NZ509631A

NZ509631A - Peptide fragments of cholera toxin B or enterotoxin B as vaccine adjuvants

Info

Publication number: NZ509631A
Application number: NZ509631A
Authority: NZ
Inventors: Timothy Raymond Hirst; Neil Andrew Williams
Original assignee: Univ Bristol
Priority date: 1998-09-07
Filing date: 1999-09-07
Publication date: 2003-11-28
Also published as: EA200100314A1; MXPA01002387A; ZA200100758B; GB2357507A; GB0107375D0; JP2002524469A; AU5751699A; PL346549A1; CA2338384A1; NO20011075D0; GB9819484D0; IL141695A0; NO20011075L; CZ2001839A3; KR20010044842A; WO2000014114A1; CN1317013A; EP1109828A1; BR9913501A; HUP0104090A2

Abstract

The use of a peptide derived from CtxB or EtxB in the manufacture of a medicament for the treatment and/or prevention of a condition wherein the peptide: i) comprises the sequence of EVPGSQH; ii) has fewer than 40 amino acids; and iii) acts as an immunomodulator in the treatment and/or prevention of the condition. Also covered is a screening method for identifying an agent which is capable of interacting with said peptide, which involves contacting the peptide with a candidate agent and determining whether or not the candidate agent interacts with the peptide.

