AU2006203495A1 - Mimotopes - Google Patents

Description

AUSTRALIA

Patents Act 1990 DIATECH PTY LTD COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Mimotopes The following statement is a full description of this invention including the best method of performing it known to us:- Mimotopes Field of the Invention The present invention relates to mimotopes of natural Epstein-Barr Virus (EBV) epitopes. The invention also relates to methods of screening for mimotopes that bind to antibodies that bind to natural EBV epitopes. The invention also relates to methods of screening for anti-mimotopes that bind to natural EBV epitopes. The invention further relates to the use EBV mimotopes or anti-mimotopes in the diagnosis of EBV infection and to distinguish between infection by EBV or other Herpes Virus infections.

Background to the Invention Epstein-Barr virus (EBV) is the primary causative agent of infectious mononucleosis and in severe cases is involved in the pathogenesis of certain types of lymphomas, nasopharyngeal carcinoma and post-transplant lymphoproliferative disease The acute infection can be diagnosed serologically by measuring IgM antibodies primarily against viral capsid antigen (VCA). It is common, however, to augment this test with others to measure IgM, IgG or IgA antibody levels to VCA, EBV nuclear antigen (EBNA) and early antigen (EA) to assess staging and severity of the disease ELISA is the most sensitive method for diagnosis and current commercial tests usually employ native antigens purified from EBV-producing cultures to capture IgM or IgG antibodies in a patient's serum Some ELISA tests use recombinant antigens to EBNA and EA-D However, affinity-purified native antigens have repeatedly shown the best performance for commercial VCA assays Since all EBV antigens are, however, complexes of typically two to six proteins that are often highly glycosylated, this has hindered their use in commercial diagnostics. The development of a simpler and more uniform diagnostic test is therefore desirable.

Summary of the Invention We have now identified and isolated molecules that mimic antigenic determinants of EBV. A number of the molecular mimics identified have been synthesized and found to be capable of detecting antibodies typically produced in human subjects after infection with EBV.

Accordingly, the present invention provides a purified mimotope which is capable of binding to an antibody to EBV.

The identification and isolation of molecules that mimic antigenic determinants of EBV provides significant advantages and benefits, especially in relation to diagnostic testing. The use of molecules mimicking epitopes, and in particular peptidebased mimotopes, as diagnostic antigens is advantageous because it allows focus on relevant single specificities and avoids the diagnostically unimportant epitopes present in crude extracts or complex antigens. Peptides also have other significant advantages; unlike recombinant antigens they can mimic carbohydrate epitopes In addition, peptides of high quality and stability can be cheaply and reproducibly produced and are easily applied to ELISA as well as other formats.

The present invention also provides a mimotope of the present invention conjugated to a fusion partner or fusion partners.

The present invention also provides an isolated nucleic acid encoding a peptide mimotope in accordance with the invention.

The invention also provides a diagnostic reagent comprising at least one mimotope according to the invention.

The present invention also provides an assay device comprising at least one mimotope of the present invention.

The present invention also provides for the use of a mimotope of the present invention to assay for the presence and/or amount of EBV antibodies in a sample to be tested.

Accordingly, the present invention provides a method of diagnosing EBV infection in a subject which comprises: contacting a biological sample from the subject with at least one mimotope according to the invention; and assaying for the presence or absence of a mimotope-antibody complex, wherein the presence of the mimotope-antibody complex is indicative of EBV infection.

The present invention also provides a diagnostic kit comprising at least one mimotope of the present invention.

The present invention also provides a method for selecting a mimotope of the present invention which method comprises panning a phage display library encoding a plurality of randomised peptides with an antibody that binds specifically to EBV.

The present invention also provides a peptide mimotope selected by said methods.

The mimotopes of the invention may advantageously be used to generate immune responses in animals and preferably humans.

The present invention therefore also provides a vaccine composition for providing immunological protection against EBV infection in a subject, the vaccine composition comprising a mimotope of the present invention.

The present invention also provides a method of inducing EBV neutralizing antibodies in an subject which method comprises administering to the subject a mimotope or vaccine composition of the invention.

The present invention also provides a method for isolating a molecule that binds to a mimotope of the present invention, which method comprises contacting a biological sample with a mimotope of the invention, and isolating a complex formed between the mimotope and molecules present in the sample. A molecule identified by this method is called an "anti-mimotope".

The present invention also provides a pharmaceutical composition comprising the anti-mimotope of the invention.

The present invention also provides the use of the anti-mimotope of the invention in the manufacture of the pharmaceutical composition for use in the treatment of EBV infection.

The present invention also provides an antibody raised against a mimotope of the present invention.

The present invention also concerns the use of the antibody raised against a mimotope of the present invention in the manufacture of the pharmaceutical composition for use in the treatment of EBV infection.

The present invention also provides a hybridoma cell line [F1] deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6833.

The present invention also provides a hybridoma cell line [A2] deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6832.

The present invention also provides a hybridoma cell line [A3] deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6834.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Brief Description of the Figures Figure 1: Antibody titer of a mouse immunised with crude EBV used for preparation of MAbs. ELISA showing antibody titer to EBV antigens, EBV, VCA, p 18, EAD and EBNA before and after immunisation.

Figure 2: Specificity of monoclonal antibodies for EBV antigens. Binding of mAbs gp125 (IgGI subclass), F1 (IgG2b), A2 (IgM) and A3 (IgGI) to crude EBV, VCA, EAD, EBNA and p18 by ELISA. Error bars indicate the ranges of individual values.

Figure 3: Selection of phage clones. Reactivity of selected phages from each round of panning by ELISA using MAb A2, MAb Fl, MAb A3 and (D) MAb gp 125 Error bars indicate the ranges of individual values.

Figure 4: Sequences of selected phage clones derived from panning on the corresponding MAb. All clones had the same sequence in Rounds 3 and 4 for gp125, Fl and A2, whereas 3 different clones were identified for A3, all had 2 cysteine residues underlined. *Clone 2 was selected for further study (see Figure Figure 5: The binding of 3 clones isolated from panning A3 MAb were compared by ELISA. Clone 2 (C2) showed the highest binding and was selected for further study.

Figure 6: Specificity of selected phage by ELISA. Each selected phage clone binds only to its corresponding MAb and does not bind an isotype control MAb (IC) or the other MAbs. Error bars indicate the ranges of individual values.

Figure 7: Specificity of selected phage by ELISA. A2 phage competes with p 8 antigen; FI phage compete with crude EBV antigen for binding to the parent MAb; A3 phage compete with crude EBV antigen for binding to the parent MAb; gp125 phage compete with crude EBV antigen for binding to the parent MAb. Error bars indicate the ranges of individual values.

Figure 8: Reactivities of gp125, Fl A2 and A3 peptides with individual EBV IgM positive (41) and negative sera (21) as determined by ELISA. The mean of triplicate absorbance reading values is plotted. The cut-off level is defined as the mean of the negative population +3 SD.

Figure 9: Reactivity of biotinylated gp125 peptide mimotope (N-terminus) with EBV positive and negative serum (1/100 dilution) comparison of binding with the a VCA IgM commercial diagnostic kit.

Figure 10: Reactivity of the gp125 peptide mimotope (peptide) with Q fever positive sera defined as seropositive with a diagnostic kit (IgM). The peptide crossreacts with some of the Q fever seropositive samples.

Figure 11: Antibody titer of a rabbit immunised with EBV. ELISA showing the rabbit antibody titer to crude EBV, VCA, p18, EBNA and EAD antigens before, and after immunisation.

Figure 12: Specificity of human EBV positive purified IgG for EBV antigens.

ELISA showing the binding of human serum purified on protein G to EBV antigens.

Figure 13: Selection of phage clones for rabbit anti-EBV IgG and (B) human anti-EBV IgG. The reactivity of selected phages from each round of panning is shown by ELISA. Error bars indicate the ranges of individual values.

Figure 14: Reactivity of individual phage clones isolated after panning on rabbit EBV IgG with immune and pre-immune IgG. Reactivity of isolated phage clone after panning on human EBV IgG with the same IgG and a non-specific human IgG preparation. Bars show the mean ELISA signal of duplicate wells and the bars indicate errors.

Figure 15: Human phage clone binds to 4 different preparations of human EBV positive IgG, indicating specificity for a common epitope typically present in individuals infected with EBV.

Figure 16: Human serum previously analysed using a diagnostic test for EBV was allowed to bind to the A3(SSGC) peptide and the bound IgM antibodies were detected using anti-human IgM-Horse radish peroxidase conjugate (HRP). The absorbance readings (abs 450nm) for positive, negative and putative cross-reactive sera for Parvo virus (Parvo), herpes simplex virus (HSV), cytomegalovirus (CMV) and rheumatoid factor (RF) are plotted for Rab 1(GC), Rab 6/1, Rab 6/10, (D) Rab 4/9 and hum 1. The cut-off value is defined as the mean of the negative population =3SD indicated by a solid horizontal line, the sensitivity of detection is shown and the specificity was 100% Figure 17: Binding of Fl-BSA conjugate to Fl MAb, coating at 10 and 50pg/ml. The Fl-BSA conjugate binds to Fl MAb but not to another MAb of the same isotype indicating the peptide Fl has successfully been conjugated to BSA and retains its binding capacity.

Figure 18: Comparison of Fl-BSA with BSA alone and Fl peptide alone for a set of EBV IgM positive and negative serum samples.

Figure 19: Comparison of Fl-BSA and VCA IgM diagnostic kit (VCA IgM commercial diagnostic kit) for detection of serum IgM antibodies typically produced after infection with EBV.

Figure 20: Reactivities of peptides gp125 and Fl conjugated to BSA with 64 individual EBV IgM positive (Pos), and 118 negative sera (Neg) as determined by ELISA. An additional 8 sera indicative of past infection, either seropositive for VCA IgG and/or seropositive for EBNA and negative for VCA IgM were analysed and added to the negative population. Putative cross-reactive sera which were IgM positive by ELISA were also analysed. Nineteen samples for cytomegalovirus (CMV), 3 for Herpes Simplex virus (HSV), 3 for Varicella Zoster 3 for Parvovirus (Parvo) and 6 for Rheumatoid factor The mean BSA level was subtracted from the value obtained for the BSA conjugated peptide. The cut-off level is defined as the mean of the negative population +3 SD indicated by a horizontal line. Cut-off: gpl25-BSA 0.35, Fl-BSA 0.36.

Figure 21: Gp and Fl peptides were conjugated to high purity BSA and human serum albumin (HSA) and analysed for reactivity by comparison with the previous BSA conjugated material.

Figure 22: Adhesive peptide enhancement of EBV mimotope Fl binding to standard ELISA plates.

Figure 23: Improvement by adhesive peptide of presentation of peptides to microtiter plates for ELISA gp-WC Fl-WN.

Figure 24: Analysis of the ability of A3 and A3(red/alk) peptides to bind to the parent mAb. Assessment of A3red/alk's ability to recognise EBV IgM antibodies in patients sera.

Figure 25: A: Binding of Fl antibody to S-layer fusion protein A (Fl at Cterminus) and B: Fl at the N-terminus and wild type (wt) no Fl fusion. Binding of EBV sdera to Fl mimotope as an S-layer fusion protein. Comparison of detection of IgM antibodies in clinical samples using F1 coupled to SbpA, BSA and adhesive peptide as solid phases.

Detailed Description of Preferred Embodiments Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in molecular biology and biochemistry). Standard techniques are used for molecular and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4 h Ed, John Wiley Sons, Inc. and the full version entitled Current Protocols in Molecular Biology, which are incorporated herein by reference) and chemical methods.

Furthermore, throughout this application various publications are referenced, many in parenthesis. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.

Mimotopes In this specification, by "mimotope" we mean a molecule, and preferably a molecular sequence, which "mimics" the epitopic region of a particular antigen, but which does not contain the specific amino acid sequence which comprises the epitope.

Thus, a mimotope is structurally distinct from an epitope, though functionally it is very similar as it is capable of binding in a similar fashion to the binding cleft of the antibody directed to the antigen containing the particular epitope.

As a matter of general definition, an epitope is that region of a particular antigen which contains the critical binding region of the antigen necessary for triggering an immunity-related antibody binding response. Epitopes are also often referred to in the alternative as "antigenic determinants".

