC (Frankel et al, 1996b; Higgins et al, 1999b), but not intimin γ from EHEC (Hartland et al, 2000), restored the ability of a C. rodentium eae deletion mutant, strain DBS255, to colonise the colon of orally challenged C3H/Hej. This model provides an opportunity to evaluate the in vivo biological properties of EPEC intimin mutants in mice.

Mice were challenged orally with DBS255 alone, DBS255 harbouring pCVD438 or with DBS255 harbouring the different pCVD438-derivatives. Twelve days post-challenge, measurement of pathogen burden in the colons of these mice revealed marked differences in the ability of each strain to colonise the colonic epithelium. Mice infected with either DBS255(pCVD438) or DBS255(pICC83) had equivalently high numbers of challenge bacteria in their colons (Fig Table 2), and induced colonic hypeφlasia as measured by colonic weight (Fig. 7A). In contrast, mice infected with DBS255(pICC84) or DBS255(pICC85) had significantly fewer challenge bacteria in their colons (Fig.7A ) and had colonic weights similar to those of mice challenged with the avirulent strain DBS255 (Fig. 7B). Collectively, these data imply that residues V911 and T914 of intimin are important for either initiation or maintenance of C. rodentium colonisation ofthe colonic epithelia.

Discussion

The first gene to be associated with A/E activity was eae encoding the intimate EPEC and EHEC adhesin, intimin (Jerse et al, 1990). Intimin exists as at least five antigenically distinct subtypes that have been named intimin α, β, γ, δ and ε (Adu-Bobie et al, 1998; Oswald et al, 2000). EPEC/EHEC intimins exhibit homology at their amino-termini to the invasin polypeptides of Yersinia (Isberg et al, 1987) and like Yersinia invasin (Leong et al, 1990), intimin harbours receptor binding activity at the C-terminus of the polypeptide (Frankel et al, 1994). 76-amino acid motifs , enclosed by a disulphide bridge between two cysteines, lie within the C-terminal domains of intimin and invasin. This is absolutely required for invasin binding to βl integrin (Leong et al, 1993) and for intimin binding to the host cell and βl integrin, A/E lesion formation and colonisation of mucosal surfaces (Frankel et al, 1995; Frankel et al, 1996a; Frankel et al, 1998a; Hicks et al, 1998; Higgins et al, 1999a; Higgins et al, 1999b). However, this disulphide bridge is not required for intimin-Tir interaction (Hartland et al, 1999).

Recently we have determined the global fold of the C-terminal 280 amino acids of intimin α (Int280α) by a combination of perdueteration, site- specific protonation and multidimensional nuclear magnetic resonance (NMR) (Kelly et al, 1999). The structure shows that Int280α is approximately 90 A in length and is built from three globular domains. Modelling other intimin types (including the EHEC intimin γ) (Adu-Bobie et al, 1998; Yu and Kaper, 1992) would suggest they have similar structures, and define a new family of bacterial adhesion molecules. Despite this we have recently demonstrated significant differences in the biological activities between intimin α and intimin γ. We have shown that while strains expressing intimin α, either in EPEC or EHEC backgrounds, colonise and induce A/E lesion on all human small intestinal explants, strains expressing intimin γ, in both EPEC and EHEC, are restricted in their tropism to follicle associated epithelium of the Peyer's patches (Phillips and Frankel, 2000; Phillips et al, 2000). Moreover, C. rodentium expressing either intimin β or intimin α from EPEC colonise and induce A/E lesions and colonic hypeφlasia in the mouse model (Frankel et al, 1996b; Higgins et al, 1999b), while C. rodentium expressing intimin γ from EHEC were found incapable of colonising the mouse colon (Hartland et al, 2000), similarly to an eae negative C. rodentium strain (Schauer and Falkow, 1993b). The molecular basis for these differences is not defined.

The Tir-binding site of intimin comprises a 20 A by 8A surface-exposed patch within the C-terminal 100 amino acids that lies at the tip of the intimin structure and is presented for interaction with Tir. Recently the structure of the intimin-Tir complexes was determined by NMR (Kelly et al, 1999) and crystallography (Luo et al, 2000). These studied identified a number of Int280 residues involved in Tir binding, include Y230, K232, 1237, 1238, S239, W240, T244, Q246, D247, A248, V252, A253, S254, T255, K260, Q261, N266, 1267, S270, E271, N273, A274, Y275, T277, and V279. In this study, three solvent expose residues, 1237/897, V252/911 and T255/914 were replaced with alanine and the outcome of the mutagenesis on the biological activity of intimin was determined using intimin Tir- binding assays and in vitro, ex vivo and in vivo infection models.