Description

<div class="application article clearfix" id="description"> 5096 31 WO 00/14114 JV/ J U J I PCT/GB99/02970 PEPTIDE FRAGMENTS OF CHOLERA TOXIN B OR ENTEROTOXIN B USEFUL AS VACCINE ADJUVANTS FIELD OF THE INVENTION The present invention relates to peptides consisting of the amino acid sequence of SEQ ID 5 No: 2, 3, 4 or 5 and functionally equivalent variants thereof. In particular, the present invention relates to a peptide that is capable of displaying one or more properties that are useful in medicine. 10 By way of example, the peptides disclosed are useful for use as an immunomodulator and/or an adjuvant and/or an inhibitor of toxin-induced diarrhoea. The peptides disclosed can be used as an immunomodulatory agent in the preparation of a medicament to modulate an immune response - such as that associated with an autoimmune disease. More particular, the present invention relates to the use of such peptides as an immunomodulator formulated for sequential or co-administration with a related or an unrelated antigen. 20 More in particular, the present invention relates to the use of such peptides in the preparation of a medicament for inhibiting toxin-induced diarrhoea. Disclosed is an assay for screening for agents capable of interacting with peptide of the present invention. 25 BACKGROUND OF THE INVENTION Escherichia coli (E. coli) heat labile enterotoxin (Etx) and its closely related homologue, cholera toxin (Ctx) from Vibrio cholerae, are examples of protein toxins 30 which bind to glycolipid receptors on host cell surfaces. Each toxin consists of six noncovalently linked polypeptide chains, including a single A subunit (27 kDa) and five identical B subunits (11.6 kDa) which bind to GM-1 ganglioside receptors found "Intellectual property OFHOF OF n.2 19 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 2 on the surfaces of mammalian cells (Nashar et al 1996 Proc Natl Acad Sci 93: 226-230). The A subunit is responsible for toxicity possessing adenosine diphosphate (ADP) ADP-ribosyltransferase activity, whereas the B subunits (EtxB and CtxB) are non-toxic oligomers which bind and cross-link a ubiquitous cell surface glycolipid 5 ganglioside, called GM-1, thus facilitating A subunit entry into the cell. In contrast to the poor immunogenicity of the A subunit alone, both EtxB and CtxB are exceptionally potent immunogens and their respective holotoxins, Etx and Ctx (which comprise the A and B subunits) are known to be potent adjuvants when given 10 orally in combination with unrelated antigens (Ruedl et al 1996 Vaccine 14: 792-798; Nashar et al 1993 Vaccine 11: 235; Nashar and Hirst 1995 Vaccine 13: 803; Elson and Ealding 1984 J Immunol 133: 2892; Lycke and Holmgren 1986 Immunology 59: 301). Because of their immunogenicity, both EtxB and CtxB have been used as carriers for other epitopes and antigens (Nashar et al 1993 ibid) and have been used as 15 components of vaccines against cholera and E.coli mediated diarrhoeal diseases (Jetbom etal 1992 Vaccine 10:130). Several studies have been carried out on the immunodominant epitope of the CtxB and EtxB subunits with a view to developing a vaccine against the cholera toxin and 20 heat labile E.coli toxin. By way of example, the following disclosures represent some of the work that has been carried out in this area. UK Patent Application No. 2 415 419A discloses a synthetic vaccine against cholera and against heat labile toxin of E. coli comprising a conjugate of a carrier with a 25 synthetic polypeptide corresponding to part of the sequence of CtxB. WO 85/02611 discloses synthetic polypeptides corresponding to particular sequences of EtxB which are deemed useful as conjugates or as an active ingredient to raise antibodies against the B subunit and for protecting a host animal against infection by 30 enterotoxins. WO 00/14114 PCT/GB99/02970 3 WO 89/10967 discloses an amino acid sequence which represents residue numbers 50-64 of the CtxB which can be used in combination with an epitopes of a heterologous organism, such as Flagellum and/or Salmonella, in vaccine formulations with a view to providing protection against infection by the heterologous organism or 5 to providing protection against conditions or disorders caused by an antigen of the organism. WO 90/03437 relates to a hybrid protein which fuses the CtxB subunit with the active sequence of a heterologous antigen which is deemed useful for vaccination purposes, 10 particularly to help the stabilisation of heterologous antigens in the intestinal environment. WO 94/06465 relates to amino acid fragments which are linked, either with or without a linker, to an appropriate carrier such that the amino acid fragment linked to the 15 carrier generates an opsonic or protective immune response to the epitopes of the fragment. W095/29701 discloses a vaccine against Vibrio cholera which comprises a conjugate of cholera toxin B subunit (CTB) or a synthetic fragment peptide which consists of a 20 portion thereof, such as peptide CTP 3 comprising the 50-64 amino acid sequence of the B chain linked to an inert carrier. W096/26282 discloses an expression systems for expressing gene products from recombinant Bordetella strains wherein the gene product may be a cholera toxin 25 molecule. W096/34893 discloses hybrid molecules between EtxB and CtxB which may be useful as a vaccine and to prevent and/or treat enterotoxin-induced illness in an individual. 30 WO 00/14114 PCT/GB99/02970 WO 98/21344 discloses an EtxB subunit which is modified to include an inserted antigenic peptide. The chimeric antigen-EtxB molecule is used to elicit an antibody response against an antigenic peptide in host animals. 5 These studies related to either the use of (i) a peptide comprising part of the sequence of CtxB/EtxB or (ii) a peptide comprising part of the sequence of CtxB/EtxB coupled to a second entity (such as an antigen) to induce and/or maximise the immunological response to that peptide. Accordingly, these documents relate to the use of an immunodominant epitope of CtxB/EtxB or parts thereof as an immunogen in inducing 10 an immunological response against these subunits with a view to developing immunity against cholera and/or E.coli mediated diarrhoea diseases. WO 91/07979 discloses a chimeric protein which includes a portion of CtxB and an epitope region of a desired antigen which are designed for use as a vaccine to elicit an immune response in a subject to a desired antigen. In this regard, the portion of CtxB is being used as 15 an adjuvant but not as an immunomodulator. Accordingly, none of the above cited documents relates to the use of a CtxB/EtxB peptide or part thereof as an immunotherapeutic capable of modulating the immune response. We have shown that the EtxB subunit is capable of acting as an immunomodulator in 20 immune disorders. By way of example, we have disclosed in WO 97/02045 that EtxB binds to GM-1 ganglioside receptors which are found on the surfaces of mammalian cells and that this binding induces differential effects on lymphocyte populations including a specific depletion of CD8+ T cells and an associated activation of B cells. These effects are absent when a mutant EtxB protein (G33D) (lacking GM-1 binding 25 activity) is employed. Consequently, these experimental results would suggest that all of the functionalities associated with EtxB and CtxB are attributable to the capacity of the EtxB and CtxB subunits to bind to the GM-1 receptor since mutants lacking the capacity to bind GM-1 (such as EtxB (G33D)) fail to act as adjuvants or immunomodulators. Thus, the prior art to date has suggested that immunomodulation 30 and other effects of Etx and Ctx are mediated through GM-1 binding. However, until now, no investigations have been carried out on CtxB/EtxB mutants which retain the capacity to bind to the GM-1 receptor but which lack an immunomodulatory effect. WO 00/14114 PCT/GB99/02970 (followed by page 5a) 5 We have suprisingly found that not all of the effects of Etx and Ctx are mediated through GM-1 binding. SUMMARY ASPECTS OF THE INVENTION 5 In accordance with the present invention we have now found that the immunomodulation and some other effects of Etx and Ctx are not mediated through GM-1 binding. jq In one aspect, the present invention provides the use of an agent in the manufacture of a medicament for the treatment and/or prevention of a condition, wherein the agent: (i) comprises the sequence of SEQ ID No. 2; (ii) has fewer than 40 amino acids; and (iii) acts an immunomodulator in the treatment and/or prevention of the condition. If In a further aspect, the present invention provides a peptide consisting of the amino acids sequence of SEQ ID No. 2, 3, 4 or 5. 20 hi a still further aspect, the present invention provides a peptide having fewer than 40 amino acids which comprises the sequence of SEQ ID No. 4 or 5. In another aspect, the present invention provides for the use of a peptide of the invention to identity mimetics which have an immunomodulatory effect. In yet another aspect, the present invention provides a screening method for identifying an agent which is capable of interacting with a peptide according to the present invention, which comprises the step of contacting the peptide with a candidate agent and determining whether or not the candidate agent interacts with the peptide. intellectual property ofrce of n.z 19 SEP 2003 RECEIVED 5a Described is a substance comprising any one or more of: an amino acid sequence comprising the sequence of SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB; but wherein the substance is not capable of exhibiting GM-1 binding activity. Also described is a substance comprising any one or more of: an amino acid sequence comprising the sequence of SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to loop of EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity. Preferably, the substance comprises any one or more of: an amino acid sequence comprising the sequence of SEQ ID No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as or is similar to the )34-a2 loop of EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity. intellectual property*"* qfrof of n.z 19 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 The substance 6 can be an amino acid sequence or a chemical derivative thereof. The substance may be a synthetic peptide or a synthetic peptide variation - such as a retroinverso D peptide. The substance may even be an organic compound or other chemical. The latter examples are example of mimetics of SEQ ID The peptide of the present invention is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB. 10 The term "same as or is similar to" is a qualitative term rather than a quantitative term. In this respect, it may be desirable to have an increased binding affinity. An assay for determining whether a substance is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB would be readily determinable to those 15 skilled in the art. For example, the assay may measure and/or determine an effect on cell populations, such as lymphocyte cell populations. These effects can include but are not limited to an induction of apotosis in CD8+ T cells , the enhanced activation of CD4+ T cells and the polyclonal activation of B cells. In addition, or in the alternative, the assay could be based on determining and/or measuring particular cell 20 surface marker(s) indicative of activation of certain intracellular events (e.g. meauring an increase in CD25 expression). The peptide of the present invention is not capable of exhibiting GM-1 binding activity. An assay for determining the lack of GM-1 binding activity would be readily determinable to those skilled in the art. For example, the assay may utilise GM-1 bound to a solid support and wherein the substance is then passed across the bound GM-1. Elution of the substance is indicative that it does not bind to GM-1. In a more 30 preferred aspect, the assay is that described in WO 97/02045. 