EBV mimotopes of the invention are compounds that are structural mimics of an epitope of EBV but which differ in their composition. Thus mimotopes of the present invention do not include natural EBV epitopes such as EBV VCA, EBV NA, EBV EA- D or fragments thereof.

Accordingly, the present invention provides a purified mimotope which is capable of binding to an antibody to EBV.

In one embodiment, the mimotope is capable of specific binding to an antibody to EBV.

In another embodiment of the invention, the antibody is specific to EBV.

By "specific binding" we mean that the mimotope is capable of being bound to the antigen-binding site of an antibody in a selective fashion in the presence of excess quantities of other materials not of interest, and tightly enough with high enough affinity) that when used in an immunoassay, it provides a useful assay result.

Similarly, an antibody "specific to EB" is one which is capable of binding to EBV in a selective fashion in the presence of excess quantities of other materials not of interest, and tightly enough that when used in an immunoassay it provides a useful assay result.

The antibody to EBV can be any antibody, fragment or construct thereof, that binds to EBV. Various forms of such antibodies are contemplated which may include Fv, Fab, ScFv and the like. Also contemplated are multivalent and/or multispecific constructions which have been described in the literature and which comprise two or more polypeptide chains (see, for example, WO 94/09131 and WO 97/14719) or are based on a 'double ScFv' approach, wherein the multivalency arises when two or more monovalent ScFv molecules are linked together, providing a single chain molecule comprising at least four variable domains (see, for example, WO 93/11161 and WO 94/13806).

The antibody to EBV may bind to any EBV antigen. For example, the antibody may bind to the viral capsid antigen (VCA), EBV nuclear antigen (EBNA) or early antigen (EA) or a combination of these antigens. In one particular embodiment, the antibody binds to gp125 and/or p 8 which are epitopes of VCA.

Gp125 (BALF 4) is a major antigen of EBV VCA that has been proposed as a single antigen for diagnosis of infectious mononucleosis Currently EBV crude cell extracts are purified using an immunoaffinity column prepared with an antibody to the gp125 antigen and this preparation is used as a solid phase antigen to capture serum antibodies in commercial diagnostic tests As an alternative, other kits use p 1 8, which is an 18kD immunodominant region of VCA Gpl25 is clearly an important diagnostic epitope and the present invention provides a peptide selected from a random peptide library that mimics this epitope.

To select for peptides that mimic further epitopes, the present inventors have also developed a screening strategy for polyclonal serum. Since MAbs represent only a small portion of the immune response to the antigen, polyclonal sera may cover different epitopes simultaneously including the epitopes missed by screening with MAbs. This approach could also lead to identifying peptides not only specific for EBV-IgM but also for EBV-IgG antibodies.

Thus, in one embodiment, the antibody is a polyclonal antibody. For example, the polyclonal antibody may be IgG or IgM comprising complementarity determining regions complementary to the topology of an EBV epitope.

In another embodiment, the antibody is a monoclonal antibody.

For example, the monoclonal antibody .may be MAb gp125 (clone L2), commercially available from Chemicon International (Temecula, CA). Other monoclonal antibodies directed against gp125 are also available (for example, Clones 2Q1919 and 8.F.96, United States Biologicals) and are suitable for use in the present invention.

In another example, the monoclonal antibody is directed against p 8. Examples of monoclonal antibodies directed against p18 are described in US 5,424,398. A polyclonal anti-pl8 antibody is available from Biodesign International (Maine, United states).

In one particular embodiment, the monoclonal antibody is MAb F produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6833.

In another embodiment, the monoclonal antibody is MAb A2 produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6832.

In another embodiment, the monoclonal antibody is MAb A3 produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6834.

The mimotope of the present invention can be any molecule (peptide or nonpeptide) or sequence of molecules, such as, for example, a peptide, a carbohydrate, a lipid, a DNA molecule, an RNA molecule, a chemically synthesized molecule, or a combination of such molecules. In a particularly preferred embodiment, it is a short low molecular weight peptide. Suitably, a peptide mimotope is no greater than 30, and preferably no greater than 20, amino acids in length. Suitably, the peptide is at least 3, preferably at least 4, more preferably at least 5 and yet more preferably at least 6 amino acids in length.

In one embodiment, the mimotope comprises the tripeptide sequence motif SIK.

For example, the peptide may comprise a sequence selected from the group consisting of: SSSIKIWNKLGWNTVIAGTR (SEQ ID NO: and AITCAHTLSIKSRRCQYVFK (SEQ ID NO: 4).

In another embodiment, the mimotope comprises a peptide sequence selected from the group consisting of: AASYASRTVGFASVYWFSRP (SEQ ID NO: 6); NGPSYHVAVHFKNSRGLRHS (SEQ ID NO: 7); DNYWSFSDSTYWTLRYSSG (SEQ ID NO: 8); DQFAQAYRGDRNFFNELTST (SEQ ID NO: 9); ELISSCLVWSARGCLFGGGI (SEQ ID NO: FVNAFQNANFMRPRELFALA (SEQ ID NO: 11); GGWYSFDSPYLMSITEMRLR (SEQ ID NO: 12); LRGTHDFYLQVDMSDLSDLR (SEQ ID NO: 13); NGALYPRFFPDYSILMFPII (SEQ ID NO: 14); RLRGDYNVGPIRFGWPVAPN (SEQ ID NO: RQFSKFKDASDRYGNYLHFF (SEQ ID NO: 16); SANLNFFSPDFGLYTPNASA (SEQ ID NO: 17); SKLLYNYGACRTGCYMAGR (SEQ ID NO: 18); TELSLFCDSHGLGLSPYRQC (SEQ ID NO: 19); TPNTVRDFYYNVSLPSYMLI (SEQ ID NO: VMDECVFSSISVLFCNHMLH (SEQ ID NO: 21); and YTDSSMAVTLMKFASNFLF (SEQ ID NO: 22).

In another embodiment, the mimotope comprises a retro, inverso, or retroinverso sequence of SEQ ID NOs: 3, 4 and 6-22.

By "retro sequences" with reference to a peptide sequence it is meant peptide sequences where the sequence orientation is reversed. Thus a retro sequence of the peptide AGDT is TDGA. It has been found in the art that retro sequences of peptide mimotopes are often peptide mimotopes themselves. Peptide mimotope sequences of the invention may be entirely or at least in part comprised of D-stereo isomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are of the Dstereoisomer form. Such retro, inverso or retro-inverso peptides have the advantage of potentially being more stable and/or immunogenic in a host when administered as an immunogen. Methods to make D amino acids and incorporate them into proteins are well known in the art [see, for example, Thorson et al. (1998) Methods Mol. Biol.

77:43-73, Chartrain et al. (2000) Curr. Opin. Biotechnol. 11:209-14].

By "amino acid" we mean any naturally or non-naturally occurring amino or imino acid.

In a preferred embodiment, the mimotope is a C-terminus-amidated peptide.

The present invention also encompasses variants and derivatives of the peptide mimotope sequences disclosed herein. The terms "variant" or "derivative" in relation to an amino acid sequence of the present invention 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 amino acid sequence retains its ability to bind to an antibody to EBV.

Thus, the mimotopes disclosed herein may be modified for use in the present invention. Typically, modifications are made that maintain biological properties/activity such as the ability to bind to an antibody to EBV. Thus, in one embodiment, amino acid substitutions may be made, for example from 1, 2 or 3 to or 30 substitutions provided that the modified sequence retains at least about 25 to of, or substantially the same activity.

In general, preferably less than 20%, 10% or 5% of the amino acid residues of a variant or derivative are altered as compared with the corresponding region depicted in the sequence listing.

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 in the third column may be substituted for each other: ALIPHATIC Non-polar

GAP

ILV

Polar uncharged

CSTM

NQ

Polar charged

DE

KR

AROMATIC_

HFWY

Mimotope peptides of the invention may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, preferably the N-terminus.

Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences). Heterologous sequences may also include sequences that increase the affinity for an antibody to EBV and/or which facilitate identification, extraction and/or purification of the polypeptides.

The present invention also provides non-peptide mimetics ("peptidomimetic") of the EBV peptide mimotopes of the invention. A peptidomimetic is a molecule that mimics the biological activity of a peptide but is no longer peptidic in chemical nature.

By strict definition, a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids). However, the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Whether completely or partially non-peptide, peptidomimetics for use in the methods of the invention provide a spatial arrangement of reactive chemical moieties that closely resembles the threedimensional arrangement of active groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems which are similar to the biological activity of the peptide.

Peptidomimetics may be advantageous in that molecules concerned are typically small enough to be both orally active and have a long duration of action. There are also considerable cost savings and improved patient compliance associated with peptidomimetics, since they can be administered orally compared with parenteral administration for peptides. Furthermore, peptidomimetics are much cheaper to produce than peptides.

Suitable peptidomimetics based on EBV peptide mimotopes of the invention and having similar biological activities, and therefore similar utilities, can be developed using readily available techniques. Thus, for example, peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics derived from EBV peptide mimotopes can be aided by determining the tertiary structure of the original peptide by NMR spectroscopy, crystallography and/or computer-aided molecular modelling.

These techniques aid in the development of analogues/derivatives of higher potency and/or greater bioavailability and/or greater stability than the original peptide (Dean, 1994, BioEssays, 16: 683-687; Cohen and Shatzmiller, 1993, J. Mol. Graph., 11: 166- 173; Wiley and Rich, 1993, Med. Res. Rev., 13: 327-384; Moore, 1994, Trends Pharmacol. Sci., 15: 124-129; Hruby, 1993, Biopolymers, 33: 1073-1082; Bugg et al., 1993, Sci. Am., 269: 92-98).

Information on the structure of an EBV peptide mimotope can be used to search three-dimensional databases to identify molecules having a similar structure, using programs such as MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, CA), ChemDBS-3D (Chemical Design Ltd., Oxford, and Sybyl/3DB Unity (Tripos Associates, St. Louis, MO).

Databases of chemical structures are available from a number of sources including Cambridge Crystallographic Data Centre (Cambridge, Chemical Abstracts Service (Columbus, OH), and ACD-3D (Molecular Design Ltd).

De novo design programs include Ludi (Accelrys), Leapfrog (Tripos Associates) and Aladdin (Daylight Chemical Information Systems, Irvine, CA).

Those skilled in the art will recognize that the design of a mimotope may require slight structural alteration or adjustment of a chemical structure designed or identified using these databases.

The mimotopes of the invention may be conformationally constrained.

Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a compound. Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.

The active conformation of the mimotope of the invention may be stabilized by a covalent modification, such as cyclization or by incorporation of gamma-lactam or other types of bridges. For example, side chains of peptides can be cyclized to the backbone so as create a L-gamma-lactam moiety on each side of the interaction site.

See, generally, Hruby et al., 1992, "Applications of Synthetic Peptides," in Synthetic Peptides: A User's Guide: 259-345 H. Freeman Cyclization of peptides also can be achieved, for example, by formation of cystine bridges, coupling of amino and carboxy terminus groups of respective terminus amino acids, or coupling of the amino group of a Lys residue or a related homolog with a carboxy group of Asp, Glu or a related homolog. Coupling of the alpha-amino group of a polypeptide with the epsilon-amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken. See, for example, Wood and Wetzel, 1992, Intl J. Peptide Protein Res.

39: 533-39.

Another approach described in US 5,891,418 is to include a metal-ion complexing backbone in the peptide structure. Typically, the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the coordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a coordination number of four to six. The nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulfur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities. In addition, the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino.

A further approach approach is to use bifunctional crosslinkers, such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl pyridyldithio) propion amido] hexanoate, and sulfosuccinimidyl pyridyldithio) propionamido]hexanoate (see US Patent 5,580,853).

Techniques for chemically synthesising the peptides and derivatives described above are described in the above references and also reviewed by Borgia and Fields, 2000, TibTech 18: 243-251 and described in detail in the references contained therein.