Residues 1237/897, V252/911 and T255/914 lay in exposed environments within the binding site and are therefore ideally positioned to make key contacts with Tir (Kelly et al, 1999; Luo et al, 2000). The hydrophobic residues V252/911 and 1237/897 lie at opposite ends of the Tir-binding site: V252/911 at the top and 1237/897 at the bottom (Fig. 1). They both make important hydrophobic contributions to the binding of Tir; V252/911 contacts the side-chains of two lysine residues (314 and 298) and 1237/897 interacts with 1301 within Tir. Despite T255/914 lying centrally within Tir binding region, it is not in direct contact with Tir but, based on NMR titration studies, shows an altered environment. Using the yeast two-hybrid system to investigate Int280-Tir interaction we found that the three site-directed Int280 mutants bound Tir at a similar level to the native Int280. In contrast, the gel overlay assay revealed that while Int280i_237A and Int280_T255A bound to the immobilised Tir-M, Int280_v252 showed an impaired Tir binding activity. The reason for the difference is not know, although it may suggest that the mutation in V252 restricted binding of Int280 to folded Tir-M only. Interestingly, Liu et al (Liu et al, 1999) reported recently on a fortuitous PCR-generated alanine substitution for V906 in intimin γ, the equivalent of V911 in intimin α reported here, that disrupted Tir binding activity. In their paper Liu et al described results with this mutation only in the context of an MBP fusion protein hence making any direct comparison with our study difficult. Nevertheless, it is possible that the apparent inconsistency is due to the different intimin type studied.

Mutating the V911 residue in the whole intimin did not abolish A/E lesion activity on HEp-2 cells, supporting the results of the yeast two-hybrid system. Moreover, CVD206(pICC84) was still capable of colonising and inducing A/E lesions on the human intestinal explants, although adhesion was restricted to the follicle associated epithelium of the Peyer's patch. Significantly, this kind of restricted tissue tropism is one of the major characteristics of intimin γ (Phillips and Frankel, 2000) In addition, like intimin γ (Hartland et al, 2000), expressing pICC84 in the C. rodentium eae knockout background did not restore mouse virulence. Hence, by altering only a single amino acid in intimin α we were able to generate an intimin γ- like molecule. This suggests that the V911A substitution might affect the structural integrity of the binding site or that this residue is involved, in addition to Tir binding, in interaction, either directly or indirectly, with a receptor encoded by the host. This putative receptor might be present on the plasma membrane of the epithelial cell itself or be associated with either the mucus or glycocalyx layers. This putative host cell receptor might be a protein or, based on the similarity of Int280 D3 to CTLD, it could be a carbohydrate. Indeed, we have recently reported that sugars extracted from human milk are capable of blocking Int280 binding to HEp-2 cells (Sarney et al, 2000).

Unlike V252, substitution of 1237 or T255 did not affect binding of Int280 to Tir in both the yeast two hybrid system or gel overlay. Moreover, expressing the mutations in CVD206 did not effect A/E lesion formation on HEp-2 cells or tropism to the small intestinal explants. The fact that all three mutations were capable of mediating A/E lesion formation on Hep-2 cells and the human intestinal explants implies that, individually, the alterations are not sufficient to disrupt Tir binding. Because the intimin-Tir interaction is intrinsically rather weak (micromolar binding constant (Luo et al (2000))), perhaps individual binding contributions are not large enough for activity to be removed when they are altered.

Substitution of the 1897 in the whole intimin, when expressed in DBS255, resulted in intimin capable of restoring mouse virulence to

DBS255(pICC82). In contrast, BDS255(pICC85) was avirulent.

Importantly, we have reported before that deleting the last amino acid

(L939) resulted in an intimin α derivative capable of mediated A/E lesion formation on HEp-2 and IVOC when expressed in CVD206 but incapable of restoring mouse virulence when expressed in DBS255 (Frankel et al,

1998a). These results show that these single amino acid mutations might interfere in binding to a mouse encoded intimin receptor. In summary, our results show that by altering single residues in intimin α we were able to segregate different intimin functions. One of the mutations (V252/V911A) had a more global effect (mainly seen in the ex vivo and in vivo models). In contrast, the I237/I897A mutation had no effect in any of the models and the T255/914A mutation did not affect binding to the human explants but was inactive in the mouse. This study not only provides an additional layer of evidence for intimin-mediated tissue and host specificity, but also strengthens the concept that intimin binds to both Tir and a host encoded receptor. Further mutagenesis studies, combined with a range of binding assays and infection models, are required to unravel the molecular basis for these distinct functions of intimin.

Table 1. List of plasmids used. Plasmid Description Reference pGBT9 A yeast GAL4 DNA-BD Clontech cloning vector pICCIO PGBT9 expressing Tir (Hartland et al, 1999) pGAD424 A yeast GAL4 DNA- AD Clontech cloning vector pICC19 pGAD424 expressing Int280α (Hartland et al, 1999) pICC77 ρGAD424 expressing Int280i237A TS¹ pICC78 ρGAD424 expressing Int280_V252A TS pICC79 pGAD424 expressing Int280_T25_5A TS pICC22 pMal encoding MBP-Int280α (Frankel et al, 199 A) pICC80 pMal encoding MBP-Int280_I237A TS pICC81 pMal encoding MBP-Int280_V252 TS pICC82 pMal encoding MBP-Int280_T255A TS pICC18 pET28a encoding Tir-M (Hartland et al, 1999) pCVD438 pACYC184 encoding intimin α (Donnenberg and

Kaper, 1991) ρICC83 pACYC 184 encoding intimin α_I237 TS ρICC84 pACYCl 84 encoding intimin α_V252A TS ρICC85 pACYCl 84 encoding intimin α_T255A TS

!TS = this study

Table 2. Summary of the effect of site directed mutagenesis of intimin on binding and biological activities.

lumbers in brackets are total number of positive over total number of incubations.

²ND = not done.