5 No. 2. 25 intellectual property OFRCF- OP IM.z 19 SEP 2033 received OS WO 00/14114 PCT/GB99/02970 7 It is to be noted that the binding activity of the peptide is not necessarily dependent on a primary binding event as is found with full length Ctx and EtxB subunits. With full length Ctx and EtxB, the primary binding activity is GM-1 binding activity. In this regard, the substance may exhibit a single binding event. However, for some cases, the 5 substance may possess the capability of having more than one binding activity. Preferably, the peptide is substantially isolated and/or substantially pure. As used herein, the terms "isolated" and "purified" refer to molecules, either nucleic 10 or amino acid sequences, that are removed from their natural environment and/or isolated or separated from at least one other component with which they are naturally associated. A protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the peptide and still be regarded as substantially isolated. The present invention is based on the suprising finding that there are mutants which are capable of binding to the GM-l receptor but which lack an immunomodulatory effect. These mutants facilitate the elucidatation of the mechanism by which the B subunits of Ctx and/or Etx act, particularly vis-a-vis an immunomodulatory effect. 20 Other aspects of the present invention are as follows. A peptide according to the present invention employed as a medicinal agent and more particularly for use as an immunomodulator or as an adjuvant. A peptide according to the present invention is also useful as an inhibitor of toxin-induced diarrhoea. A peptide according to the present invention may be used as an agent in a medicament 30 wherein the medicament additionally comprises an antigen or an antigenic determinant. 15 25 19 SEP 2003 WO 00/14114 PCT/GB99/02970 8 Disclosed is a pharmaceutical composition comprising the peptide according to the present invention, optionally admixed with one or more pharmaceutically acceptable carrier(s), diluent(s) or excipient(s). One embodiment relates to the use of a peptide according to the present invention in the 5 manufacture of a medicament that is capable of treating and/or preventing and/or modulating a disease and/or condition associated with an immune disorder and/or a toxin mediated disorder. I Also described is an assay method for determining one or agents that are capable of 10 interacting with and/or affecting the peptide according to the present invention; wherein the assay comprises contacting the peptide with an agent to be tested, and then determining whether or not the agent affects the substance. 15 An agent identified by the assay method is also described. Also described is a method of treatment, comprising administering to a subject in need of treatment of and/or prevention of and/or modulation of a disease and/or condition associated with an immune disorder and/or a toxin mediated disorder a peptide according to the 20 present invention. These aspects are presented under separate section headings. However, it is to be understood that the teachings under each section heading are not necessarily limited to that particular section heading. Mrr^lBag . , i intellectual property I of=!op of n.z 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 9 IMMUNOMODULATOR As used herein, the terra "immunomodulator" means a substance that is capable of modulating the immune response by inducing, for example, a differential effect on 5 cells, such as lymphocyte cells - preferably leading to induction of apoptosis in CD8+ T cells and/or enhanced activation of CD4+ cells and/or the polyclonal activation fo B cells. The term "differential effect on leukocyte cells" may include but is not limited to a 10 specific depletion of CD8+ cells (through for example apotosis), the enhanced activation of CD4+ T cells and/or an associated activation of B cells. Preferably the immunomodulator is capable of downregulating the pathological response of Thl and/or Th2-associated immune responses. 15 Preferably the immunomodulator is capable of upregulating the production of antibodies at mucosal surfaces. ADJUVANT 20 As used herein, an "adjuvant" is a substance which non-specifically enhances the immune response to an antigen. It also includes any substance which is capable of affecting the extent of the immune 25 response to an entity such as an antigen and/or an antigenic determinant, by altering the antigenicity of the antigen or by altering the specific reactivity or the nonspecific effector associated mechanisms of the host such that an immune response is induced in a host cell and/or is guided in a particular direction. In one preferred aspect, the adjuvant is capable of acting as a mucosal adjuvant. 30 Preferably the adjuvant is capable of prolonging antigen presentation and providing a sustained immunologic memory in a mammalian subject. WO 00/14114 PCT/GB99/02970 10 ANTIGEN As used herein, an "antigen" means an entity which, when introduced into an immunocompetent host, stimulates the production of a specific antibody or antibodies 5 that can combine with the entity. The antigen may be a pure substance, a mixture of substance or soluble or particulate material (including cells or cell fragments). In this sense, the term includes any suitable antigenic determinant, auto-antigen, self-antigen, cross reacting antigen, alloantigen, xenoantigen, tolerogen, allergen, hapten, and immunogen, or parts thereof, as well as any combination thereof, and these terms are 10 used interchangeably throughout the text. An "allergen" includes any antigen that stimulates an allergic reaction, inducing a Type I hypersensitivity reaction. 15 Examples of common allergen sources include but are not limited to ragweed, rye, couch, wild oat, timothy, Bermuda, Kentucky blue, mugwortalder, birch, hazel, beech, Cupressae, oak, olive, Aspergillus spp., Cladosporium spp., Alternaria spp., Basidospores, Ascomyceteswheat, rye, oatcat, dog, horse, rabbit, guinea pig, hamsterbudgerigar, parrot, pigeon, duck, chicken, Dermatophagoides pteronyssinus, 20 D.farinae, Euroglyphus maynei, cockroach, fly, locust, midge, seafood, legumes, peanuts, nuts, cereals, dairy products, eggs, fruits, tomatoes, mushrooms, alcoholic beverages, coffee, chocolate, penicillins, sulphonamides and other antibiotics, sulphasalazine, carbamazepine, bee and wasp stings, ant and mosquito bitesblood products, sera, vaccines, contrast media, drugs (including anti-asthma drugs and 25 antibiotics). ANTIGENIC DETERMINANT The term "antigenic determinant" as used herein refers to a site on an antigen which is 30 recognised by an antibody or T-cell receptor. Preferably it is a short peptide derived from or as part of a protein antigen. However the term is also intended to include glycopeptides and carbohydrate epitopes. The term also includes modified sequences WO 00/14114 PCT/GB99/02970 11 of amino acids or carbohydrates which stimulate responses which recognise the whole organism. It is advantageous if the antigenic determinant is an antigenic determinant of the 5 infectious agent (such as a bacterium or virus) which causes the infectious disease. By way of example, if the infectious agent is EBV, the antigenic determinant may be an antigenic determinant of gp340 or gp350 or of a latent protein (for example EBNAs 1,2 3A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER). If the infectious 10 agent is an influenza virus, the antigenic determinant may be an antigenic determinant of a viral coat protein (for example haemagglutinin and neuraminidase) or of an internal protein (for example, nucleoprotein). if the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic and enteroaggregative E.coli, then the antigenic determinant may 15 be an antigenic determinant of a bacterial toxin or adhesion factor. It is also advantageous if the antigenic determinant is an antigenic determinant from an autoantigen. 20 AGENT The agent can be an amino acid sequence or a chemical derivative thereof. The substance may even be an organic compound or other chemical. The agent may be a nucleotide sequences - which may be sense or anti-sense sequences. The agent may 25 be an antibody. In one preferred aspect, the agent is a cell receptor that is engageable by the peptide. I intellectual property 1 OFRCB OF N.Z I 19 SEP 2003 RECEIVED I WO 00/14114 PCT/GB99/02970 12 INHIBITOR OF TOXIN-INDUCED DIARRHOEA The term "inhibitor of toxin-induced diarrhoea" includes any substance which is capable of affecting the activity of Etx/Ctx holotoxins such that the pathological 5 consequences of Etx/Ctx, such as diarrhoea, may be avoided. DETAILED DESCRIPTION OF THE INVENTION The present invention demonstrates the highly surprising finding that: 10 (i) the peptide of the present invention is capable of acting as an immunomodulator and/or an adjuvant and/or an inhibitor of toxin-induced diarrhoea which is capable of affecting enterotoxin mediated diarrhoeal diseases. 15 (ii) the peptide of the present invention is capable of acting in a manner that is the same as or is similar to EtxB and/or CtxB. The activity of the peptide of the present invention may be mediated by the "so-called" B4-a2 loop of EtxB and CtxB, which is a flexible loop included within amino acid residues 45-65. 