Peptides used in the present invention can be readily synthesised by solid phase procedures well known in the art. Suitable syntheses may be performed by utilising "Tboc" or "F-moc" procedures. Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Alternatively, the peptides may be produced by recombinant methods, including expressing nucleic acid molecules encoding the mimotopes in a bacterial or mammalian cell line, followed by purification of the expressed mimotope. Techniques for recombinant expression of peptides and proteins are known in the art, and are described in Maniatis, Fritsch, E.

F. and Sambrook et al., Molecular cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Although the mimotopes of the invention can be structural mimics of either linear or conformational epitopes, preferably the epitope is a conformational epitope.

Further EBV peptide mimotopes can be isolated using the methods described below.

The mimotopes of the present invention may be modified for the purposes of ease of conjugation to a carrier compound. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine to the peptide. In addition it may be desirable for peptides conjugated to a carrier compound to include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier compound. For example, the peptidic mimotopes may be modified to have an Nterminal cysteine and/or a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide. Furthermore, one or more amino acids may be deleted from the peptidic mimotopes of the invention, as long as a mimotope is retained which is capable of cross-reacting with the antibodies of present interest.

In one embodiment the mimotope is biotinylated at the N-terminus and/or Cterminus of the peptide to allow coupling to avidin/streptavidin coated solid supports.

In another embodiment, the mimotope is conjugated to a linker group.

Preferably, the linker group comprises at least one glycine or at least one serine residue.

For example, the coupling group may comprise three or four glycine residues or three or four serine residues or a combination of glycine and serine residues.

In another embodiment, the mimotope is conjugated to an adhesive compound.

Preferably, the adhesive compound is an adhesive peptide. Suitable adhesive peptides include those described in copending International Patent Application Number PCT/AU2005/001775, the entire contents of which are incorporated by reference.

Examples of suitable adhesive peptides are those comprising an amino acid sequence according to one of the following formulas: Xal A 1 Xaa2 A 2 Xaa3 A 3 Xaa 4 Xaa5 A 4 or a part thereof (ii) [Xj]nl X1 X2 [Xj]n2 (iii) [Xj]nl X, X2 X3 [Xj]n2 (iv) [Xj]n X1 X 2

X

3 X4 X 5 [Xj]n2 wherein, where present: Xaa any amino acid; and At to A 4 is independently selected from a hydrophobic amino acid selected from; Ala, Gly, Ile, Phe, Pro, Met, Trp, Tyr, Val; D or L isomers thereof; and a functional analog thereof as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substitutent; Xi is Phe, Tyr or Trp;

X

2 is His;

X

3 is Phe, Tyr or Trp;

X

4 is any naturally or non-naturally occurring amino acid;

X

5 is Phe, Tyr or Trp; and [Xj]n is a sequence ofn amino acids wherein nl n2 is from 0 to 50 amino acids and wherein the sequence Xj may comprise the same or different amino acids selected from any naturally or non-naturally occurring amino acid; or a functional derivative thereof comprising at least about 20% amino acid sequence similarity thereto or a homolog, mimetic or analog thereof.

The sequence Xj may also comprise at least one proline. The recited amino acid sequences may also be repeated. Repetitions include single or multiple repeats of the recited sequence. In other embodiments, nl n2 is from 2 to 50, or any number from 0 to As used with reference to adhesive peptides, the term "alkyl" denotes saturated straight chain, branched or cyclic hydrocarbon groups, preferably CI- 20 alkyl, e.g. Cl- 1 0 or C.- 6 Examples of straight chain and branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, n-pentyl and branched isomers thereof, nhexyl and branched isomers thereof, n-heptyl and branched isomers thereof, n-octyl and branched isomers thereof, n-nonyl and branched isomers thereof and n-decyl and branched isomers thereof. Examples of cyclic alkyl groups include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.

As used with reference to adhesive peptides the term "alkynyl" denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethynically mono-, di- or poly- unsaturated alkyl or cycloalkyl groups as previously defined. The term preferably refers to C2- 20 alkynyl. Examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers.

As used with reference to adhesive peptides, the term "aryl" denotes a C6-C14 aromatic hydrocarbon group. Suitable aryl groups include phenyl, biphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl and phenanthrenyl. Preferred aryl groups include phenyl, biphenyl and naphthyl.

The used with reference to adhesive peptide the term "heterocyclyl" denotes monocyclic, polycyclic or fused, saturated, unsaturated or aromatic hydrocarbon residues, wherein one or more carbon atoms (and where appropriate, hydrogen atoms attached thereto) are replaced by a heteroatom. Suitable heteroatoms include, 0, N, S, and Se. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable examples of heterocyclic groups may include pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholino, indolinyl, imidazolidinyl, pyrazolidinyl, thiomorpholino, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, pyridyl, thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. Preferred heterocyclyl groups include but are not limited to indolinyl, pyridyl, indolyl, quinolinyl, isoquinolinyl and quinazolinyl.

In an illustrative example, Al to A 4 is independently selected from one of Trp, Tyr, Phe, Ala, DY, DW, DF, 60 (L-1-beta-Naphthylalanine), 103 (Beta-(3-Pyridyl)-Lalanine), 161 (Nz-Dabcyl-L-lysine), 264 (beta-(2-quinolyl)-alanine) and 389 (Phenylphenylalanine; Biphenylalanine).

Preferred adhesive peptides comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 33 to 51 and SEQ ID NOs: 52 to 88 or a functional derivative thereof having at least 20% similarity to any one of SEQ ID NOS: 33 to 51 and SEQ ID Nos: 52 to 88.

In some embodiments, the adhesive peptide is selected from the group comprising SEQ ID NOs: 58, 59, 60, 61, 66, 70, 74, 75 and 80 or a part thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide. Preferred adhesive peptide is selected from the group consisting SEQ ID NOs: 58, 59, 75, 76, 78, 80, 81, 85, 86 and 88 or apart thereof or a functional form thereof having at least about 40% sequence identity over the full length of the adhesive peptide.

In an illustrative embodiment, the adhesive peptide is selected from the group consisting of SEQ ID NO: 58 wherein Xa any amino acid and wherein A, to A 4 is independently selected from a hydrophobic amino acid selected from: Ala, Gly, Ile, Phe, Pro, Met, Trp, Tyr, Val; and D isomers of any of these; and any of these hydrophobic amino acids as substituted with a non-polar substituent such as, for example, an alkyl, alkenyl, alkynyl, aryl or heterocyclyl substitutent.

In a further illustrative embodiment, the adhesive peptide has the sequence SEQ ID NO: 89.

In some embodiments, the substituted hydrophobic amino acid is alanine. In other particular embodiments, the D isomer is D-tryptophan or D-tyrosine.

In some embodiments, the adhesive peptides are further modified in accordance with the present invention in order to serve a wide range of functions. For example, for different chemical or physical properties (solubility, strength, length weight etc) or in order to immobilize attached molecules or to serve to identify the adhesive peptides or the attached molecule or even the attached molecule bound or otherwise attached to a further one ore more molecules.

In a further embodiment, the adhesive peptide is conjugated to the N-terminus and/or C-terminus of a peptide mimotope according to the present invention, preferably via a coupling or linker group.

In another embodiment, the mimotope is conjugated to a carrier compound.

Preferably, the carrier compound is a carrier protein or a lipid vesicle. Preferably, the carrier protein is keyhole limpet haemocyanin (KLH), serum albumins such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diptheria toxins 19 (TT and DT), or recombinant fragments thereof (for example, Domain 1 of Fragment C of TT, or the translocation domain of DT), CRM197, or the purified protein derivative of tuberculin (PPD).

In one example, the mimotope is covalently conjugated to the carrier compound.

Suitable techniques for such covalent conjugation are commonplace in the art and may be readily adapted for the particular reagents employed. In one embodiment, the mimotope is conjugated to the carrier compound via a coupling or linker group.

The present invention also provides a mimotope of the present invention conjugated to a fusion partner or fusion partners. In a preferred embodiment, the fusion partner or fusion partners comprise the coupling group, the adhesive compound and/or the carrier compound.

Table 1 Codes for non-conventional amino acids Non-conventional Code Non-conventional Code amino acid amino acid cc-aminobutyric acid L-amno-x-methylbutyrate aminocyclopropanecarboxyl ate arninoisobutyric acid aminonorbornylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-glutamic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-ornithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan D-tyrosine D-valine D-ot-methylalanine D-cc-methylargi nine D-ca-methylasparagine D-c-methylaspartate D-cx-methylcysteine D-ca-methylglutamine D-ax-methylhistidine D-cc-methylisoleucine D-cc-methylleucine D-a-methyllysine D-cc-methylmethionine Abu Mgabu Cpro, Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dile Dleu Dlys Dmet Dom Dphe Dpro Dser Dthr Dtrp Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-Nmethylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylomithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline c-methyl-aminoisobutyrate ax-methyl-y-aminobutyrate ac-methylcyclohexylalanine cc-methylcylcopentylalanine ac-methyl-a-napthylalanine cc-methylpenicillaniine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3 -aminopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbarnylethyl)glycine Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nmile Nmleu Nmlys Nrnmet Nmnle Nmnva Nmomn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva.

Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Nomn Nmaabu Anap Nphe Ngln D-oc-methylomithine Dmom D-ca-methylphenylalanine Dmnphe D-oc-methylproline Dmnpro D-c-methylserine Dmnser D-ec-methylthreonine Dmthr D-c-methyltryptophan Dmtrp D-c-methyltyrosine Dmnty D-cx-methylvaline Dmval D-N-methylalanine Dnmala D-N-methylargi nine Dnmarg D-N-methylasparagine Dnmasn D-N-methylaspartate Dnmasp D-N-methylcysteine Dnmcys D-N-methylglutamine Dnmgln D-N-methylglutamate Dnmnglu D-N-methylhistidine Dnmhis D-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysine Dnmlys N-methylcyclohexylalanine Nmchexa D-N-methylomnithine Dnmom N-methylglycine Nala N-methylaminoisobutyrate Nmaib N-(1 -methylpropyl)glycine Nile N-(2-methylpropyl)glycine Nleu D-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvaline Dnmval y-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine Etg L-homnophenylalanine Hphe L-cc-methylarginine Marg L-cc-methylaspar-tate Masp L-cz-methylcysteine Mcys L-a-methylglutamine Mgln L-cc-methylhistidine Mhis L-c-methylisoleucine Mile L-cx-methylleucine Mleu L-cx-methylmethionine Mmet L-ot-methylnorvaline Mnva L-c-methylphenylaianine Mphe L-ax-methylserine Mser L-cx-methyltryptophan Mtrp L-c-methylvaline Mval N-(carbamnylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycine N-(3 ,3-diphenylpropyl)glycine N-(3-guaridinopropyl)glycine N-(1 -hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazolylethyl))glycine N-(3 -indolylyethyl)glycine N-methyl-y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1 -methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamnine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamnine L-cc-methylalanine L-ax-methylasparagine L-oc-methyl-t-butylglycine L-methylethylglycine L-ec-methylglutamate L-cc-methylhomophenylalanine N-(2-methylthioethyl)glycine L-cc-methyllysine L-cc-methylnorleucine L-oc-methylomithine L-ac-methylproline L-a-methylthreonine L-cc-methyltyrosine L-N-methylhomophenylalanine Nasn Nglu Nasp Ncbut Nehep Nchex Nedec Ncdod Ncoct Nepro Ncund Nbhmn Nbhe Narg Nthr Nser Nhis Nhtrp Nmgabu Dnmmet Nmcpen Dnmphe Dnmpro Dnmser Dnmthr Nval Nmanap Nmnpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys Mnle Mom Mpro Mthr Mtyr Nmhphe N-(N-(2,2-diphenylethyl) carbamiylmethyl)glycine I -carboxy- 1 -(2,2-diphenylethylamino)cyclopropane Nz-Dabcyl-L-lysine Phenyl-phenylalanine; Biphenylalanine Nnbhni Nmbc 161 ,3 -diphenyipropyl) arbamylmethyl)glycine L- I -beta-Naphthylalanine Beta-(3-Pyridyl)-L-alanine beta-(2-quinolyi)-alanine Nnbhe 103 264 389 Table 2 Some adhesive peptide sequences Sequence SEQ ID NO: GWTWQWHPW 58 G(DW)TWQWHPW 59 GYTWQWHPW 160 G(DY)TWQWHPW 61 GFTWQWHPW 62 1G(DF)TWQWHPW 63 64 G(103)TWQWHPW G(264)TWQWHPW 66 G(389)TWQWHPW 67 GATWQWHPW 68 GWT(DW)QWHPw 69 GWTYQWHPW GWT(DY)QWHPw 71 GWTFQWHPW 72 GWT(DF)QWHPw 73 74 GWT(103)QWHPW GWT(264)QWHPW 76 GWT(389)QWHPW 77 GWTAQWHPW 78 G(DW)T(DW)QWHPw 79 GYTYQWHPW G(DY)T(DY)QWHPW 81 GFTFQWHPW 82 G(DF)T(DF)QWHPw 83 G(60)T(60)QWHPW 84 G(1 03)T(I 03)QWHPW G(264)T(264)QWHPw 86 G(389)T(389)QWHPW 87 GATAQWHPW 88 [WQWTPWS 89 7 The invention also provides an isolated nucleic acid encoding a peptide mimotope in accordance with the invention. The nucleic acid may be prepared by cloning from a library of sequences or from an organism a phage or a bacterium), or prepared by in vitro synthesis using standard techniques automated solid phase oligonucleotide synthesisers, which are commercially available from many sources) or, less conveniently, by performance of ligation reactions, ligating together component nucleic acid sequences from different sources. Typically the isolated nucleic acid sequence will be a DNA sequence (but could, conceivably, be a sense RNA sequence) and will comprise a minimum of 9 bases. Typically, the nucleic acid (or rather, that portion thereof which encodes the peptide mimotopes) will comprise from 12 to bases, preferably from 15 to 90 bases and more preferably from 15 to 60 bases. The nucleic acid may advantageously comprise other components, such as promoter, enhancer and terminator sequences, one or more origins of replication, and the like. In addition, the isolated nucleic acid may encode a fusion protein, in which the peptide mimotope is fused (at either the 5' or 3' terminus) to another polypeptide moiety such as a polypeptide label. In such embodiments as aforesaid, whilst that portion of the nucleic acid which encodes the mimotope will generally comprise a number of bases within the ranges identified above, the nucleic acid as a whole may be considerably larger. It will be understood that the nucleic acid molecule may, in some embodiments, encode a peptide which consists solely of the peptide mimotope without any extraneous amino acid residues the mimotope will be in isolation from the sequences adjacent thereto in any naturally-occurring molecule from which the mimotope may be derived).