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Nataro, J. P., and Kaper, J. B. (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11: 142-201. Oswald, E., Schmidt, H, Morabito, S., Karch, H, Marches, O., and Caprioli, A. (2000) Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect Immun. 68: 64-71. Perna, N. T., Mayhew, G. F., Posfai, G., Elliott, S., Donnenberg, M. S., Kaper, J. B., and Blattner, F.R. (1998) Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli 0151 :W. Infect Immun 66: 3810-3817.

Phillips, A. D., and Frankel, G. (2000) Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures. J Infect Dis 181: 1496-500.

Phillips, A. D., Navabpor, S., Hicks, S., Dougan, G., Wallis, T., and Frankel, G. (2000) Enterohaemorrhagic Escherichia coli O157:H7 targets Peyer's patches in man and causes attaching-effacing lesions in both human and bovine intestine. Gut 47: 377-381.

Robins-Browne, R. M., Tokhi, A. M., Adams, L. M., Bennett- Wood, V., Moisidis, A. V., Krejany, E. O., and O'Gorman, L. E. (1994) Adherence characteristics of attaching and effacing strains of Escherichia coli from rabbits. Infect Immun 62: 1584-1592. Sarney, D. B., Hale, C, Frankel, G., and Vulfson, E. N. (2000) A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration. Biotechnol Bioeng 20: 461-467.

Schauer, D. B., and Falkow, S. (1993a) Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hypeφlasia. Infect Immun 61: 2486-2492.

Schauer, D. B., and Falkow, S. (1993b) The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hypeφlasia. Infect Immun 61: 4654-61. Tzipori, S., Gunzer, F., Donnenberg, M. S., de Montigny, L., Kaper, J. B., and Donohue-Rolfe, A. (1995) The role of the eaeA gene in diarrhea and neurological complications in a gnotobiotic piglet model of enterohemorrhagic Escherichia coli infection. Infect Immun 63: 3621-3627.

Tzipori, S., Wachsmuth, I. K., Chapman, C, Birden, R., Brittingham, X, Jackson, C, and Hogg, J. (1986) The pathogenesis of hemorrhagic colitis caused by Escherichia coli O157:H7 in gnotobiotic piglets. J Infect Dis 154: 112-11A.

Ulshen, M. H, and Rollo, J. L. (1980) Pathogenesis of Escherichia coli gastroenteritis in man—another mechanism. N Engl J Med 302: 99- 101.

Yu, X, and Kaper, J.B. (1992) Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 6: 411-417.

Example 2: Further site-directed mutagenesis of intimin α

Table 3 shows the results of further mutagenesis of intimin α. Mutagenesis and investigation of the properties of the mutated polypeptides was performed as described in Example 1.

Table 3. Summary of the effect of site directed mutagenesis of intimin on binding and biological activities.

Δ loop* = deletion ofthe 4 amino acids loop (YEYY) -** = No bugs, some changes in the tissue. +*** Intermediate colonisation no hypeφlasia.

lumbers are position in Int280

Example 3: Raising an antibody response in a patient

Active immunisation of a patient is preferred. In this approach, one or more detoxified intimin polypeptides, for example comprising in combination detoxified polypeptide sequences of two or more of intimin types α, β, γ, δ and ε (or further intimin types) are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the patient. It is preferred that the polypeptide or polypeptides comprise a mutated Tir binding site or a mutated neighbouring solvent exposed loop, as discussed above, derived from one or more intimin polypeptides. It is preferred that the patient is administered two or more of detoxified Int280α, Int280β, Int280γ, Int280δ and Int280ε.

By polypeptides is included peptidomimmetic molecules, fusion polypeptides containing intimin peptides or full length intimin or chimaeric polypeptides of the invention. Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Lscoms" 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.

Alternatively, as discussed above, a DNA vaccine may be administered.

Suitable formulations and methods for preparing same will be apparent to those skilled in the art, and are summarised in, for example, PCT/GBOO/00254.

Preferred formulations include those suitable for oral administration, including topical oral administration, intranasal (mucosal) administration and parenteral administration, including intramuscular or subcutaneous injection.

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 Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35-40°C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals. Example B : Intramuscular inj ection

Active ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glucofurol 75® 1.45 g Water for Injection q.s. to 3.00 ml

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). Example C: Syrup Suspension

Active ingredient 0.2500 g

Sorbitol Solution 1.5000 g

Glycerol 2.0000 g

Dispersible Cellulose 0.0750 g

Sodium Benzoate 0.0050 g

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In 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 ofthe suspension.

Example D: Suppository mg/suppository Active ingredient (63 :m)* 250 Hard Fat, BP (Witepsol HI 5 - Dynamit Nobel) 1770

2020

*The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm 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 μm 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 stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm 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 ofthe mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

Example E: Pessaries mg/pessary

Active ingredient 250

Anhydrate Dextrose 380

Potato Starch 363

Magnesium Stearate 7

1000

The above ingredients are mixed directly and pessaries prepared by direct compression ofthe resulting mixture.

Use in medicine

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) ofthe recipient (which may be an animal, and which may have no symptoms of disease) and/or the state ofthe particular bacterial disease against which the treatment (which may be prophylactic treatment) is directed. Diagnosis of disease

The 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.

Prevention of disease

The agents of the invention may find particular utility in the prevention of bacterial infections. For example, the agents can be administered to humans or animals at particular risk of exposure to bacterial infections. Such risks may arise when an individual or group of humans or animals is likely to, or has already, come into contact with a source of infection, for example an affected individual.

It will be appreciated that the agents of the invention can be used to treat humans and animals.