20 (iii) EtxB molecules with point mutations at three separate sites within the 134-a2 loop (positions 51, 56 and 57) retain GM-1 binding activity, but lack other activities, such as toxicity and the capacity to upregulate CD25 and trigger apoptosis of CD8-positive T-cells. In addition, Ctx holotoxins comprising B subunits with mutations also show a defect in an ability to trigger electrogenic chloride secretion, the primary 25 secretory event responsible for mediating diarrhorea. These finding are particularly surprising, since flexible loops are usually thought to serve only to join two elements of secondary structure together, and rarely have an important function themselves. (iv) the binding activity of the peptide is not necessarily dependent on a primary 30 binding event as is found with full length Ctx and EtxB. With full length Ctx and EtxB, the primary binding activity is GM-1 binding activity. intellectual property of^cf of i\j.z 1 9 SEP 2C03 RECEIVED WO 00/14114 PCT/GB99/02970 13 Ctx/Etx TOXINS As used herein, the term "Ctx" refers to the cholera toxin and the term "CtxB" refers to the B subunit of the cholera toxin. In other texts, these may sometimes be 5 identified as CT or Ct or CTB or CtB respectively. 10 As used herein, the term "Etx" herein means the E. coli heat labile enterotoxin and the term "EtxB" is the B subunit of Etx. In other texts, these may sometimes be identified as LT or Lt and LTB or LtB respectively. J34-cc2 LOOP One embodiment relates to a peptide comprising the sequence EVPGSQH (SEQ ID No 2) which is capable of acting in a manner that is the same or 15 is similiar to EtxB and/or CtxB or a variant thereof, or a homologue thereof, or a fragment thereof or a derivative thereof or a mimetic thereof but which is not capable of exhibiting GM-1 binding activity. Without wishing to be bound by theory, we believe that the binding of the five 20 Etx/Ctx B subunits to GM-1 is a high affinity interaction, which allows a relatively low affinity secondary binding activity of EtxB/CtxB to occur. This binding is mediated by the the B4-a2 loop of EtxB/CtxB. The structure of the B4-a2 loop of EtxB/CtxB can be understood by reference to the molecular structure of Etx as described in detail in Sixma et al. J. Mol. Biol. (1993) 230; 890-918) and as illustrated 25 in Figure 1. In summary, each B subunit of Etx or Ctx consists of a small N-terminal helix (al), two three-stranded anti-parallel sheets (sheet I, composed of strands B2, 63, B4 and sheet II, composed of strands Bl, B5 and B6), and a long a-helix (a2). The two B 30 sheets form a B barrel. The loops joining these elements of secondary structure in the B subunit can be divided into two classes, referring to the two ends of the sheets.. tinteu-ECTUAA proper"™ one end of the subunit, the "narrow" (or "A") end, the loops are |enerall^'Wortf '7 1 9 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 14 involving the connections CI-132, B3-B4, and a2-B5 as well as the C-terminus. The subunit widens at the other end, with much longer loops connecting secondary structure elements ctl-Bl, B2-B3, B4-a2. The longest loop connects 64 and a2 (hereinafter the "B4-a2 loop"), includes the residues Glu 51 to Asp 59, and extends 5 below the plane of the B sheets. This loop is quite flexible, but according to Sixma et al (Nature (1992) 355; 561-564) becomes distinctly less mobile after lactose binding. The present invention demonstrates that the B4-a2 loop of EtxB/CtxB is responsible for the secondary binding activity and so the use of this loop in isolation from the rest of the EtxB/CtxB molecule (for example as a peptide), may permit the secondary 10 binding activity to occur in the absence of the first. Selective mutation of the B4-a2 loop, or a peptide derived from this loop, may be exploited with a view to increasing the affinity of the secondary binding activity. By increasing the affinity of the secondary binding activity, the interaction with GM-1 may be further obviated. 15 As used herein, the term "B4-a2 loop of EtxB/CtxB" is the entity which is responsible for the secondary binding activity of the B subunits of toxins such as the cholera toxin and heat labile E.coli toxin. When the B4-a2 loop is used in isolation from the rest of the EtxB and/or CtxB molecule (for example as a peptide), the secondary binding activity may occur in the absence of the first and is herein after referred to as an 20 activity or binding activity. Preferably the peptide comprises an isolated |34-a2 loop of EtxB/CtxB. 25 The peptide of the present invention may therefore be used to identify a mimetic of the isolated (34-a2 loop of EtxB/CtxB. Particularly, a mimetic of the isolated (34-a2 loop of EtxB/CtxB with a high affinity binding activity. Preferably the peptide comprises from about 5 to about 40 amino acids, which comprises the sequence of iSEQ ID No. 4 or 5. ELLE OFFICE OF N.z' 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 5 10 15 Preferably the peptide has less than 25 amino acids. If the peptide is a fusion protein, preferably the peptide has greater than 25 amino acids. Preferably the peptide comprises the sequence VEVPGSQHDDSQ (SEQ ID No 3). Preferably the peptide comprises the sequence GATFQVEVPGSQHIDSQKKAI (SEQ ID No 4). Preferably the peptide comprises the sequence GETFQVEVPGSQHIDSQKKAI (SEQ ID No 5) derivable from residues 45-65 of porcine E.coli. Preferably the peptide comprises residues 45-65 derivable from EtxB of the human 15 variant of E.coli derivable from EtxB of the porcine variant of E.coli. AMINO ACID SEQUENCE Disclosed is a peptide comprising the amino acid sequences of 20 the present invention which is capable of acting as an immunomodulator and/or an adjuvant and/or an an inhibitor of toxin-induced diarrhoea which is capable of affecting enterotoxin mediated diarrhoeal diseases. The peptide may also be used in assays for the identification of one or more agents capable of interacting with and/or affecting the peptide activity. 25 As used herein, the term "amino acid sequence" refers to peptide, polypeptide sequences, protein sequences or portions thereof. OFFICE OF N.Z 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 16 AFFECT The term "affect" includes modulation, such as treatment, prevention, suppression, alleviation, restoration, elevation, modification of the substance activity. 5 The term "modification" includes but is not limited to disabling, silencing, mutating, removing, enhancing, increasing, agonising, antagonising, decreasing or blocking the substance activity. 10 VARIANTS/HOMOLOGUES/DERTVATIVES Preferred amino acid sequences of the invention are SEQ ID No 2 or SEQ ID No 3 or SEQ ID No 4 or SEQ ID No 5 or sequences obtainable from the substance of the present invention but also include homologous sequences obtained from any source, 15 for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof. Thus, the present invention covers variants, homologues or derivatives of the amino acid sequences presented herein, as well as variants, homologues or derivatives of the 20 nucleotide sequence coding for those amino acid sequences. In the context of the present invention, a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least the 7 amino acids of SEQ ID 25 No 2, for example as shown in the sequence listing herein. In particular, homology should typically be considered with respect to those regions of the sequence (such as amino acids at positions 51, 56 and 57) known to be essential for an activity which is the same or is similiar to EtxB and/or CtxB rather than non-essential neighbouring sequences. Although homology can also be considered in terms of similarity (i.e. 30 amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. WO 00/14114 PCT/GB99/02970 17 Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. 5 % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. 10 Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. 15 Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. 20 However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and 25 a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit 30 package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. WO 00/14114 PCT/GB99/02970 18 Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 5 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is 10 preferred to use the GCG Bestfit program. Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise 15 comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, 20 or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. 25 The terms "variant" or "derivative" in relation to the amino acid sequences of the present invention presented as SEQ ID No 2, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5 includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant 30 entity retains an activity, preferably having at least the same and/or similiar activity as CtxB and/or EtxB. WO 00/14114 PCT/GB99/02970 19 SEQ ED No 2 or SEQ ED No 3 or SEQ ED No 4 or SEQ ID No 5 may be modified for use in the present invention. Typically, modifications are made that maintain the activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the 5 activity. The peptide of the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made 10 on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar 15 hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line 20 in the third column may be substituted for each other: ALIPHATIC Non-polar GAP IL V Polar - uncharged CSTM NQ Polar - charged DE KR AROMATIC HF W Y rintellectual property i off/cf of n.z 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 20 NUCLEOTIDE SEQUENCE Disclosed are nucleotide sequences encoding the peptide of the present invention capable of acting as a template or as targets in assays (such as a yeast two hybrid assay) for the identification of one or more agents 5 and/or derivatives thereof capable of affecting the substance. As used herein, the term "nucleotide sequence" refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence 10 may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof. Preferably, the term nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). 15 Preferably, the term "nucleotide sequence" means DNA. The peptide ■ encoding nucleotide sequence may be the same as the naturally occuring form for this aspect. Preferably the nucleotide sequence encoding the peptide is a non-native nucleotide sequence - or is a variant, homologue, fragment or 20 derivative thereof. Thus, in a preferred embodiment, the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. For ease of reference, we have called this preferred embodiment the "non-native nucleotide sequence". 25 As used herein "naturally occurring" refers to an substance with an amino acid sequence found in nature. As used herein "biologically active" refers to an substance having regulatory or 30 biochemical functions of the naturally occurring substance. wellk^twimy 19 SEP 2003 Received WO 00/14114 PCT/GB99/02970 21 VARIANTS/HOMOLOGUES/DERIVATIVES The terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence 5 modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for an substance having an activity, preferably having at least the same activity as the SEQ SEQ ID No 2, SEQ ID No 3, SEQ ID No 4 and SEQ ID No 5 presented in the sequence listings. As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology. Nucleotide homology comparisons may be 15 conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide. 20 The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length. As used herein a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. 