The isolated nucleic acid molecule may conveniently take the form of a plasmid or other replicable moiety.

According to the invention there is also provided a diagnostic reagent comprising at least one mimotope of the invention. In one embodiment, the diagnostic reagents comprises a combination of two, three, four or more mimotopes of the present invention. For example, the diagnostic reagent may comprise two or more peptide mimotopes selected from peptides comprising the sequence SEQ ID NOs: 8, 10, 12 or 22.

In one example, the diagnostic reagent comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10 and a peptide mimotope comprising the sequence SEQ ID NO: 12.

In another example, the diagnostic reagent comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10 and a peptide mimotope comprising the sequence SEQ ID NO: 22.

In another example, the diagnostic reagent comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10, a peptide mimotope comprising the sequence SEQ ID NO: 22 and a peptide mimotope comprising the sequence SEQ ID NO: 12.

In one embodiment of the diagnostic reagent of the invention, a combination of two or more mimotopes are conjugated to a single carrier compound or other scaffold.

In one example, the diagnostic reagent comprises at least one mimotope of the invention conjugated to a label. Preferably, the label is any entity the presence of which can be readily detected. Preferably the label is a direct label, such as those described in detail in U.S. Pat. No. 5,656,503. Direct labels are entities which, in their natural state, are readily visible either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. UV light to promote fluorescence. Examples include radioactive, chemiluminescent, electroactive (such as redox labels), and fluorescent compounds. Direct particulate labels, such as dye sols, metallic sols (e.g.

gold) and coloured latex particles, are also very suitable and are, along with fluorescent compounds, preferred. Of these options, coloured latex particles and fluorescent compounds are most preferred. Concentration of the label into a small zone or volume should give rise to a readily detectable signal, e.g. a strongly coloured area. Indirect labels, such as enzymes, e.g. alkaline phosphatase and horseradish peroxidase, can also be used, although these usually require the addition of one or more developing reagents such as substrates before a visible signal can be detected.

Conjugation of the label to the mimotope of the invention can be by covalent or non-covalent (including hydrophobic) bonding, or by adsorption. Techniques for such conjugation are commonplace in the art and may be readily adapted for the particular reagents employed.

According to the invention there is provided an assay device comprising at least one mimotope of the present invention. In one embodiment, the assay device is selected from the group consisting of a direct enzyme-linked immunosorbent assay device, an indirect enzyme-linked immunosorbent assay device, a direct sandwich enzyme-linked immunosorbent assay device, an indirect sandwich enzyme-linked immunosorbent assay device, a competitive enzyme-linked immunosorbent assay device, biacore device, SAW device, lateral flow assay device or any combination thereof.

Such a device can take different forms, and it can be varied depending on the precise nature of the assay being performed. For example, the mimotope of the invention may be coated onto a solid support, typically nitrocellulose or other hydrophobic porous material. Alternatively, the mimotope may be coated on a synthetic plastics material, microtitre assay plate, microarray chip, latex bead, filter comprising a cellulosic or synthetic polymeric material, glass or plastic slide, dipstick, capillary fill device and the like.

Coating of the mimotopes to these surfaces can be accomplished by methods known in the art and described in, for example, EP-B-0291194. Protein carriers are typically used for such complexing, with BSA or adhesive peptides being the most preferred.

In one embodiment, the mimotope of the invention is releasably immobilised on the solid support. In a further preferred embodiment, the diagnostic reagent is nonreleasably immobilised on the solid support.

Selection, production and use of mimotopes and anti-mimotopes The mimotopes of the invention are typically obtained using screening techniques that involve screening randomised libraries with an antibody that binds to an EBV epitope, preferably a conformational epitope. The antibody may be a polyclonal antibody or a monoclonal antibody, the latter being preferred.

A preferred approach for identifying mimotopes of EBV is the phage-display approach in which peptides are identified and isolated from a large library that bind to EBV antibody-binding sites thereby mimicking the 3-D conformational features of linear or conformational EBV epitopes. These peptides are defined as 'mimotopes' as they mimic the essential features of EBV epitopes but do not necessarily have the same sequence. This approach requires only antibodies for screening purposes and does not require any structural data on the target antigen. The success of this approach is dependent on the ability of peptides to mimic immunodominant epitopes within the native antigens.

The generation and expression of libraries of nucleotide sequences that express peptides/polypeptides is well known in the art. Typically the library comprises a plurality of random nucleotide sequences generated by synthesis of a collection of random synthetic oligonucleotides. A suitable method is described in W097/27213 where degenerate oligonucleotides are produced by adding more than one nucleotide precursor to the reaction at each step. The advantage of this method is that there is complete control over the extent to which each nucleotide position is held constant or randomised. Furthermore, if only G or T are allowed at the third base of each codon, the likelihood of producing premature stop codons is significantly reduced since two of the three stop codons have an A at this position (TAA and TGA).

Oligonucleotide synthesis is performed using techniques that are well known in the art (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, IRL Press at Oxford University Press 1991). Libraries can also be specified and purchased commercially. The synthetic process can be performed to allow the generation of all or most possible combinations over the length of the nucleic acid, thus generating a library of randomised nucleic acids. These randomised sequences are synthesised such that they allow in frame expression of the randomised peptide with any fusion partner.

In one embodiment, the library is fully randomised, with no sequence preferences or constants at any position. In another embodiment, the library is biased, i.e. partially randomised. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. Thus some nucleic acid or amino acid positions are kept constant with a view to maintaining certain structural or chemical characteristics.

Generally the library of peptide sequences will be large enough such that a structurally diverse population of random sequences is presented. This ensures that a large subset of shapes and structures is represented and maximises the probability of a functional interaction.

It is preferred that the library comprises at least 1000 different nucleotide sequences, more preferably at least 104, 105, 106 or 108 different sequences. Preferably at least 5, 10, 15 or 20 amino acid residues of the peptides encoded by the nucleotide sequences are randomised.

Typically, the peptides encoded by the randomised nucleotide sequences comprise no greater than 30 amino acids. In one embodiment, they also comprise no greater than 20 amino acids.

Whilst any suitable technique can be used to screen the library for peptides that bind to an EBV specific antibody of interest, a particularly preferred method is to use phage display technology. In a phage display library, the randomised sequences are cloned as an in-frame fusion with a phage coat protein such that the randomised peptide is displayed on the surface of the resulting phage. The library is then contacted with the antibody of interest and 'binders', i.e. phage that bind to the antibody, are recovered. A specific protocol that may be used to 'pan' a phage display library is described in the experimental section.

Phage recovered in one round of selection may be subject to further rounds of selection. The sequence of peptides identified in the screen can conveniently be determined using standard DNA sequencing techniques.

Mimotopes identified by the above methods may be subject to affinity maturation, using for example error-prone PCR, to produce mimotopes with an increased affinity for the antibody of interest e.g. greater than 5 x 10 7 preferably greater than 108 M 1 In addition, or alternatively, peptide mimotopes may be used to design nonpeptide mimetics, as described above.

The present invention also provides a method for selecting a mimotope of the present invention which method comprises panning a phage display library encoding a plurality of randomised peptides with an antibody that binds specifically to EBV.

In one embodiment, the antibody that binds specifically with EBV is a monoclonal antibody.

For example, the monoclonal antibody may be MAb gpl25 (clone L2), commercially available from Chemicon International (Temecula, CA). Other monoclonal antibodies directed against gpl25 are also available (for example, Clones 2Q1919 and 8.F.96, United States Biologicals) and are suitable for use in the present invention.

In another example, the monoclonal antibody is directed against pl 8. Examples of monoclonal antibodies directed against p18 are described in US 5,424,398. A polyclonal anti-pl8 antibody is available from Biodesign International (Maine, United states).

In one particular embodiment, the monoclonal antibody is MAb Fl produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6833.

In another embodiment, the monoclonal antibody is MAb A2 produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6832.

In another embodiment, the monoclonal antibody is MAb A3 produced by a hybridoma cell line deposited on 6 July 2005 with American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA, under the provisions of the Budapest Treaty under deposit number PTA-6834.

The present invention also provides a peptide selected by said methods.

The present invention also provides a method for isolating a molecule that binds to a mimotope of the present invention, which method comprises contacting a biological sample with a mimotope of the invention, and isolating any complexes formed between the mimotope and molecules present in the sample.

A molecule identified by this method is called an "anti-mimotope". The antimimotope molecule can be any suitable molecule, such as, for example, an antibody, a second peptide, a carbohydrate, a lipid, a DNA molecule, an RNA molecule, a chemically synthesized molecule or a combination of any of these compounds. Such peptides, proteins, or other biological, synthetic, or semi-synthetic molecules that are capable of binding to the mimotope can be identified by: raising antibodies against the mimotope; selecting from bacteriophage, chemical, hybridoma cell, or other types of libraries, cells, or chemical syntheses that might produce a set or subset of molecules having high affinity for the mimotope sequence; or designing molecules intended to have a high affinity for the mimotope sequences using computer-assisted or other theoretical approaches. Suitable anti-mimotopes can also be developed using in vitro evolution of nucleic acids capable of binding to the peptide mimotope (see Joyce 1994). Such anti-mimotopes can be generated in an animal as polyclonal or monoclonal antibodies. Preferably, they are in the form of monoclonal antibodies.

Preferably, the monoclonal anti-mimotopes are human or murine in origin.