Example 4: Assessing conferral of protective immunity This example describes methods by which conferral of protective immunity may be assessed, for example in mice.

Materials and Methods Mice Female, specific pathogen free C3H/Hej mice (6-8 weeks old) are purchased from Harlan Olac (Bichester, United Kingdom). All mice are housed in individual ventilated cages with free access to food and water.

Bacterial strains Suitable test and comparison strains are described in Example 1.

Immunisation and oral infection of mice

For intranasal (i.n.) immunisations, groups of mice are lightly anaesthetised with gaseous halothane and 30 μl of Ag in PBS applied to the nasal nares. Mice are i.n. immunised on day 0, 14 and 28 and orally challenged between days 42-44. For subcutaneous (s.c.) immunisation, groups of mice (n=5 or 6) are injected s.c. on the left side of the abdomen with 150 μl of Ag mixture in PBS. As per i.n. immunisation, mice are s.c. immunised on 0, 14 and 28 and orally challenged between day 42-44. Bacterial inoculums are prepared by culturing bacteria overnight at 37°C in L-broth containing nali dixie acid (100 μg/ml) (C. rodentium) or L-broth containing nalidixic acid (lOOμg/ml) plus chloramphenicol (50μg/ml) (DBS255(pCVD438) or DBS255 carrying another plasmid, as discussed in Example 1). After incubation, bacteria are harvested by centrifugation and resuspended in an equal volume of PBS. A 1/10 dilution of bacteria in PBS is then prepared and mice orally inoculated, without anaesthetic, using a gavage needle with 200μl of the bacterial suspension. The viable count of the inoculum is determined by retrospective plating on L-agar containing appropriate antibiotics.

Enterotoxins and recombinant proteins

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 Int280α, which represents the C-terminal 280 amino acids of intimin (Int_660-939) from EPEC strain E2348/69, is purified as described previously [Kelly, 1998]. Int_388-667, which corresponds to two putative Ig-like domains upstream of Int280, and Tir-M, the intimin-binding domain of Tir, are purified as poly-histidine tagged polypeptides as described, [Batchelor, 1999]. EspA is 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].

Measurement of pathogen burden

At selected time points post-infection, mice are killed by cardiac exsanguination under terminal anaesthesia or by cervical dislocation. Spleens, livers, mediastinal and caudal lymph nodes are then aseptically removed. The distal 6cm of the colon is also removed and the colon weighed after removal of faecal pellets. In some experiments, the distal 1cm of colon is removed for histological analysis. Spleens, livers, lymph nodes and colons are then homogenised mechanically using a Seward 80 stomacher (London, England) and the number of viable bacteria in organ homogenates determined by viable count.

Analysis of humoral immune responses

At selected times post-immunisation, 0.2 ml of blood is collected from the tail vein of immununised mice, sera collected and stored at 20°C until analysed. For analysis of antigen-specific antibody responses, wells of microtitre plates (Maxisorb plates, Nunc™) are coated overnight at 4°C with lOOμl of a bicarbonate solution (pH 9.6) containing Int280α (2.5μg/ml), EspA (1.5 μg/ml), EspB (1.5 μg/ml), Tir-M (1 μg/ml) or C. rodentium lysate (20μg/ml). After washing with PBS/Tween20, wells are blocked by addition of 1.5% (w/v) BSA in PBS for 1 h. Plates are then washed twice with PBS/Tween-20 before sera from individual mice was added and serially diluted in PBS/Tween-20 containing 0.2% (w/v) BSA and incubated for 2 h at 37°C. For the determination of IgA antibody titres, wells are washed with PBS/Tween-20 before addition of 100 μl of an IgA horseradish peroxidase (HRP) conjugate (Dako, Buckinghampshire, UK) diluted 1/1000 in PBS/Tween-20 containing 0.2% (w/v) BSA for 2 h at 37°C. For the determination of antigen-specific IgGl and IgG2a antibody titres in mouse sera, biotinylated rat mAbs against IgGl and IgG2a (Pharmingen, Hull, United Kingdom), used at concentrations previously shown to give equivalent optical densities when assayed against identical amounts of purified IgGl or IgG2a respectively, are used as secondary antibodies. After washing with PBS/Tween-20, a 1/1000 dilution of strepavidin-HRP is added for 2 h. Finally, after washing with PBS/Tween- 20, bound antibody is detected by addition of o-phenylenediamine substrate (Sigma) and the A₄₉₀ measured. Titres are determined arbitrarily as the reciprocal of the serum dilution corresponding to an optical density of 0.3. The minimum detectable titre is about 100.