30 As used herein an "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance. SEQ ID No 1 include any substitution of, variation of, 10 25 WO 00/14114 PCT/GB99/02970 22 As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. HYBRIDISATION The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies. Nucleotide sequences of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred nucleotide sequences will comprise regions homologous to nucleotides comprising SEQ ID No 1 preferably at least 80 or 90% and more preferably at least 95% homologous to SEQ ID No 1. The term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P. Hybridization conditions' are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular 19 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 23 Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below. Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the 5 probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related 10 nucleotide sequences. Disclosed are nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC {lxSSC = 0.15 M NaCl, 0.015 M Na3 Citrate pH 7.0). Where 15 the nucleotide sequence is double-stranded, both strands of the duplex, either individually or in combination, are contemplated. Where the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also contemplated. 20 EXPRESSION VECTORS The peptide of the present invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the 25 nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two 30 or more different promoters described above. ?ntellect(jal property op-/ce of n.z 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 24 The peptide . produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The peptide coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or 5 eukaiyotic cell membrane. FUSION PROTEINS The , peptide 0f the invention may also be produced as fusion proteins, for example 10 to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAM (DNA binding and/or transcriptional activation domains) and p-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein 15 will not hinder the activity of the peptide comprising the amino acid sequence. In one embodiment, the fusion protein comprises an antigen or an antigenic determinant fused to the peptide of the present invention. In this 20 embodiment, the fusion protein is a non-naturally occuring fusion protein comprising a peptide which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the peptide., 25 In another embodiment, the peptide of the invention may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognized by a commercially available antibody. 30 f intellecfual^property ofrce of m.z 13 SEP 2C03 received WO 00/14114 PCT/GB99/02970 25 In yet another embodiment, an assay for identifying an agent, such as a target receptor, or for an agent capable of regulating CD25 transcriptional activity may be conducted using a bound fusion protein. 5 ANTIBODIES The substance disclosed herein may be an antibody. This antibody may be capable of acting as a mimetic of the present invention. 10 Antibodies may be produced by standard techniques, such as by immunisation with the substance of the invention or by using a phage display library. For the purposes of this invention, the term "antibody", unless specified to the contrary, 15 includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site 20 of the antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in substance-A-239400. Neutralizing antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics. 25 In one embodiment, monoclonal or polyclonal antibodies to peptides of the invention such as polypeptides or fragments thereof are disclosed. Also disclosed is a process for the production of monoclonal or polyclonal antibodies to peptides of the invention. ■ rimlectual property OF-F/cp of N.Z 19 SEP 2003 Received WO 00/14114 PCT/GB99/02970 26 POLYCLONAL ANTIBODIES If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing a epitope(s) obtainable from an identifed agent and/or peptide of the present invention. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (.Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from an identifed agent and/or peptide of the present invention contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides or fragments thereof haptenised to another polypeptide for use as immunogens in animals or humans. MONOCLONAL ANTIBODIES Monoclonal antibodies directed against epitopes obtainable from an identifed agent and/or peptide ■ 0f the present invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of !NTELLECtUAL property ore,'of- qf ^2 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 27 monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity. Monoclonal antibodies to the peptide and/or identified agent of the present 5 invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and 10 the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-15 6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the substance specific single chain antibodies. 20 Antibodies, both monoclonal and polyclonal, which are directed against epitopes obtainable from an identifed agent and/or substance of the present invention are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise antiidiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an 25 "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These antiidiotype antibodies may also be useful in therapy. Antibodies may also be produced by inducing in vivo production in the lymphocyte 30 population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299). ifm^cttwi^opfotr owce OF fiz 19 SEP 2003 WO 00/14114 PCT/GB99/02970 28 Antibody fragments which contain specific binding sites for the substance may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1). ASSAYS FOR IMMUNOMODULATORY SUBSTANCES The immunomodulation of the immune response may be measured by transcriptional profiling, for example, by assaying for activation of transcription of CD25 cell surface marker by measuring the signal from a linked reporter gene. REPORTERS Preferably a wide variety of reporters may be used in the assay methods disclosed with preferred reporters providing conveniently detectable signals (eg. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties. Examples of reporter molecules include but are not limited to P-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (3-glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabeled or fluorescent tag-labeled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes. In one preferred embodiment, the production of the reporter molecule is measured by the enzymatic activity of the reporter gene product, such as (3-galactosidase. i intellectual property! ofrcb of n.z 19 SEP 2033 RECEIVED WO 00/14114 PCT/GB99/02970 29 ASSAYS FOR INHIBITORS OF TOXIN-INDUCED DIARRHOEA The peptide of the present invention or a derivative or homologue thereof and/or a cell line that expresses the peptide of the present invention or a derivative or 5 homologue thereof may be used to screen for agents (such as antibodies, peptides, organic or inorganic molecules) capable of affecting the activity of the peptide • By way of example, any agent capable of inhibiting the activity of the peptide may be screened for inhibitors of toxin-induced diarrhoea thereby identifying agents capable of affecting the cholera and/or enterotoxin mediated diarrhoeal diseases. the peptide of the present invention, such as antibodies, peptides or small organic molecules which are capable of acting as inhibitors of toxin-induced diarrhoea 15 ASSAYS FOR AGENTS Phage display may be employed in the identification of agents, such as a cell surface receptor that is engageable by the peptide of the present invention. The positive identification of such a receptor may faciliate the use of combinatorial libraries to 20 identify mimetics capable of acting in the same or a similiar manner as the peptide of the present invention. Phage display is a protocol of molecular screening which utilises recombinant bacteriophage. The technology involves transforming bacteriophage with a gene that 25 encodes an appropriate ligand (in this case a candidate agent) capable of reacting with a target substance (or a derivative or homologue thereof) or the nucleotide sequence (or a derivative or homologue thereof) encoding same. The transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate ligand (such as the candidate agent) and displays it on their phage coat. 30 The entity or entities (such as cells) bearing the target molecules which recognises the candidate agent are isolated and amplified. The successful candidate agents are then characterised. Phage display has advantages over standard affinity 10 In one embodiment, the screens may identify antagonists of WO 00/14114 PCT/GB99/02970 30 ligand screening technologies. The phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occuring conformation. This allows for more specific and higher affinity binding for screening purposes. 5 ASSAYS FOR MIMETICS In one embodiment, the screens . may identify mimetics of the peptide of the present invention, such as antibodies, or other chemical compounds 10 which have an immunomodulatory effect. Such mimetics can be administered alone or in combination with other therapeutics for the treatment of diseases.- 15 SCREENS The peptide of the present invention to be used for identifying immunomodulators, adjuvants, mimetics and/or inhibitors of toxin-induced diarrhoea hi any of a variety of drug screening techniques. The peptide employed in such a test may be free in 20 solution, affixed to a solid support, bome on a cell surface, or located intracellularly. The abolition of > activity or the formation of binding complexes between the peptide and the agent being tested may be measured. Another technique for screening provides for high throughput screening (HTS) of 25 agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564. It is expected that the assay methods disclosed will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays. ' QF ,^2 I 1S SSP 2003 WO 00/14114 PCT/GB99/02970 31 PHARMACEUTICAL COMPOSITIONS Described is a pharmaceutical composition comprising administering a therapeutically effective amount of the peptide of the present 5 invention and a pharmaceutical^ acceptable carrier, diluent or excipients (including combinations thereof). The peptides of the invention are especially useful as agents in the preparation of pharmaceuticals. Such pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a 10 pharmaceutically acceptable diluent, earner, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and 15 standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binders), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the 20 pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. There may be different composition/formulation requirements dependent oil the 25 different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. 