Polyclonal anti-mimotopes can be prepared by administering a mimotope of the invention, preferably using an adjuvant, to an animal, preferably a non-human mammal, and collecting the resultant antiserum. .Improved titres can be obtained by repeated injections over a period of time. There is no particular limitation to the species of animals which may be used for eliciting anti-mimotopes; it is generally preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, rats, cows, sheep, etc., can also be used. In the production of anti-mimotopes, a definite amount of a mimotope of the invention is e.g. diluted with physiological saline solution to a suitable concentration and the resulting diluted solution is mixed with complete Freund's adjuvant to prepare a suspension. The suspension is administered to the animal, e.g. intra-peritoneally, e.g. to a rabbit, using from about 50 jtg to about 2500 tg of the mimotope of the invention per administration. The suspension is preferably administered about every two weeks over a period of up to about 2-3 months, preferably about 1 month, to effect immunization. Anti-mimotope is recovered by collecting blood from the immunized animal after the passage of 1 to 2 weeks subsequently to the last administration, centrifuging the blood and isolating serum from the blood.

The anti-mimotopes of the invention are capable of being used in passive prophylaxis or therapy, by administration of the antibodies into a patient, for the amelioration or prevention of disease. Furthermore, the anti-mimotopes of the invention may be humanised or CDR grafted for therapeutic use using techniques known in the art [see, for example, Holliger and Bohlen (1999) Cancer Metastasis Rev.

18:411-9, Gavilondo and Larrick (2000) Biotechniques 29:128-32, 134-6, 138, and Kipriyanov and Little (1999) Mol. Biotechnol. 12:173-201].

Preferably, the patient will be treated with an Fab' fragment preparation from murine monoclonal anti-mimotope or a chimeric human-mouse anti-mimotope (comprising human Fc region and mouse Fab' region) so as to minimize any adverse reaction to the foreign animal immunoglobulin. Murine monoclonal anti-mimotopes may be prepared by the method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature 256 [1975] 495), e.g. by fusion of spleen cells of hyper-immunized mice with an appropriate mouse myeloma cell line. Numerous methods known in the art may be utilized to raise human monoclonal anti-mimotopes, including production by cell line for B lymphocyte hybridization; (ii) human-murine hybridomas; (iii) humanhuman hybridomas; (iv) human x human-mouse heterohybridomas; and repertoire cloning (phage display).

Human x human-mouse heterohybridomas are the most preferred, and involve combining favourable characteristics of both human and murine parental cell types.

Human-mouse heterohybridoma cell lines have been rendered suitable for B cell fusion (Teng, N. N. M. et al., Proc. Natl. Acad. Sci. USA 80 [1983] 7308).

The present invention also provides a pharmaceutical composition comprising the anti-mimotope of the invention. The present invention also provides the use of the anti-mimotope of the invention in the manufacture of the pharmaceutical composition for use in the treatment of EBV infection.

Furthermore, if one has a mimotope it should be possible to select a binding partner, i.e. a compounds that binds to native EBV or artefacts thereof, for drug discovery purposes. Alternatively if one has a binding partner it should be possible to select for a mimotope for, for example, diagnostic, screening or vaccine studies.

Diagnostic methods and kits Mimotopes of the invention may also be used in diagnostic methods for detecting the presence of antibodies against EBV in biological samples. Suitable samples include blood samples and samples taken from the respiratory tract of an animal or human.

Accordingly, the present invention provides a method of diagnosing EBV infection in a subject which comprises: contacting a biological sample from the subject with at least one mimotope according to the invention; and assaying for the presence or absence of a mimotope-antibody complex, wherein the presence of the mimotope-antibody complex is indicative of EBV infection.

In one embodiment, the biological sample is selected from the group consisting whole blood, serum, plasma and saliva.

The design of suitable immunoassays to put this method of diagnosis into effect may be subject to a great deal of variation, and a variety of these immunoassays are known in the art. For example, the immunoassay may utilise one mimotope of the invention; or alternatively, the immunoassay may use a combination of mimotopes of the invention and optionally other known EBV antigens.

Suitable immunoassay protocols may be based, for example, upon competition, or direct reaction, or sandwich type assays. The immunoassay protocols used may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labelled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilise biotin and avidin, and enzyme-labelled and mediated immunoassays, such as ELISA assays.

Mimotopes of the invention may be bound to a solid support, for example the surface of an immunoassay well or dipstick, and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

Accordingly the present invention also provides a diagnostic kit comprising at least one mimotope of the present invention. In a preferred embodiment, the kit further comprises at least one additional such as one or more suitable reagents for performing an immunoassay, a control, or instructions for use of the kit.

In one embodiment, the kit of the present invention comprises a combination of two, three, four or more mimotopes of the present invention. For example, the kit may comprise two or more peptide mimotopes selected from peptides comprising the sequence SEQ ID NOs: 8, 10, 12 or 22.

In one example, the kit comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10 and a peptide mimotope comprising the sequence SEQ ID NO: 12.

In another example, the kit comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10 and a peptide mimotope comprising the sequence SEQ ID NO: 22.

In another example, the kit comprises the combination of a peptide mimotope comprising the sequence SEQ ID NO: 10, a peptide mimotope comprising the sequenceSEQ ID NO: 22 and a peptide mimotope comprising the sequenceSEQ ID NO: 12.

EBV has been identified as the primary cause of infectious mononucleosis.

Latent EBV infection has also be associated with numerous other illnesses. Some of these illnesses are restricted geographically, such as Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) which occur mainly in central Africa and southern China respectively. Until recently, this virus was considered the cause of chronic fatigue syndrome (CFS) sometimes referred to as "yuppie disease", due to its prevalence in more affluent individuals in the United States. The virus has also been implicated in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome and multiple sclerosis. EBV infection has been identified with the generalized lymphoproliferative disease seen in transplant patients and leukoplakia seen in AIDS patients. It has been the suspected cause of Hodgkins and Graves diseases. The common denominator for all these illnesses is the presence of elevated antibody titers to EBV and/or the recovery of virus or identifiable EBV DNA from patients with these illnesses. Numerous investigators have reported connections between EBV-associated illnesses and defects of cellular immunity. Other investigators have reported connections between EBV-associated illnesses and environmental factors.

Therefore, it will be appreciated by the skilled person that the mimotopes of the present invention may also be used in diagnostic methods for detecting the presence of antibodies against EBV in biological samples in order to diagnose or at least to assist in the diagnosis of a disease or condition associated with EBV infection. In particular, the mimotopes may be useful in diagnosing methods associated with the detection of nasopharyngeal carcinoma (NPC).

As will be appreciated by the skilled person, other aspects of the present invention, including for example, vaccines based on the mimotopes of present invention, may be useful in treating other conditions, such as NPC though to be associated with EBV infection.

Administration The mimotopes of the invention may be combined with various components to produce compositions of the invention. Preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition of the invention may be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration. Typically, each mimotope may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.

Vaccines Vaccines may be prepared from one or more mimotopes of the invention.

Preferably, the vaccine composition comprises at least one mimotope that is capable of generating EBV neutralizing antibodies in a subject.

In a further preferred embodiment the vaccine composition comprises at least one mimotope that is capable of specific binding to an antibody that binds to an EBV antigen other than gp125.

The preparation of vaccines which contain an immunogenic mimotope as active ingredient, is known to one skilled in the art. Typically, such vaccines are prepared 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 mimotopes encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for 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, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-Disoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-Disoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero -3-hydroxyphosphoryloxy)ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

Further examples of adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, 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, 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., Rahway, or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, 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.

The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (A1 2 0 3 basis).

Conveniently, the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 pg/ml, preferably 5 to 50 pig/ml, most preferably 15 jtg/ml.

After formulation, the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4 0 C, or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.

The vaccines 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 glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to Oral formulations include such normally employed excipients as, for example, 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% 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.

Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit Eudragit cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

The mimotopes of the invention may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.

The present invention will now be described in more detail with reference to the following non-limiting Examples.

EXAMPLES

EXAMPLE 1 Screening for EBV peptide mimotopes using EBV MAbs Materials and Methods Human sera Individual human serum samples were provided by Queensland Medical laboratory, (Brisbane, Australia). The positive sera were collected from individuals with recent or early stage of active infectious mononucleosis and were tested for the presence of IgM antibodies to EBV VCA using a commercial diagnostic test. The negative sera were collected from patients having no previous exposure to EBV infection and were defined as seronegative using the commercial diagnostic test.

For screening of peptides alone: We screened 41 positive VCA IgM samples, 17 negative VCA IgM samples and 4 additional samples that were IgM seropositive for cytomegalovirus (CMV) and negative for IgM-EBV.

Monoclonal antibodies antibody L2 was purchased from ABI (Maryland, USA). Fl, A2 and A3 monoclonal antibodies (MAb) were raised in-house by immunising mice with partially purified EBV-infected cell extract or crude EBV (ABI, Maryland, USA). This crude EBV preparation was shown to have high reactivity with antibodies to viral capsid antigen (VCA), EBV nuclear antigen (EBNA) and early antigen (EA-D) (Figure Mice were immunised subcutaneously with 20pg EBV extract emulsified in Freund's complete adjuvant. Two bi-weekly booster doses diluted 1:1 in Freund's incomplete adjuvant followed by a final double-dose boost without adjuvant 3 days before culling, were performed. Spleen cells were harvested and hybridomas were generated using standard procedures Clones were assayed for their reactivity to EBV antigens using ELISA (refer to ELISA section).

Phage library and selection We constructed a linear peptide library of 20 random amino acids displayed as N-terminus fusions to protein III of filamentous phage M13, using the Fuse 5 vector The library of>5 x 108 random peptides was screened for peptides that bound to MAbs gp125, Fl, A2 and A3 using microtiter plates as described with the following modifications. Coated wells were blocked with 5% blotto (milk powder diluted in PBS) and the 20-mer phage peptide library was pre-incubated in 1% blotto for 15 mins to remove any milk-binding phage before being added to the MAb coated wells. Phage was amplified, titrated and DNA sequencing was performed using similar procedures as described previously [12].

ELISAs Phage ELISAs were performed by coating 10 tg/ml of antibody in PBS to a microtiter plate (Nunc, Maxisorp) overnight at 4 0 C and subsequent blocking with skimmed milk for 2h. Phage dilutions (100 pil) were prepared in PBS, transferred in duplicate to the coated blocked wells and incubated for I h shaking. Wells were washed times with PBS containing 0.05% Tween 20 (PBST) and 100 Pl of anti-M13 antibody conjugated to horseradish peroxidase (Pharmacia) at 1/5000 dilution was added to each well. After a further Ih incubation and washing as above, bound phage were detected with o-phenylenediamine (OPD) substrate (Sigma). For competition experiments with crude EBV and p18 antigens, the same concentration of phage (50 il) were mixed with varying concentrations of competitor (50 pl) for 30 mins before adding to the coated wells and the same procedure was followed.

A similar format was used for EBV antigen ELISAs, using crude EBV, p18, EBNA, EA-D and VCA antigens for coating (commercially available), and anti-human IgM-HRP (at 1/1000; Silenus) conjugate was added for lh. Binding was detected with 3,3',5,5'-tetramethylbenzidine substrate (TMB, Sigma).

ELISAs with synthetic peptides were performed using peptide immobilizer plates (Exiqon, Vedbaek, Denmark). Briefly, wells were coated with 10tg/ml peptide in 0.1 M sodium carbonate buffer overnight at 4 0 C with gentle agitation. Control wells without any peptide coated were used for each sample. The plate was washed 3 times with PBS-Tween and serum samples (100l) in triplicate at 1/100 dilution in fish gelatin diluent fish gelatin [Sigma], 1% Tween-20, 1% bovine serum albumin, in PBS) were added for 1h. Following a further 5 washes, a 1:1000 dilution of sheep anti-human IgM conjugated to horseradish peroxidase (HRP, Silenus) was added for lh. The wells were washed 5 times and peptide-reactive antibody was detected. All sera were assayed twice to ensure reproducible results. The cut-off for screening ELISAs was determined from the mean of the negative population +3 SD of the mean.

All ELISAs were performed in duplicate or triplicate and the mean individual values are shown as vertical error bars on each of the relevant figures.

Peptide synthesis and reconstitution Peptides were synthesized to >70% purity by AusPep Pty Ltd. (Melbourne, Australia). For A3 peptide an intramolecular disulphide bond was formed between the cysteine residues. Gp125 and F1 peptides were dissolved in dimethyl formamide, A3 in 20% ethanol and A2 in dimethylsulphoxide, at 1-5mg/ml and stored in aliquots at 0

C.