Detection of intimin-specific T cell responses by ELISPOT Spleens from immunised mice (n=3) are aseptically removed and single cell suspensions from prepared by passing organs through 100 μm nylon sieves (Marathon Laboratories, London, UK). After lysis of splenic erythrocytes with Tris-ammonium chloride, a total of 10⁶ leukocytes are cultured in the presence of 1 μg/ml Int280α 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 μg/ml streptomycin (cRPMI) in triplicate for 24 h in wells of a 24 well plate (Costar). After incubation, cells are removed by gentle pippeting, washed twice with cRPMI and graded numbers of effector cells, consisting of cells which remained viable after the culture period, plated onto ELISPOT plates. ELISPOT plates are prepared as follows: nitrocellulose-based 96-well microtitre plates (Multiscreen-HA, Millipore, Hertfordshire, UK) are coated overnight at 4°C with 50 μl/well of either anti-IFN-γ (4 μg/ml) (R46A2) or anti-IL-4 (4 μg/ml)(HBl l) mAb diluted in carbonate buffer pH 9.6. After washing 3 times with filtered PBS, all wells are blocked with 200 μl of cRPMI for 2-3 h at 37°C. Following removal of the blocking media, threefold serial dilutions of spleen cells from individual mice are added to the wells in duplicate (maximum 5x10⁵ cells/well in 200μl of media) and incubated for 20 h at 37°C in 5% CO₂. Cells are removed by washing 3 times with PBS, followed by a further 3 times with PBS/Tween 20 (0.05% v/v), then 50 μl of the biotinylated anti- IFN-γ (XMG1.2) or anti-IL-4 (BVD6-24G2) antibodies (1 μg/ml in filtered PBS/Tween 20) is added to each well for 2 h. After washing plates 5 times with filtered PBS/Tween-20, a 1/1000 dilution of Extravadin-alkaline phosphatase (Sigma) is added to all wells for 1-2 h at room temperature. Finally, after washing 3 times with PBS/Tween-20 and once with PBS alone, a solution of 5-bromo-4 chloro-3 -indolyl phosphate/nitro blue tetiazolium (Fast BCIP/NBT; Sigma) is added as substrate. Spots, representing single IFN-γ or IL-4 producing cells are counted using a dissecting microscope. The number of mt280α-specific spot-forming cells is determined by subtracting the number of spots obtained with cells stimulated with media from those stimulated with Int280α.

Immunohistochemistry

In some challenge experiments, the distal 1cm of colon is removed, cut longitudinally and rolled, then snap frozen in liquid nitrogen. From frozen colonic tissue, 5μm thick, cryostat cut sections were mounted on poly-L- lysine coated glass microscope slides. Staining for bacterially expressed intiminα in frozen tissue is performed as previously described [Higgins, 1999]. Statistical analysis

The non-parametric Mann- Whitney test is employed for all statistical analysis.

Expected Results

Mice infected with recombinant C. rodentium mount immune responses to detoxified intimin antigens and develop acquired immunity

Sera from mice infected with C. rodentium expressing detoxified intimin-α cross-reacts with Int280α from EPEC strain E2348/69. Serum IgG antibody responses are detectable 2 weeks post-infection and are maximal 4-6 weeks post-infection. IgA responses to all antigens remained detectable 8 weeks post-infection.

Mice infected with recombinant C. rodentium develop acquired immunity

Two groups of C3H/Hej mice are orally infected with 7x10⁷ cfu of recombinant C. rodentium expressing detoxified intiminα or intiminβ. Three months later, one group of convalescent mice is re-challenged with 8x10⁸ cfu of wild-type C. rodentium (expressing intiminβ) and the second group with 2xl0⁹ cfu of a C. rodentium strain expressing α intimin (DBS255(ρCVD438)). Age and sex-matched naive mice are orally challenged in parallel with convalescent mice. Fourteen days after oral challenge the pathogen burden in mouse tissues is determined in all groups. Compared to naive animals, convalescent mice harbour significantly fewer challenge bacteria in colons and draining lymph nodes. Further, the colon weights of challenged mice, a good indicator of the degree of infection- driven pathology in the mucosa [Higgins, 1999], is substantially lower in convalescent mice compared to the naive animals. Mice infected with C. rodentium develop acquired immunity to re-infection with C. rodentium strains expressing either homologous or heterologous intimin types.

Induction of Int280α-specific immune responses using mucosal or parenteral immunisation strategies

A highly purified preparation of recombinant detoxified Int280α is used as an immunogen in mucosal and parenteral vaccination regimes. Mice are vaccinated intranasally or subcutaneously with or without the use of Escherichia coli heat-labile toxin (LT) or mutant derivatives as adjuvants.

Mice are s.c. immunised three times, on day 0, 14 and 28, with lOmg of Int280α with or without adjuvant. Mice immunised with detoxified Int280α, in the absence of adjuvant, mount serum IgGl and IgG2a but not IgA antibody responses to Int280α. The co-administration of LT or LTR72 with detoxified Int280α prompts a more rapid Ig response to Int280α, but does not, however, increase the magnitude of the final Int280α-specific IgGl or IgG2a titre compared to that obtained in mice s.c. immunised with detoxified Int280α alone. Suφrisingly, s.c. co-administration of LT or LTR72 with detoxified Int280α prompts a weak Int280α-specific serum IgA response, although this occurrs in only a small number of mice. Int280α-specific IgGl is the predominant IgG subclass elicited by parenteral vaccination, although the ratio of IgGl :IgG2a is reduced when detoxified Int280α is co-administered with the adjuvants LT or LTR72.

In mucosal immunisation regimes, mice are immunised three times, on day 0, 14 and 28, with lOmg of detoxified Int280α with or without an enterotoxin-based adjuvant. Mice i.n. administered lOmg of detoxified Int280α mount serum IgGl and IgG2a, but not IgA, antibody responses to Int280α. Co-delivery of lmg of LT, LTR72 or LTK63 with detoxified Int280α significantly increases the serum IgGl and IgG2a antibody response to Int280α. Moreover, the addition of a mucosal adjuvant results in the induction of Int280α-specific serum IgA responses. Analysis of Int280α-specific IgG subclasses in i.n. immunised mice shows a predominance of IgGl over IgG2a. As occurrs in s.c. immunised mice, the ratio of IgGl :IgG2a is reduced when detoxified Lnt280α is co-administered with an enterotoxin-based adjuvant.