30 Alternatively, the formulation may be designed to be delivered by both routes. intellectual property i OFRCE OF M.Z I 19 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 32 Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile. 5 Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules 10 either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or 15 monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner. 20 25 VACCINES In one embodiment of the present invention, the peptide is incorporated into a vaccine composition used to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection or graft-versus-host disease (GVHD), allergic or infectious disease. In another embodiment the vaccine composition may additionally comprise an antigen(s) or antigenic determinant(s). Suitable such antigens and/or antigenic determinants are disclosed in WO 99/34817. 30 Preferably the vaccine composition comprises an antigen and/or antigenic determinant. intellectual*p*^5perty ofrcf OF M.Z 19 SEP 2003 received WO 00/14114 PCT/GB99/02970 33 Preferably the antigen is a self-antigen or a homologue thereof. Preferably, the one or more peptides of the present invention is used in the preparation of a therapeutic or prophylactic vaccine. 5 A "prophylactic vaccine" is a vaccine which is administered to naive individuals to prevent disease development. A "therapeutic vaccine" is a vaccine which is administered to individuals with an 10 existing infection to reduce or minimise the infection or to abrogate the immunopathological consequences of the disease. The preparation of vaccines which contain one or more substances as an active ingredient(s), is known to one skilled in the art. Typically, such vaccines are prepared 15 as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for 20 example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents. The vaccine composition may also comprise a combination of adjuvants which enhance the effectiveness of the vaccine. Examples of additional adjuvants which , in combination, may be effective include but are not limited to: aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, 30 kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, 25 jj iNTEL'cr,"r"*' "' ^ERTY 19 SE? 2003 RECFIVFD WO 00/14114 PCT/GB99/02970 34 vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Railway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, 5 Detroit, Michigan). Typically, adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use. 10 ADMINISTRATION Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the 15 particular patient. The dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited. The compositions may be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, 20 intravenous, subcutaneous, intraocular or transdermal administration. Typically, each protein may be administered at a dose of from 0.01 to 30 mg'kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. The term "administered" includes delivery by viral or non-viral techniques. Viral 25 delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof. The routes for such delivery mechanisms include but are not 30 limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. OFRCF OP iM.Z j 19 SEP 2003 f i received property WO 00/14114 PCT/GB99/02970 35 The term "administered" includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route. The term "co-administered" means that the site and time of administration of each of the peptide of the present invention and an additional entity such as an antigen and/or antigenic determinants are such that the necessary modulation of the immune system is achieved. Thus, whilst the peptide and the antigen may be administered 10 at the same moment in time and at the same site, there may be advantages in administering the peptide at a different time and to a different site from the antigen. The peptide and antigen may even be delivered in the same delivery vehicle - and the peptide and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled. The antigenic determinant and peptide or homologue or mimetic thereof may be administered separately or co-administered to the host subject as a single dose or in multiple doses. different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra-vaginal, urethral or ocular administration. 25 ' The vaccines comprising the peptide agent of the present invention are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene 30 glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, may be 1% to 2%. Oral formulations include such normally employed excipients as, for example, 5 15 20 The vaccine composition may be administered by a number of WO 00/14114 PCT/GB99/02970 36 pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% 5 to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer DISEASES 10 The peptide of the present invention is useful as a medicinal agent to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection, allogeneic or xenogeneic transplant, graft-versus-host disease (GVHD), allergic or infectious diseases. Within the group "infectious diseases", are diseases in which, during infection, the infectious agent 15 binds to, colonises or gains access across the mucosa are particularly preferred, as are diseases in which immunopathological mechanisms are commonly involved. Examples of infectious diseases • include but are not limited to HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and HHV-8, hepatitis A, B, C, D 20 and E, Neisseria meningitides, Haemophilus influenzae type B and Streptococcus pneumoniae, Legionella pneumophila and Mycobacterium tuberculosis, Neisseria gonnorheae, HIV-1, HTV-2 and Chlamydia trachomatism, E.coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae, Staphylococcus aureus, Streptococcus 25 pyogenes and Streptococcus mutans, malaria, Trypanasoma spp., Taxoplasma gondii, Leishmania donovani and Oncocerca spp. Examples of allergic disorders include but are not limited to diseases include asthma, allergic cough, allergic rhinitis and conjunctivitis, atopic 30 eczema and dermatitis, urticaria, hives, insect bite allergy, dietary and certain drug allergies. | INTELLECTUAL property j ofrce of n.z 19 SEP 2883 RECEIVED WO 00/14114 PCT/GB99/02970 37 Examples of autoimmune diseases include but are not limited to diseases such as rheumatoid arthritis, multiple sclerosis and diabetes. KITS 5 Described are diagnostic assays and kits comprising the substance of the present invention. Such kits may be used to prevent and/or treat and/or modulate the diseases of the present invention. 10 In one embodiment of the present invention, the kit may also comprise an antigen and/or antigenic determinant and/or a separate adjuvant for coadministration with said therapeutic or prophylactic composition. Alternatively, a kit may be provided comprising mimetics of the present invention in 15 the form of antibodies of the invention bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. In summary, disclosed is a substance comprising any one or more of: an amino acid sequence comprising the sequence of SEQ ED No. 2, or a variant thereof, or a homologue thereof, or a fragment thereof, or a derivative thereof, or a mimetic thereof; which substance is capable of acting in a manner that is the same as 25 or is similar to EtxB and/or CtxB; but wherein the substance does not exhibit GM-1 binding activity. Also disclosed is an assay method for determing one or agents that are capable of interacting with and/or affecting the peptide of the present invention 30 wherein the assay comprises contacting the peptide with an agent to be tested, and then determining whether or not the agent affects the peptide. r^as r"— intellectual property office of n.z 19 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 38 Other disclosures made are presented below by way of numbered paragraphs which include: 1. A peptide which comprises the sequence EVPGSQH, or a homologue or mimetic thereof. 2. A peptide according to paragraph 1, which comprises the sequence VEVPGSQHIDSQ. 3. A peptide according to paragraph 2 which comprises the sequence GATFQVEVPGSQHIDSQKKAI or the sequence GETFQVEVPGSQHIDSQKKAI. 4. A prophylactic or therapeutic composition which comprises a peptide according to any preceding paragraph or a homologue or mimetic thereof. 5. A prophylactic or therapeutic composition according to paragraph 4, which also comprises an antigen or an antigenic determinant. 6. A prophylactic or therapeutic composition according to paragraph 4 or 5, wherein the therapeutic or prophylactic agent is used as an immunomodulator. 7. A prophylactic or therapeutic composition according to paragraph 4 or 5, wherein the therapeutic or prophylactic agent is used to upregulate the production of antibodies at mucosal surfaces. 8. A prophylactic or therapeutic composition according to paragraph 4 or 5, wherein the therapeutic or prophylactic agent is used to prolong antigen presentation and give sustained immunological memory in a mammalian subject. * intellectual property i of^hf- of n.z 1 9 SEP 2003 RECEIVED WO 00/14114 PCT/GB99/02970 39 9. A prophylactic or therapeutic composition according to paragraph 4 or 5, wherein the therapeutic or prophylactic agent is used to downregulate the pathological components of Thl and Th2-associated immune responses. 5 10. A prophylactic or therapeutic composition according to any of paragraph 4 to 9, which is used to treat or prevent autoimmune disease, human T cell leukaemia, transplant rejection, graft-versus-host disease or infectious diseases. 11. A prophylactic or therapeutic composition which comprises an agent which 10 binds specifically to the B4-a2 loop of EtxB or CtxB. 12. A prophylactic or therapeutic composition according to paragraph 11, wherein the agent is an antibody. 15 13. A prophylactic or therapeutic composition according to paragraph 11 or 12, which is used to treat diarrhoea. 14. A vaccine composition for use against a disease, comprising a peptide according to any of paragraphs 1 to 3 or a homologue or a mimetic thereof. 20 15. A vaccine composition according to paragraph 14 which also comprises an antigenic determinant. 16. A vaccine composition according to paragraph 14 or 15 which is used to treat 25 or prevent infections diseases, autoimmune disease, human T cell leukaemia, transplant rejection or graft-versus-host disease (GVHD) diseases. 