Results MAbs covering different EBV epitopes We raised MAbs by immunising mice with -crude EBV and in order to select for hybridoma's expressing antibodies to various EBV epitopes screening involved selection of clones with different binding specificities. Three MAbs Fl, A2 and A3 exhibiting different binding profiles to EBV components (Figure 2) were selected for use as screening reagents. MAb A2 reacted predominantly with p 1 8, whereas A3 only recognised an epitope present in crude EBV. In contrast, MAb Fl strongly bound both EBV and VCA. The commercial anti-gpl25 MAb reacted predominantly with VCA, with lower binding to crude EBV.

Screening for EBV mimotopes Peptides mimicking the MAbs gp125, Fl, A2 and A3 were isolated by screening a 20 amino acid random linear peptide library using multiple rounds of panning on MAb-coated microtiter plates. To bias selection towards peptides binding with high affinity the stringency of washing was increased with successive rounds of panning [see 6, Parmley and Smith 1988; Cwirla et al. 1990; Scott and Smith 1990; Christian et al. 1992; Smith and Scott 1993]. For each selection an increased number of bound phage was detected after the 3rd round of panning with a further increase in round 4 (Figure 3).

DNA sequencing of 20 clones from rounds 3 and 4 revealed only one peptide was isolated for gp125, Fl and A2 MAbs whereas 3 different sequences were isolated for A3 MAb (Figure No consensus sequence was apparent for all A3 clones although all 3 peptides contained two cysteine residues, which could form an intramolecular disulphide bond. Clone 2 (C2) repeatedly demonstrated the highest binding compared with Cl and C3 at the same phage concentration (Figure therefore A3 C2 was selected for further study. There was little homology between any of the isolated clones as would be expected since the peptides should recognise different epitopes. In addition none of the peptides had a convincing match with EBV proteins using blast analysis. This is perhaps because many epitopes rely on conformation and therefore peptides mimicking these would not necessarily be an exact sequence match as the corresponding epitope on the antigen.

Fl, A2 A3 and gp125 isolated clones bound only to their corresponding MAb indicating binding was specific for individual epitopes and these were not overlapping (Figure Importantly, these results show that all selected phage clones failed to react with the relevant isotype control MAb (IC).

Further, for all selected phage clones, binding to the parent MAb was inhibited by crude EBV antigen (Figure Interestingly clone A2 competed with p18 antigen but required a very high concentration to compete with the EBV antigen (Figure 7A).

This is consistent with the specificity of the parent A2 MAb binding strongly to p18 and weakly to crude EBV (Figure Thus, the peptides we have selected appear to be true epitope mimics, which share the antibody binding specificity of the corresponding epitope on EBV or p 8 in the case of A2.

Peptides corresponding to each of the selected clones were found to bind to the corresponding MAb (data not shown), confirming that the MAb epitopes were contained within the peptide sequence and were not dependent on the phage context.

EBV peptide mimotopes as diagnostic reagents To assess the diagnostic potential of our panel of peptides, we assayed their ability to recognise EBV specific antibodies in clinical samples. An ELISA was designed to capture EBV antibodies in patient's serum by coating peptides in separate wells as solid phase antigens. To ensure blocking was adequate each serum sample was analysed for binding to wells without peptide. We analysed the reactivities of 41 EBV seropositive and 21 EBV seronegative sera for all four peptides individually. The cut-off level was evaluated for each data set by taking the mean OD450nm value of the negatives and adding 3 standard deviations. Using this criterion, any value above the cut-off was defined as positive and below this a negative result and each individual absorbance reading is plotted in Figure 8. Although the majority of absorbance readings were <0.8 OD, the data clearly shows a difference between the EBV positive and negative sera readings. For the negative population, there were no false positives resulting in 100% specificity for all four peptides. Importantly, the peptides did not recognise serum samples that had previously tested positive for reactivity with cytomegalovirus (CMV), which is a common cross-reaction for existing EBV diagnostics For the same set of serum samples we also measured the ability of the peptides to recognise IgG antibodies. Using the ELISA conditions described here the results revealed comparatively low ELISA signals and no clear distinction between positive and negative samples, resulting in very low specificity of detection for IgG (data not shown). Therefore no further diagnostic analysis was performed using the peptides alone for diagnosis of EBV-IgG.

Peptide gp125 was biotinylated at the N'-terminus and the peptide was bound to streptavidin coated microtiter plates. To assess the ability of the peptide to recognise IgM antibodies a small number of clinical samples were screened (Figure This initial screen showed the peptide recognised antibodies in the positive samples and not the negatives. No further analysis was performed using biotinylated peptides however biotin could be attached to both the N' or C' terminus by addition of a lysine residue and preferably with a glycine linker region (eg. 3 or 4 glycine residues) such that the biotin is not directly attached to the N' or C' terminus.

A common cross-reaction with commercial EBV kits using VCA antigen is detection of Q fever. We analysed 17 Q fever samples positive for IgM and 2 negative samples (Figure 10). It was apparent that the peptide gp125 cross reacted with 4/17 of the positive samples. This may have implications for a diagnostic kit if the peptide was combined with other peptides specific for Q fever in order to achieve complete coverage of the IgM antibodies typically produced after infection with Q fever.

Summary and Discussion Our aim was to select peptides using a random peptide library displayed on phage that can mimic key diagnostic epitopes using monoclonal antibodies specific for EBV infection. We screened the library for peptides specific for 4 EBV monoclonal antibodies (Table 3) and as peptides bound to microtiter plates the peptides were able to detect IgM antibodies typically generated after infection with EBV.

Table 3. Sequences of EBV peptides that have been shown to recognise IgM antibodies in patient's sera specific for EBV infection.

Peptide Amino Acid Sequence Gp125 GGWYSFDSPYLMSITEMRLR (SEQ ID NO: 12) F1 YTDSSMAVTLMKFASNFLF (SEQ ID NO: 22) A2 DNYWSFSDSTYWTLRYSSG (SEQ ID NO: 8) A3 ELISSCLVWSARGCLFGGGI (SEQ ID NO: The sensitivity of detection for the IgM-positive samples is shown in Table 4.

As a single peptide Fl had the highest sensitivity of 88% correctly detecting IgM antibodies in 36 out of the 41 positive individual serum samples. A3 peptide had the next highest sensitivity followed by gpl25 and lastly A2 Only one of the EBV IgM-positive serum samples did not give an ELISA signal with any of the peptide mimics, however this sample gave a low signal in the commercial EBV diagnostic test.

A combination of any of the peptides resulted in an increase in the sensitivity (Table since each peptide effectively recognises a different subset of antibodies thus indicating that antibodies to different epitopes are being detected. The best combination of two peptides for recognising EBV-IgM antibodies was gpl25 and A3 with a maximum sensitivity of 97.5%. Overall, the sensitivity was generally slightly higher by combining three peptides but combining all four peptides did not further improve the sensitivity.

Table 4. Detection of EBV IgM-positive sera by the panel of peptides. Each individual serum sample was tested for reactivity with each peptide. The number of positives the peptides detected greater than the cut-off value was recorded out of a maximum of 41 seropositive samples and the sensitivity is shown where 41/41 100%. The sensitivity is also tabulated for possible combinations of 2, 3 or 4 peptides.

1 peptide 2 peptides 71% Gp125 FI 93% F1 88% Gp25 A2 76% A2 54% Gpl25 A3 97.5% A3 85% Fl A2 88% F1 A3 A2 A3 88% 3 peptides 4 peptides F1 +A2 93% Gp125 +F1 +A2 97.5% +A3 Gp125 F +A3 97.5% Gp125 A2 A3 Fl A2 +A3 Diagnosis of EBV-related diseases relies on detection of EBV-specific antibodies in the serum of infected individuals. Current commercial tests usually employ native antigens purified from EBV-producing cultures to capture serum antibodies. As possible replacements for these complex antigens we have generated EBV antigen mimics by selecting peptides from a phage display library. In this study we demonstrated that our panel of peptides can detect IgM antibodies typically produced in human subjects after infection with EBV. One of the peptides was selected against a commercial MAb to the gp125 antigen of VCA, since this antigen is known to be diagnostically important. The other peptides (Fl, A2 and A3) were isolated by screening the library against novel MAbs, generated by immunising mice with the crude EBV antigen, and although the exact antigen the corresponding peptides mimic is not known they all appear to be diagnostically important.

The highest sensitivity of detection of IgM in EBV-positive sera was achieved using the Fl peptide alone and this was further improved when coupled to the carrier molecule BSA It is possible therefore that Fl-BSA could be used alone for diagnosis, however further characterisation of the identity of the epitope and importantly a much larger set of clinical samples must be screened before combinations of peptides be eliminated from our studies.

A high sensitivity of detection was also achieved using A3 peptide although the identity of the A3 epitope is also unknown, this data indicates that the antigen this peptide mimics is important diagnostically. The sensitivity for the peptide was slightly lower 71% and again this was significantly improved when coupled to the carrier protein BSA It is known that this epitope on VCA is important diagnostically and the high level of sensitivity reflects this. Peptide A2 had the lowest sensitivity of detection of 54%; this peptide competed only with p 18 and not with the EBV complex indicating specificity for this antigen. Serum antibodies reactive with this epitope could be less common resulting in the lower detection rate.

The parent antibody A2 is an IgM and this subclass tends to have lower affinity and often higher cross-reactivity than IgG subclasses. Therefore these characteristics could also be reflected in the corresponding A2 peptide.

Exposure to EBV infection will stimulate the immune system to generate antibodies towards multiple exposed surface epitopes and individual serum samples are likely to have antibodies to several immunodominant regions EBV. An effective diagnostic would aim to cover as many of these immunodominant epitopes as necessary to recognise every EBV-positive sample. Each peptide effectively represents a different epitope therefore will recognise a different subset of antibodies, so combining the peptides should lead to greater coverage of these important epitopes and an increase in sensitivity of detection. Our results showed that this phenomenon was observed when any of the peptides were combined the sensitivity increased (Table 4).

The greatest sensitivity was achieved with a combination of only two peptides A3 and gp125, and this was not further improved when 3 or 4 peptides were added.

When we increased the stringency of the cut-off level from the mean of the negative population +3 SD to +5 SD, the sensitivity was highest for A3 and Fl peptides This was further improved when gp125 peptide was added therefore indicating combinations of peptides can achieve a high level of sensitivity using more stringent conditions and there is an advantage of using more than 1 or 2 peptides.

Only one positive sample contained antibodies that were undetectable by any of the peptides alone, which was in fact a weakly positive sample as measured by a commercial ELISA (VCA IgM commercial diagnostic kit). This suggests further peptides are required to achieve 100% coverage of the IgM repertoire and/or an increase in sensitivity of detection is required.

The ELISA plates we used in this study were specifically designed for immobilization of peptides; the free amino groups were adsorbed via an ethylene glycol spacer. Our preliminary experiments showed that this was preferable to adsorption directly onto microtiter plates (data not shown). However, when peptides are displayed on phage they may adopt a more rigid conformation than as a free peptide, as they are anchored at the C-terminus to the pll protein leaving the N-terminus free. We therefore chose to couple the peptides via the C-terminus to BSA resembling their orientation on phage and this led to improved presentation of the peptides and increased their diagnostic sensitivity. In addition more than one peptide may be coupled to the same microtiter well or several different peptides conjugated to the same carrier molecule.

It is also unlikely that all 20 amino acid residues of the peptides will be involved in binding and it is possible by alanine scanning to identify the residues essential for binding Once the essential amino acids have been identified non-essential residues could be removed resulting in shorter peptides that could also be combined. A similar technique was used for the p18 peptide when a "mixotope" representing a collection of mimotopes was synthesized and improved the sensitivity and specificity of detection of EBV antibodies in clinical samples [16].

In this study the peptides appear to be EBV-specific mimotopes and did not cross-react with the four CMV positive sera, resulting in 100% specificity. However, since one of the purposes of a peptide ELISA is to increase specificity by eliminating potentially cross-reactive epitopes present in the full length protein or antigen extract and in remaining contaminants in the purified protein; if these peptides are to be developed for a diagnostic test further screening against all typically cross-reactive species is required. Common cross-reactions of current EBV diagnostic kits are rheumatoid factor and other herpes viruses such as CMV Routine diagnostics have been developed to remove cross-reacting rheumatoid factor by pre-absorption with anti-Ig. Although only 62 samples were used in this study there appeared to be no false positives due to rheumatoid factor.