^■n Collectively, these data show that detoxified Int280α is immunogenic in vivo and that enterotoxin-based adjuvants can modulate the kinetic and isotype ofthe elicited humoral immune response.

Vaccine-elicited T cell responses to Int280α The extent and type of Int280α-specific T cells elicited by selected immunisation strategies is evaluated using cytokine-specific ELISPOT. Int280α-specific T cell responses re compared in mice i.n. immunised with PBS, lmg of LTR72, lmg of LTR72 plus lOmg of detoxified Int280α or lOmg of detoxified Int280α alone. For comparison, Int280α-specific T cell responses are also assessed in mice s.c. immunised with lmg of LTR72 plus lOmg of detoxified Int280α or lOmg of detoxified Int280α alone. Mice are immunised on day 0, 14 and 28 and killed on day 42. Splenocytes from immunised mice are stimulated with media or 1 ug/ml of recombinant Int280α for 18 hrs before being washed and cultured in the absence of antigen for another 18 hrs on IL-4 or IFN-γ ELISPOT plates. ELISPOT plates are then developed and counted. In mice immunised with detoxified Int280α, irrespective of the vaccination route, there is a predominance of IFN-γ SFC's over IL-4 SFC's. Mice vaccinated i.n. three times with lOmg of detoxified Int280α alone have the highest number of IFN-γ SFC's. The ratio of IFN-γ SFC's over IL-4 SFC's is greatest in mice immunised s.c. with lmg of LTR72 plus lOmg of Int280α followed by mice immunised i.n. with lOmg of detoxified Int280α alone. Although these data suggest that vaccination with detoxified Int280α predominantly elicits T cells which produce IFN-γ upon antigenic stimulation, there are no marked relationships between a particular vaccination regime and the magnitude or type of T cell response elicited.

Efficacy of Int280α-based vaccination strategies for the prevention of C. rodentium colonisation in C3H/Hej mice

DBS255(pCVD438), a recombinant C. rodentium strain which only expresses intimin α, is virulent in mice and induces similar mucosal pathology in the distal colon as wild-type C. rodentium [Higgins, 1999]. To determine whether vaccination with detoxified Int280α could modulate the outcome of infection with DBS255(pCVD438), mice are i.n. or s.c. immunised three times, on day 0, 14 and 28, with lOmg of detoxified Int280α with or without adjuvant. In separate experiments, mice are orally challenged with between 2-3x10⁷ cfu of DBS255(pCVD438) 13 or 16 days after the last immunisation. Mice are killed 14 days post-challenge, the colon is weighed, homogenised and the pathogen burden determined by viable count. Mice immunised s.c. with PBS or adjuvant alone have uniformly high C. rodentium counts in the colon. In contrast, the colons of mice immunised s.c. with detoxified Int280α alone harboured significantly fewer challenge bacteria than the colons of naive or control animals. Suφrisingly, mice immunised with detoxified Int280α together with a mucosal adjuvant are more susceptible to colonic infection than mice which received detoxified Int280α alone. Similar results are obtained in i.n. immunised mice. Mice immunised i.n. with PBS or an adjuvant have uniformly high C. rodentium counts in the colon. The pathogen burden is reduced, however, if mice are immunised i.n. with detoxified Int280α alone. As occurrs in s.c. immunised animals, the addition of a mucosal adjuvant with detoxified Int280α negates some of the protective efficacy of i.n. vaccination using detoxified Int280α alone.

Susceptible mice infected with C. rodentium develop colitis and have colons which are heavier than those in age-matched uninfected control mice. Measurement of colon weights shows that animals immunised either s.c. or i.n. with detoxified Ϊnt280α alone are also more resistant to the colitis which develops during C. rodentium infection.

In selected groups of mucosally and parenterally immunised mice, the number of DBS255(pCVD438) present in the mediastinal lymph nodes and spleen is also determined. Compared to PBS immunised control mice, animals vaccinated s.c. or i.n. with detoxified Int280a alone have significantly fewer challenge bacteria in spleens and draining lymph nodes.

An appropriately administered detoxified Int280α-based vaccine modulates the severity of a C. rodentium infection. A vaccination strategy that limits pathogen colonisation may also modulate the extent of colitis in infected animals.

Efficacy of detoxified Int280α-based vaccination strategies for the prevention of C. rodentium infection in C3H Hej mice

The vaccine efficacy attained by s.c. immunisation with detoxified Int280α alone is verified in further experiments. Groups of C3H/Hej mice are vaccinated s.c. three times, two weeks apart, with lOmg of the irrelevant antigen ovalbumin (OVA), lOmg of detoxified Int280α or PBS. All mice are orally challenged with 3xl0⁷ cfu of DBS255(pCVD438) 14 days after the last immunisation. C3H/Hej mice which received lOmg of detoxified Int280α s.c. have significantly fewer challenge bacteria in colons, spleens and mediastinal lymph nodes compared to either PBS or OVA immunised mice. Such results confirm that s.c. vaccination with detoxified Int280α is efficacious in C3H/Hej mice.