17. A kit comprising a therapeutic or prophylactic composition according to any of paragraphs 4 to 13. 30 The present invention will now be described only by way of example in which reference is made to the following Figures: WO 00/14114 PCT/GB99/02970 40 Figure 1 which shows a stereo ribbon drawing showing a B subunit of Etx/Ctx (from Sixma et al. J. Mol. Biol. (1993) 230:890-918, labels added); Figure 2 which shows the identification of loop residues in CtxB involved in CD8+ T-5 cells apoptosis; Figure 3 which shows mutant B subunits defective in CD8+ T-cell apoptosis retain ability to bind to cell surface receptors; 10 Figure 4 which shows total immunoglobulin levels against EtxB and EtxB (H57S) in sera from mice immunised intranasally with 1 Oug of each B-subunit; Figure 5 which shows that His-57 in CtxB and EtxB defines a region necessary for adjuvanticity; 15 Figure 6 which shows E51-158 B subunit peptide exhibits an ability to induce immunomodulation of CD 8+ T-cells. EXAMPLES 20 Example 1 Identification of residues in the Glu-51 to Ile-58 loop of CtxB that trigger immunomodulatory effects on leukocytes 25 NIH male mice were sacrificed and mesenteric lymph node tissue was subsequently removed into Hanks balanced salt solution (HBSS without Calcium and Magnesium and supplemented with 20mM Hepes). Lymphocytes were then dispersed into the solution and away from fibrous tissue by gently pressing the tissue through a wire 30 mesh. Following 3 washes in HBSS, the lymph node cells were resuspended in modified Eagle's medium (Gibco) (a-MEM) containing 20 mM Hepes, 4mM L-Glutamine, 100 IU/ml penicillin, lOOug/ml streptomycin, and 5x10*5 M2- WO 00/14114 PCT/GB99/02970 41 mercaptoethanol, at a concentration 2 x 10^ cells/ml and then mixed without (PBS control) or with 3.45uM (40 ug/ml) of wild-type CtxB or wild-type EtxB, or various mutant B-subunits, namely EtxB(G33D), CtxB(E51A), CtxB(V52A), CtxB(P53A), CtxB(G54A), CtxB(S55A), CtxB(Q56A), CtxB(H57A), or CtxB(I58A) and incubated 5 at 37°C for 96h. Thereafter, the cells were washed and resuspended in 0.4ml HBSS/20mM Hepes/0.1% sodium azide/10% rat serum. Phycoerythrin (PE) conjugated anti-CD8 (PharMingen) and FITC-conjugated anti-CD4 (PharMingen) were added at a dilution of 1/400 and the cells incubated on ice for a period of 30 min. Following antibody incubations, the cell suspensions were washed once in ISOTON 10 (Becton-Dickinson) and resuspended in 0.4ml ISOTON. FACS analysis was carried out, with 10,000 events collected for each sample and then plotted using WinMDI software. The cells with FITC-bound anti-CD4 are depicted in the top-left hand quadrant of each figure; the cells with PE-bound anti-CD8 are depicted in the bottom right-hand quadrant, with the percentage number of events in each quadrant shown. 15 Results 1 The results in Figure 2 demonstrate, that incubation of MLN cells with wild-type CtxB or wild-type EtxB causes depletion of CD8+ T-cells; which does not occur if the 20 cells are incubated in the presence of PBS (control) or a mutant form of EtxB, EtxB(G33D) which lacks an ability to bind to cell surface GM-1 ganglioside. An analysis of the CtxB mutants containing Ala substitutions in residues E51 to 158 revealed that CtxB(E51A) and CtxB(H57A) also failed to trigger CD8+ T-cell depletion. In addition, CtxB(V52A) and CtxB(I58A) exhibited a partial defect in 25 triggering CD8+ T-cell depletion. These findings indicate that residues, E51 and H57 play an essential role in triggering modulatory effects on lymphocytes with a contributory role for residues V52 and 158. 30 WO 00/14114 PCT/GB99/02970 42 Example 2 Mutant B-subunits defective in CD8+ T-cell apoptosis retain ability to bind to cell surface receptors 5 NIH male mice were sacrificed and mesenteric lymph node tissue was subsequently removed into Hanks balanced salt solution (HBSS without Calcium and Magnesium and supplemented with 20mM Hepes). Lymphocytes were then dispersed into the solution and away from fibrous tissue by gently pressing the tissue through a wire 10 mesh. Following 3 washes in HBSS, the lymph cells were resuspended in 300 ml of pre-cooled and de-gassed MACS buffer (PBS, 5mM EDTA, 0.5% BSA, pH7.2). 50ml each of anti CD4 and anti-B220 MACS antibodies were added to the cells and the CD8 T-cell population purified by negative selection in a magnetic MACS column. The CD8+ T-cells containing population were washed and resuspended in 15 modified Eagle's medium (Gibco) (a-MEM) containing 20 mM Hepes, 4mM L-Glutamine, 100 IU/ml penicillin, lOOug/ml streptomycin, and 5x10"^ M2- mercaptoethanol, at a concentration 2 x 10^ cells/ml and then mixed without (PBS control) or with 3.45uM (40 ug/ml) of wild-type CtxB or wild-type EtxB, or various mutant B-subunits, namely EtxB(G33D), CtxB(E51A), CtxB(V52A), CtxB(P53A), 20 CtxB(G54A), CtxB(S55A), CtxB(Q56A), CtxB(H57A), EtxB(H57S), or CtxB(I58A) and incubated on ice for 20 min. Thereafter, the cells were washed and resuspended in ice-cold 0.4ml HBSS/20mM Hepes/0.1% sodium azide/10% rat serum. Anti-EtxB monoclonal antibody 118-8 was added at a dilution of 1/500 to cells incubated EtxB, EtxB(G33D) or EtxB(H57S) and anti-CtxB monoclonal antibody LT-39 was added at 25 a dilution of 1/800 to cells incubated with CtxB and CtxB mutants. After 30 min, the cells were washed and resuspended in HBSS/20mM Hepes/0.1% sodium azide/10% rat serum followed by addition of a FITC-labelled anti-mouse IgG antibody. Following incubation with the secondary antibody for 30 min, the cell suspensions were washed once in ISOTON (Becton-Dickinson) and resuspended in 0.4ml 30 ISOTON. Levels of FITC fluorescence as a representative of the extent of binding of EtxB, CtxB and the various mutants to CD8+ T-cells was assessed by FACS analysis. The results of 10,000 events from each sample are plotted showing the fluorescence WO 00/14114 PCT/GB99/02970 43 intensity of CDS+ T-cells incubated in the absence of B-subunits (ie PBS contol; red line) versus the fluorescence attributable to bound B-subunits (black line) (Figure 3). Results 2 5 The results in Figure 3 show that all B-subunits, except the non-binding mutant EtxB(G33D) bound to CD8+ T-cells to a similar extent. The fluorescence intensity detected after binding CtxB(H57A) and EtxB(H57S) was somewhat higher than that exhibited by wild-type B-subunits indicating that the two mutants have a greater 10 avidity for the cell surface. This result is consistent with the finding that both CtxB(H57A) and EtxB(H57S) bind with a slightly higher avidity to GM-1-coated microtitre plates and exhibit a slightly higher Kd for GM-1 as determined by plasmon surface resonance (data not shown). 15 Example 3 Residue His-57 in EtxB is required to induce a potent anti-EtxB response Groups of NIH female mice (n = 8) were immunised intranasally with lOug EtxB or 20 EtxB(H57S) in a volume of 20ul on 3 occasions at one week intervals. The mice were sacrificed and blood removed by cardiac puncture 14 days after the third immunisation. The sera were analysed for levels of anti-EtxB IgG antibodies by a GM-1-ELISA using microtitre plates coated with lug/ml EtxB. End point titres were determined (equivalent to a dilution giving an absorbance of 0.1 above background). 25 Results 3 The results in Figure 4 show that following intranasal immunisation with wild-type EtxB high titre serum anti-EtxB IgG antibody levels axe induced (titre = 5757+/-785) 30 whereas immunisation with EtxB(H57S) induces a significantly (p=0.001) lower response (titre 1205+/-222). WO 00/14114 PCT/GB99/02970 44 Example 4 Residue His-57 in EtxB and CtxB is necessary for the B-subunits to act as 5 mucosal adjuvants Groups of NIH female mice (n = 8) were immunised intranasally with lOug ovalbumin (Ova) alone or with lOug Ova mixed with either lOug EtxB, CtxB, EtxB(H57S) or CtxB(H57A) and administered in a volume of 20ul on 3 occasions at 10 one week intervals. In addition, two groups of mice were intranasally immunised with lOug EtxB or CtxB as negative controls. All mice were sacrificed 14 days after the third immunisation and the blood removed by cardiac puncture. The sera were then analysed for levels of anti-Ova IgG antibodies by an ELISA using microtitre plates coated with 5ug/ml Ova. End point titres were determined (equivalent to a 15 dilution giving an absorbance of 0.1 above background). Results 4 The results in Figure 5 show that both wild-type EtxB and CtxB act as mucosal 20 adjuvants, substantially augmenting the anti-Ova response compared with mice immunised with Ova alone (compare lanes 4 and 5 with lane 1). By contrast, when Ova was admixed with EtxB(H57S) (lane 6) or CtxB(H57A) (lane 7) the anti-Ova response induced was substantially less than that triggered by inclusion of the wild-type B-subunits. The data demonstrate that CtxB(H57A) lacks adjuvant activity. This 25 confirms the importance of the B-subunit E51-I58 loop, and in particular H57 in mediating the immunomodulatory properties of the molecule. 30 WO 00/14114 PCT/GB99/02970 45 Example 5 A synthetic peptide EVPGSQHI corresponding to the E51 to 158 loop of EtxB and CtxB possesses immunomodulatory properties 5 To assess whether a synthetic peptide corresponding to the E51 to 158 loop of EtxB (and CtxB) causes depletion of CD8+ T-cells from MLN cultures, mesenteric lymph node cells were isolated as described in Example 1, and incubated with various concentrations (O.luM - 20uM) of the EVPGSQHI peptide or a randomly selected 10 control peptide, LRNETTTTKGDYC. After 96h incubation at 37°C the cells were washed and resuspended in 0.4ml HBSS/20mM Hepes/0.1% sodium azide/10% rat serum and then assessed for the relative proportion of CD4+ and CD8+ cells by FACS analysis, in an identical fashion to that reported in Example 1. The percentages of CD8+ T-cells remaining in cultures treated with the EVPGSQHI peptide (closed red 15 circles and line) or the control peptide (closed black squares and line) was detennined and was plotted graphically against concentration of peptide used (Figure 6). Results 5 20 The results in Figure 6 show that incubation of MLN cultures in the presence of the EVPGSQHI peptide causes a reduction in CD8+ T-cell numbers, in contrast to treatment with a control peptide. This shows that a synthetic peptide corresponding to the E51 to 158 loop of EtxB and CtxB is active in exerting direct modulatory effects on lymphocytes. WO 00/14114 PCT/GB99/02970 46 All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. 47 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. The use of a peptide in the manufacture of a medicament for the treatment and/or prevention of a condition, wherein the peptide: (i) comprises the sequence of SEQ ID No. 2; (ii) has fewer than 40 amino acids; and (iii) acts an immunomodulator in the treatment and/or prevention of the condition.