In conclusion, the data indicates that our panel of peptides can be used in the detection of IgM antibodies typically generated after infection with EBV. This study proves the use of EBV peptide mimotopes in an ELISA system have a high diagnostic potential, despite the fact that there is no prior knowledge of the antigens nor their diagnostic potential. There are also significant advantages over existing commercial tests for EBV as peptides are of relatively low cost, highly stable and easily synthesized. Moreover, peptides can mimic carbohydrate epitopes present on many EBV antigen complexes and allow focus on single sub-specificities thereby avoiding dilution with non-informative epitopes.

EXAMPLE 2 Screening for EBV peptide mimotopes using polyclonal rabbit and human anti-EBV antibodies Methods and Materials Immunisation strategy Partially purified EBV-infected cell extract or crude EBV (ABI, Maryland, USA) was used for immunisation. This crude EBV preparation was shown to have high reactivity with antibodies to viral capsid antigen (VCA), EBV nuclear antigen (EBNA) and early antigen A New Zealand white rabbit was immunised intramuscularly with 200ug EBV extract emulsified in 0.5ml Freund's complete adjuvant. Two bi-weekly booster doses diluted 1:1 in Freund's incomplete adjuvant followed by a final double-dose boost were performed.

Affinity purification of rabbit and human IgG Rabbit antisera and human serum with a high titer of antibodies to EBV were purified using Protein G purification (Pharmacia), according to manufacturer's instructions. Serum was diluted 1:5 in PBS and passed through a 0.2gm syringe filter prior to being applied to the resin. Human and rabbit EBV-antibodies were eluted with 0.1M glycine pH 3, neutralized and dialyzed against PBS.

Phage library and selection A similar panning strategy was used as described in Example 1 for the monoclonal EBV antibodies. Briefly ELISA wells were coated with purified rabbit and human anti-EBV IgG preparations and binders were selected from the 20-mer peptide library by performing 6 rounds of panning.

Results Reactivity of affinity purified anti-EBV IgG from immune rabbit and human serum The rabbit-immunised with EBV was shown to have a high titer of antibodies to crude EBV, VCA and p 8 antigens as shown in Figure 11. The IgG fraction of the antisera was affinity-purified using protein G resin and this preparation was used for immunopanning.

Human serum that had a high titer of EBV antibodies and was highly positive as shown by an EBV diagnostic kit, was purified using protein G resin and binding of the IgG fraction is shown in Figure 12. There was high binding to crude EBV and p18 antigens slightly lower binding to p18 antigen and lower reactivity to early antigens EAD and EBNA.

Selection of peptide mimotopes Peptides mimicking anti-EBV rabbit IgG and anti-EBV human IgG were isolated by screening a 20 amino acid random linear peptide library using multiple rounds of panning in a similar manner to the MAb panning. For selection of phage binding to rabbit EBV IgG an increased number of bound phage was detected after the 2"d round of panning with a further increase in round 3 and a plateau in binding in rounds 4, 5 and 6 (Figure 13A). An increase in binding to human EBV IgG was detected after the 4 th round of panning and this increased further in subsequent rounds (Figure 13B).

Sequences of phage clones We sequenced 10 clones from each round of panning with a high ELISA signal i.e. rounds 4, 5 and 6. The sequences are summarised in Table Table 5: Sequences of clones selected after 4, 5 or 6 rounds of panning on rabbit EBV IgG and human EBV IgG. The underlined residues represent areas of homology.

AMINO ACID SEQUENCE CLONE NUMBER SANLNFFSPDFGLYTPNASA Rabbit R4/1 (SEQ ID NO: 17) FVNAFQNANFMRPRELFALA Rabbit R4/2 (SEQ ID NO: 11) MSDFDRKVYTFNFITDPQHL Rabbit (SEQ ID NO: 2) NGALYPRFFPDYSILMFPII Rabbit R4/6, R6/6 R5/7 (2) (SEQ ID NO: 14) SSSIKIWNKLGWNTVIAGTR Rabbit R4/9 (SEQ ID NO: 3) AITCAHTLSIKSRRCQYVFK Rabbit R5/1 (SEQ ID NO: 4) GVTDFDFKVFSSTFPKIFLS Rabbit R5/3 (SEQ ID NO: AMINO ACID SEQUENCE CLONE NUMBER AASYASRTVGFASVYWFSRP Rabbit R5/4 (SEQ ID NO: 6) LRGTHDFYLQVDMSDLSDLR Rabbit R5/7 (SEQ ID NO: 13) RLRGDYNVGPIRFGWPVAPN Rabbit R5/4 (SEQ ID NO: TPNTVRDFYYNVSLPSYMLI Rabbit R5/2, (SEQ ID NO: RQFSKFKDASDRYGNYLHFF Rabbit R6/4, R6/3, R6/6 (SEQ ID NO: 16) DQFAQAYRGDRNFFNELTST Rabbit R6/10 (SEQ ID NO: 9) TELSLFCDSHGLGLSPYRQC Rabbit R6/4, (SEQ ID NO: 19) NGPSYHVAVHFKNSRGLRHS ALL HUMAN SEQUENCES (SEQ ID NO: 7) Fourteen different sequences were found for selections on the EBV polyclonal anti-rabbit IgG preparation, whereas only one sequence was found after panning on the human EBV IgG preparation. A small amount of homology was observed for some of the sequences R4/5 and R5/3 had a similar region of homology "DFDXKV" (SEQ ID NO: 23), and R4/9 and R5/1 also had a small area of homology "SIK".

Binding of individual phage clones Reactivity of individual phage clones is shown in Figure 14. Individual phage clones were analysed for binding to pre-immune and immune EBV rabbit IgG (Figure 14A). Only 3/14 clones reacted with the pre-immune IgG indicating the remaining 11 clones bind to EBV-specific antibodies in the immune sera. Interestingly the 2 clones and R5/3 described above with a similar area of homology "D F D X K V" both bound to the pre-bleed. The other colony R5/5 (or R5/2) that bound to the pre-bleed also contained "D F" which may also account for binding to the pre-bleed. None of the other sequences contained this "D F" motif sequence.

The one clone selected on the human IgG EBV preparation was also shown to be specific for EBV antibodies. In particular, very low binding was observed for a nonspecific preparation of human IgG (Figure 14B).

All phage clones isolated by polyclonal antibody screening failed to bind to gp125, Fl, A2 and A3 MAbs (data not shown), indicating that they therefore importantly cover different EBV epitopes.

Further, the human IgG phage clone reacted with antibodies in the serum of 4 purified EBV positive individual sera, indicating reactivity with an antibody common to each of these sera (Figure EXAMPLE 3 Screening of EBV IgM patients' sera The following peptides were synthesised for conjugation to BSA (linker sequence GGG and additional C-terminal cysteine residue shown in bold).

SEQ ID No:14 plus GGG linker and terminal C Rabl(GC): NGALYPRFFPDYSILMFPIIGGGC SEQ ID NO:7 plus GGG linker and terminal C Huml(GC): NGPSYHVAVHFKNSRGLRHSGGGC SEQ ID NO:9 plus GGG linker and terminal C Rab6/10: DQFAQAYRGDRNFFNELTSTGGGC SEQ ID NO:16 plus GGG linker and terminal C Rab6/1: RQFSKFKDASDRYGNYLHFFGGGC SEQ ID NO:3 plus GGG linker and terminal C Rab4/9: SSSIKIWNKLGWNTVIAGTRGGGC Mimotopes derived from polyclonal rabbit or human sera (IgG fraction) were coupled to BSA and attached to microtiter plates as solid phase antigens. Human serum previously analyzed using a diagnostic test for EBV was allowed to bind to the peptides and the bound IgM antibodies were detected using anti-human IgM-Horse radish peroxidase conjugate (HRP). The absorbance readings (abs 450nm) for positive, negative and putative cross-reactive sera for Parvo virus (Parvo), herpes simplex virus (HSV), cytomegalovirus (CMV) and rheumatoid factor (RF) were determined. The results are shown in Figure 16 (A The cut-off value is defined as the mean of the negative population =3SD indicated by a solid horizontal line, the sensitivity of detection is shown and since there were no false positives the specificity for each mimotope was 100%.

The individual clones were ranked in order of binding to the immune EBV rabbit IgG and the highest 4 binders that also did not bind to the pre-bleed sera were selected for conjugation to BSA and further analysis. The one human peptide sequence was also synthesised for conjugation to BSA (Huml). Refer to Example 4 for the method of conjugation.

EXAMPLE 4 Presentation of immobilised mimotopes for diagnostic ELISA tests.

In order to improve the presentation of peptides on a solid phase for diagnostic ELISA, our aim was to conjugate peptides to a carrier protein, instead of using the free peptide. It was hoped that this method would lead to an improvement in the sensitivity of detection of IgM serum antibodies typically generated after infection with EBV.

Peptides Gp125 and Fl were synthesised with 4 additional glycine residues and a cysteine residue at the C' terminus, to allow for conjugation via a thiol group to a heterobifunctional crosslinker Succinimidyl 4-(N-maleimidomethyl)cyclohexane 1carboxylate (SMCC), which enables conjugation to the carrier protein BAS.

Protocol: 1. 100 molar excess of SMCC linker (3.25mg) was added to 5mg BSA in lml (final volume) 0.15M sodium phosphate/ 0.15M sodium chloride buffer pH for 2h mixing at room temperature with gentle agitation. *Note. the solution was a bit cloudy.

2. To remove excess linker the mixture was desalted using a PD-10 column (Amersham-Pharmacia) equilibrated with the 25ml of the same buffer (as in 1) containing 0.1M EDTA. The columns have been characterised well so 2.5ml is the void volume, then 7 x 0.5ml fractions were collected. Fractions 2-6 were pooled.

3. For conjugation Img of peptide was incubated with 2mg of BSA-SMCC in the presence of 30% DMSO for 2h at room temperature with gentle agitation.(e.g.

1.25ml BSA-SMCC with 0.5ml DMSO/peptide).

4. The final BSA-conjugated peptide was desalted using a PD-10 column into PBS. Fractions 0.5ml were collected after the 2.5ml void volume, the OD280nm was measured and fractions 2-6 retained. The extinction coefficient for BSA is 0.66.

The BSA-peptide conjugates were stored at 4 0

C.

The synthesised peptides had the following sequences (linker sequence GGGG and additional C-terminal cysteine residue shown in bold): Gp125: GGWYSFDSPYLMSITEMRLRGGGGC (SEQ ID NO: 23 plus GGGG linker and terminal C)) Fl: YTDSSMAVTLMKFASNFLFGGGGC (SEQ ID NO: 24 plus GGGG linker and terminal C)) First, to establish Fl peptide was successfully conjugated to BSA, we tested the binding to the parent MAb Fl (Figure 17).

Peptide-BSA conjugate was then coupled to microtiter plates and BSA alone was used in control wells as follows: i. Coat wells of Maxisorp microtiter plate with 2 or 5tg/ml peptide-BSA conjugate or BSA alone diluted in PBS, 100ll/well, cover and leave to coat overnight in the coldroom.

ii. Take out plate and place on the plate shaker (gentle shaking) at room temp whilst preparing serum dilutions.

iii. Prepare 1/100 dilutions of test sera (100pl/well) in duplicate or triplicate using PBS/0.1% Tween 20 or fish gelatin diluent.

iv. Wash plate 2-3 times with PBS.

v. Add serum incubate Ih room temp shaking.

vi. Wash 5 times with PBS/0.1% Tween vii. Add anti-human HRP IgM, 1001l/well, at a 1/5000 dilution followed bylh shaking.

viii. wash 4 times with PBS/Tween and once with PBS.

ix. Develop with TMB.

Figure 18 shows the dramatic improvement in sensitivity of detection using the Fl- BSA conjugate compared with unconjugated peptide, using 36 positive IgM sera and 12 negative IgM sera. Figure 19 shows a comparison F1-BSA and a VCA IgM commercial diagnostic test.