Specificity of immunity elicited by detoxified Int280α vaccination A vaccine based on detoxified Int280α 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 are vaccinated s.c. three times with lOmg of detoxified Int280α or PBS then orally challenged with 1x10^s cfu of o DBS255(pCVD438) or 1x10 cfu of wild-type C. rodentium (expresses intimin β). Mice vaccinated with detoxified Int280α are more resistant to DBS255(pCVD438) infection (although this does not reach statistical significance), but not to wild-type C. rodentium infection. Mice vaccinated with PBS are susceptible to infection with both DBS255(pCVD438) and wild-type C. rodentium. Immunity elicited by detoxified Int280α vaccination is specific for this intimin type and does not offer cross- protection against strains bearing a heterologous intimin type.

Claims

1. A method of treating a human or animal with or at risk of bacterial infection, comprising the step of administering to the human or animal an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide, wherein the intimin polypeptide has the following properties: (a) it is capable of inducing in a human or animal an immune response which is protective against infection by a bacterial strain expressing a wild- type intimin polypeptide; and (b) it is either (1) a polypeptide which has the structural domains present in a wild-type intimin but does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to induce colonic hypeφlasia in a mouse, wherein the polypeptide is not a polypeptide comprising the cell- binding domain of wild-type intimin-γ, or (2) a fragment of such a polypeptide, or fusion of said fragment which does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to induce colonic hypeφlasia in a mouse, wherein the fusion of said fragment is not a polypeptide comprising the cell-binding domain of wild-type intimin-γ.

2. A food product comprising a foodstuff and an intimin polypeptide or recombinant polynucleotide as defined in claim 1.

3. A food product as claimed in Claim 2 wherein the food is adapted for consumption by animals.

4. A food product as claimed in claim 2 wherein the food is adapted for consumption by humans.

5. A vaccine effective against bacterial infection, for example EHEC and/or EPEC infection, comprising an effective amount of the intimin polypeptide or recombinant polynucleotide as defined in claim 1.

6. A pharmaceutical composition comprising an intimin polypeptide or recombinant polynucleotide as defined in claim 1 together with a pharmaceutically acceptable diluent or carrier, wherein the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein-fusion with the C-terminal 181 residues of intimin-γ in which Val906 is replaced by an alanine residue.

7. An intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide as defined in claim 1, wherein the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein- fusion with the C-terminal 181 residues of intimin-γ in which Val906 is replaced by an alanine residue.

8. Use of an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection, wherein the intimin polypeptide or recombinant polynucleotide is as defined in claim 1, 6 or 7.

9. An intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide for use in medicine, wherein the intimin polypeptide is as defined in claim 1, 6 or 7.

10. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use of any of the preceding claims wherein the intimin polypeptide further is either (1) a polypeptide which has the structural domains present in a wild-type intimin but does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to mediate intestinal colonisation in a mouse, or (2) a fragment of such a polypeptide, or fusion of said fragment which does not confer on a strain of Citrobacter rodentium lacking a functional eae gene the ability to mediate intestinal colonisation in a mouse.

11. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use of any of the preceding claims wherein the intimin polypeptide is capable of binding to a Tir polypeptide in a yeast two hybrid screen and/or a gel overlay assay.

12. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of the preceding claims wherein the intimin polypeptide is a polypeptide which is capable of conferring on a strain of EPEC or EHEC lacking a functional eae gene the ability to colonise and/or form A/E (attachment/effacement lesions) on an intestinal in vitro organ culture, or a fragment of such a polypeptide.

13. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of the preceding claims wherein the intimin polypeptide differs from a wild- type intimin polypeptide or fragment thereof in that it is mutated at one, two, three, four, five or up to 20 residues relative to the wild-type polypeptide.

14. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 13 wherein the wild-type intimin polypeptide is intimin-α, intimin-β, intimin-γ, intimin-δ or intimin-ε.

15. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 13 or

14 wherein the mutation is within the C-tenninal 280, 190 or 188 amino acids of full-length intimin.

16. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 15 wherein the mutation is within the amino acids forming the C-type lectin domain (CTLD) of intimin (residues 183-280 of Int280).

17. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of claims 13 to 16 wherein the mutation is of a solvent-exposed residue that forms part ofthe Tir binding site.

18. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of claims 13 to 17 wherein the intimin is mutated at the residue equivalent to Y140, K142, 1147, 1148, S149, W150, T154, Q156, D157, A158, V162, A163, S164, T165, K170, Q171, N176, 1177, S180, E181, N183, A184, Y185, T187, and/or V189 of Intl90α.

19. The method, foodstuff, vaccine, phannaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 18 wherein the intimin is mutated at the residue equivalent to 1147/237/897, VI 62/252/91 land/or T165/255/914 of Intl90α/Int280α/full length intimin- α.

20. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of claims 13 to 16 wherein the mutation is of a residue (or residues) that form part of a region bordering the Tir binding site.

21. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 20 wherein the mutation is of a residue (or residues) that form part of a solvent- exposed loop bordering the Tir binding site.

22. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 20 or

21 wherein the mutation is of a residue equivalent to residue 230, 231, 232 and/or 233 of Int280α.

23. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 22 wherein the said mutation is deletion or substitution by one or more alanine residues.

24. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of claims 13 to 16, 20 wherein the mutation is of the C-terminal amino acid of Intimin (L939; numbering of full-length intiminα).

25. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to any one of claims 13 to 15 wherein the mutation is within the amino acids forming an Ig-like domain of intimin (residues 1 to 91 or 93 to 181 of Int280).

26. The method, foodstuff, vaccine, pharmaceutical composition, intimin polypeptide or recombinant polynucleotide or use according to claim 25 wherein the mutation is of a residue equivalent to residue 120 of Int280α.

27. The method or use or vaccine of any one of claims 1, 5, 8, 10 to 26 wherein the bacterial infection causes an histopathologic effect on intestinal epithelial cells, known as attachment and effacement (A/E).

28. The method or use or vaccine of any one of claims 1, 5, 8, 10 to 27 wherein the bacterial infection comprises infection by one or more of eneteropathogenic E.coli (EPEC) and/or enterohemmorrhagic E.coli (EHEC), Shiga toxigenic 7i. co li, H.alvei, and C. rodentiumi.

29. The method or use or vaccine of any one of claims 1, 5, 8, 10 to 28 wherein the bacterial infection comprises E,coli 0157:H7.

30. A chimaeric polypeptide comprising or consisting of one or more copies of at least two of (1) a polypeptide as defined in any one of claims 1 to 29 derived from α-intimin, (2) a polypeptide as defined in any one of claims 1 to 29 derived from β-intimin, (3) a polypeptide as defined in any one of claims 1 to 29 derived from γ-intimin, (4) a polypeptide as defined in any one of claims 1 to 29 derived from δ-intimin, and (5) a polypeptide as defined in any one of claims 1 to 29 derived from ε-intimin.

31. A polynucleotide encoding a chimaeric polypeptide according to claim 30.

32. A recombinant microorganism, preferably bacterium, comprising a polynucleotide (for example a replicable vector) according to claim 31.

33. A chimaeric polypeptide, polynucleotide or recombinant microorganism, preferably bacterium, according to any one of claims 30 to 32 for use in medicine.

34. Use of a chimaeric polypeptide, polynucleotide or recombinant microorganism, preferably bacterium as defined in claim 33 in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection.

35. A pharmaceutical composition comprising a chimaeric polypeptide, polynucleotide or recombinant microorganism, preferably bacterium as defined in claim 33 together with a pharmaceutically acceptable diluent or carrier.

36. A vaccine effective against bacterial infection, for example EHEC and/or EPEC infection, comprising an effective amount of the chimaeric polypeptide, polynucleotide or recombinant microorganism, preferably bacterium as defined in claim 33.

37. Use of a recombinant microorganism comprising a polynucleotide encoding an intimin polypeptide as defined in any one of claims 1 to 29 in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection.

38. A recombinant microorganism comprising a polynucleotide encoding an intimin polypeptide as defined in any one of claims 1 to 29 for use in medicine.

39. A pharmaceutical composition comprising a recombinant microorganism comprising a polynucleotide encoding an intimin polypeptide as defined in any one of claims 1 to 29 together with a pharmaceutically acceptable diluent or carrier, wherein the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein-fusion with the C-terminal 181 residues of intimin- γ in which Val906 is replaced by an alanine residue.

40. A recombinant microorganism comprising a polynucleotide encoding an intimin polypeptide as defined in any one of claims 1 to 29, wherein the intimin polypeptide is not intimin-α lacking the C-terminal residue Lys939 or a maltose binding protein- fusion with the C-terminal 181 residues of intimin-γ in which Val906 is replaced by an alanine residue.

41. A vaccine effective against bacterial infection, for example EHEC and or EPEC infection, comprising an effective amount of a recombinant microorganism as defined in any one of claims 37 to 40.

42. Use of an intimin polypeptide, recombinant polynucleotide encoding an intimin polypeptide, chimaeric polypeptide, polynucleotide or recombinant microorganism as defined in any one of the preceding claims, in the manufacture of a composition for use as a food supplement or a food additive.

43. A food product comprising a foodstuff and a chimaeric polypeptide, polynucleotide or recombinant microorganism as defined in any one of the preceding claims.

44. A food product as claimed in claim 43 wherein the food is adapted for consumption by animals.

45. A food product as claimed in claim 43 wherein the food is adapted for consumption by humans.

46. The use according to claim 42 or food product according to claims 2, 3, 4, 43, 44 or 45 wherein the food is a milk substitute.

47. Use of a peptidomimetic compound or compounds corresponding to an intimin polypeptide or chimaeric polypeptide as defined in any one of the preceding claims in the manufacture of a medicament for the treatment of a human or animal with or at risk of bacterial infection.

48. A method for treating a human or animal with or at risk of bacterial infection, the method comprising the step of administering to the human or animal a peptidomimetic compound or compounds corresponding to the intimin polypeptide or chimaeric polypeptide as defined in any of the preceding claims.

49. A vaccine effective against bacterial infection, for example EHEC and/or EPEC, comprising an effective amount of the peptidomimetic compound or compounds as defined in claim 47 or 48.

50. A pharmaceutical composition comprising a peptidomimetic compound or compounds as defined in claim 47 or 48 and a pharmaceutically acceptable diluent or carrier.

51. A method of preventing and./or treating a bacterial disease comprising administering to a subject an effective amount of an intimin polypeptide or recombinant polynucleotide encoding an intimin polypeptide, chimaeric polypeptide, polynucleotide, recombinant microorganism, peptidomimetic compound, vaccine or pharmaceutical composition as defined in any of the preceding claims.