2. The use according to claim 1, wherein the peptide comprises the sequence of SEQ ID No 2, 3, 4 or 5.

3. The use according to claim 1 or 2, wherein the peptide is part of a fusion protein.

4. The use according to any preceding claim, wherein the condition is selected from autoimmune disease, human T-cell leukaemia, transplant rejection or graft-versus-host disease, or an allergic disease.

5. The use according to any preceding claim, wherein the condition is an infectious disease.

6. The use according to any preceding claim wherein, the medicament is formulated for separate, sequential or simultaneous administration with an antigen or an antigenic determinant.

7. A peptide consisting of the amino acids sequence of SEQ ID No 2, 3, 4 or 5.

8. A peptide having fewer than 40 amino acids which comprises the sequence of SEQ ID No 4 or 5.

9. The use of a peptide as defined in claim 1, 7 or 8 to identify a mimetic of the peptide which has an immunomodulatory effect.

10. A screening method for identifying an agent which is capable of interacting with a peptide as defined in claim 1, 7 or 8, which comprises the step of contacting the peptide with a candidate agent and determining whether or not the candidate agent interacts with the peptide.

11. A phage display method according to claim 10. :ctual property" ofhce OF l\j.Z 19 SEP 20 48

12. A method according to claim 10 or 11 for identifying a receptor for the peptide.

13. A method according to claim 12, wherein the receptor is a cell surface receptor.

14. A use as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.

15. A peptide as claimed in claim 7 or claim 8 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.

16. A use as claimed in claim 9 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.

17. A method as defined in claim 10 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures. </div>