Table 6. Detection of EBV IgM-positive sera by peptides Gp125 and Fl. Each individual serum sample was tested for reactivity with peptides alone or conjugated to BSA. The number of positives the peptides detected greater than the cut-off value was recorded out of a maximum of 46 seropositive samples and the sensitivity is shown as a percentage. There were no false positives resulting in specificity of 100%.

Peptide alone BSA conjugated 71% Gpl25-BSA 92% Fl 88% Fl-BSA Comparison of using Fl or gp125 peptides alone or conjugated with BSA is shown in Figure 20 and Table 6 where there is a clear advantage of conjugating the peptides to a carrier protein such as BSA in terms of an increase in sensitivity of detection.

EXAMPLE 5 Preparation of BSA/HSA conjugated peptides Gp and Fl peptides were conjugated to high purity BSA and human serum albumin (HSA) and analysed for reactivity by comparison with the previous BSA conjugated material. Both BSA and HSA can be used as coupling agents.

Peptide-BSA conjugate was then coupled to microtiter plates and BSA alone was used in control wells using the same method as described in Example 4.

and F1 peptides were conjugated to high purity BSA and human serum albumin (HSA) as coupling agents. The data comparing the two coupling agents is shown in Figure 21. The sensitivities and cut-off levels were similar for the small number of samples analysed. In conclusion it appears that both BSA and HSA can be used as solid phases for conjugation of EBV mimotopes for diagnostic ELISA's.

EXAMPLE 6 Enhancement of EBV mimotope binding to ELISA plates using adhesive sequences Adhesive peptides have been shown to improve the presentation of peptides to microtiter plates for ELISA. It is our aim to improve the sensitivity of our diagnostic ELISA assays by attaching an adhesive peptide to our EBV mimotopes.

The following two EBV adhesive peptides were synthesised to incorporate an SLT adhesive sequence (shown in bold) with EBV mimotope Fl or gp125 joined by a GGG linker: Fl(WN): -WH WQ WT PWSG GG Y TD S SMA V T LMKFASNFLF- (SEQ ID NO: 31) -G GW Y S FDS P YLMS ITEM RL RGG GWHWQWTP W S- (SEQ ID NO: 32) ELISAs were performed to detect anti-EBV in patient sera and control sera, using plates coated with these EBV adhesive peptides and EBV peptides without adhesive sequence for comparison.

Protocol: 1. EBV peptides were coated onto MaxiSorp microtitre plates (100ul/well) at a concentration of 10pg/ml diluted in coating buffer (3.85g NaHC0 3 1.93g Na 2

CO

3 Dilute in IL dH20, filter sterilise and adjust pH to Plates were incubated overnight at 4°C. Plates were washed 6 X with PBS Tween 0.05%.

2. 200pl/well of Post Coat solution (0.4g Sodium Caseinate; 200ml PBS; Filter sterilise) was added and incubated at room temperature for 1 hour. Plates were washed 6X with PBS Tween 0.05%.

3. 100 pl/well of a 1:100 dilution in specimen diluent (5X specimen diluent obtained from Silenus Labs lot 1436108-5) of patient sera (obtained from Sullivan Nicolaides Pathology. Samples were from acute phase patients.) was added to each well, and incubated for 1 hour at room temperature. Plates were washed 6X with PBS Tween 0.05%.

4. Anti-Human IgM HRP conjugated antibodies were (Anti-Human IgM Affinity Isolated HRP conjugated (Raised in sheep) Cat# AP320P 983033020, Chemicon) diluted 1:5000 in specimen diluent, 100ul was added to each well and allowed to incubate for 1 hour. Plates were washed 6X with PBS Tween 0.05%.

100 pl/well of a 1:100 dilution of TMB substrate (100X TMB Substrate Chemicon, Cat# ESO 18-2500 pl diluted 1:100 in Substrate Buffer Chemicon, Cat# 90005935) was added to each well and incubated in the dark for 100pl/well of Stop solution (H 2 S0 4 Cone. 96% w/v (BDH 10276) 28.1mL, RO/DI water to final volume) was then added and the absorbances were read at a dual wavelength of 450-620nm.

The data demonstrate that an adhesive peptide can significantly enhance binding of EBV mimotope Fl to standard ELISA plates, leading to markedly higher detection signal for patient sera. As shown in Figure 22, ELISA using FI adhesive peptide, Fl(WN), can detect anti-EBV in 65% (13/20) of patient sera tested and only one serum in the negative group is weakly reactive, whereas Fl alone could only detect anti-EBV in one patient serum, with low assay reactivity. Similar enhancement of reactivity has been observed with the gp125 EBV mimotope (not shown).

The ELISA conditions described above were used to test the Gpl(WC) and Fl(WN) adhesive peptides on the following samples: 1. Positives: 48 IgM positive individual sera as defined by VCA IgM test, 2. Negatives: 61 IgM negative individual sera as defined by VCA IgM test.

3. Cross-reactive: 17 total (5 CMV positive, 3 V.Zoster, 2 Parvo, 3 HSV, 4 Q fever) individual sera defined as false positive by the VCA IgM test.

Results of these ELISA assays are shown in Figure 23 A (for Gpl(WC) and B (for Fl(WN). These results can be summarised as follows: Gp-WC: 98% specificity, 90% sensitivity FI-WN: 100% specificity, 91% sensitivity Combined: 100% specificity, 98% sensitivity.

EXAMPLE 7: A3 reduction and alkylation of A3 mimotope A3 mimotope: ELI S S CLV W S AR G CL F G G I (SEQ IDNO: The A3 mimotope contains 2 cysteines therefore the peptide was synthesised to contain a disulphide bond between these residues. To establish whether the disulphide bond is important functionally for binding we reduced and alkylated the peptide to break the disulphide bond and analysed the resulting peptide.

Method: 1. A3 peptide (0.6mg) was solubilised in 100tl ethanol then added to 0.4ml 0.1M sodium phosphate/0.15M sodium chloride/5mM EDTA pH 8.0 and dithiothreitol (DTT) was added to a final concentration of 50mM for with gentle agitation.

2. Iodoacetamide was then added to a final concentration of 50mM, to prevent the disulphide bond reforming (alkylate) for 2 hours with gentle agitation.

3. The resulting reduced and alkylated (red/alk) peptide was dialysed into PBS using benzoylated dialysis tubing (cut-off <1,200 kD), with 3 buffer changes.

Results: Both A3 and A3(red/alk) peptides were analysed for their ability to bind to the parent mAb as shown in Figure 24 In addition A3red/alk was also assessed for the ability to recognise EBV IgM antibodies in patients sera as shown in Figure 24 The data suggests that the disulphide bond is not required for binding and it appears that binding was improved after reduction and alkylation. Therefore for subsequent studies a peptide was synthesised containing 2 serines in place of the cysteine residues. In addition to enable conjugation to BSA a glycine linker and a free C-terminal cysteine was added to the sequence. The C-terminal Isoleucine was also omitted from the sequence the final peptide was as follows: A3(SSGC): -E L I S S S L V W S A RGS L F G G G C- (SEQ IDNO: The modified A3 peptide was effectively coupled to BSA and a small set of serum samples previously tested for EBV IgM antibodies was screened. Sixteeen positives, 16 negatives and 8 putative cross-reactive samples were analysed for reactive antibodies and the results illustrated in Figure 24. Although the signals were quite low there was a clear distinction between positive and negative samples and low crossreactivity.

EXAMPLE 8 Detection of IgM antibodies in clinical samples using Fl coupled to SbpA fusion peptides Crystalline bacterial cell surface layers (S-layers) are two-dimensional proteinaceous arrays comprising SbpA protein that are found as the outermost cell envelope component of many bacteria and archea. S-layers completely cover the cell surface during all stages of growth and division, and they are composed of identical species of protein or glycoprotein subunits with a molecular mass ranging from 40 to 200 kDa. S-layer lattices exhibit an oblique, square, or hexagonal symmetry. In bacteria, the S-layer subunits are linked to each other and to the underlying cell envelope layer by non- covalent interactions.

Even after isolation from the cell wall, many S-layer proteins maintain the ability to self-assemble in suspension or to recrystallize on solid supports, such as silicon wafers, gold chips, silanized glass, or plastic materials; on Langmuir lipid films; on liposomes;, and at the air-water interface. Together with the high density and regular arrangement of functional groups in the S-layer lattice, this specific feature was recognised to have broad potential for use in the detection of IgM antibodies in clinical samples using Fl coupled to the SbpA protein.

Nano-S has described technology for generating, producing and applying Slayer fusion proteins that crystallise on ELISA plates and can present an immobilised or genetically fused peptide in a preferential and highly stable way (WO 02/097118; Using this technology, the peptide sequence for mimotope Fl was fused to the C (rSbpA-F1) or N-terminus (rSbpB-Fl) of the S-layer protein and expressed in E.coli cells. The resulting recombinant protein was purified from induced bacterial cultures and crystallised onto microtiter plates.

ELISA Method: 1. Coated plates were blocked with 200p.l/well 2% BSA/PBS Tween 20 overnight.

2. Plates were washed twice with PBS Tween 3. Serum samples were diluted in fish gelatin diluent to 1/100 dilution and 100l applied to blocked wells in duplicate, the plate was incubated for Ih shaking.

4. The plate was washed 5 times with PBS Tween Anti-Human IgM HRP conjugate was applied at 1/3500 in half strength fish gelatin diluent/ half strength PBS Tween, 100l per well for h shaking.

6. The plate was washed 4 times with PBS Tween and once with PBS.

7. TMB substrate was used to develop the ELISA.

As shown in Figure 25 (A the rSbpA surface appears to be superior to the rSbpB surface for detection of EBV IgM antibodies. The background levels are very low giving a good signal:noise ratio, when compared with other surfaces such as BSA.

All documents cited herein are incorporated by reference in their entirety.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Furthermore, the various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently features specified in one section may be combined with features specified in other sections, as appropriate.

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Claims (43)

1. A purified mimotope capable of binding to an antibody to Epstein-Barr Virus (EBV).

2. The mimotope according to claim 1, wherein the mimotope specifically binds an antibody to EBV.

3. The mimotope according to either claim 1 or claim 2, wherein the antibody to EBV is specific to EBV.

4. The mimotope according to any one of claims 1-3, wherein the antibody binds to at least one antigen selected from the group consisting of EBV viral capsid antigen (VCA), EBV nuclear antigen (EBNA), and early antigen (EA). The mimotope according to any one of claims 1-4, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

6. The mimotope according to any one of claims 1-5, wherein the antibody is a monoclonal antibody.

7. The mimotope according to claim 6, wherein the monoclonal antibody is selected from the group consisting of MAb gpl25, a monoclonal antibody directed against p 1 8, MAb Fl produced by a hybridoma cell line deposited under ATCC deposit number PTA-6833, MAb A2 produced by a hybridoma cell line deposited under ATCC deposit number PTA-6832, or MAb A3 produced by a hybridoma cell line deposited under ATCC deposit number PTA-6834.

8. The mimotope according to any one of claims 1-7, wherein the mimotope comprises the peptide sequence SIK.

9. The mimotope according to any one of claims 1-7, wherein the mimotope comprises a sequence selected from the group consisting of: SSSIKIWNKLGWNTVIAGTR (SEQ ID NO: 3); AITCAHTLSIKSRRCQYVFK (SEQ ID NO: 4); AASYASRTVGFASVYWFSRP (SEQ ID NO: 6); NGPSYHVAVHFKNSRGLRHS (SEQ ID NO: 7); DNYWSFSDSTYWTLRYSSG (SEQ ID NO: 8); DQFAQAYRGDRNFFNELTST (SEQ ID NO: 9); ELISSCLVWSARGCLFGGGI (SEQ ID NO: FVNAFQNANFMRPRELFALA (SEQ ID NO: 11); GGWYSFDSPYLMSITEMRLR (SEQ ID NO: 12); LRGTHDFYLQVDMSDLSDLR (SEQ ID NO: 13); NGALYPRFFPDYSILMFPII (SEQ ID NO: 14); RLRGDYNVGPIRFGWPVAPN (SEQ ID NO: RQFSKFKDASDRYGNYLHFF (SEQ ID NO: 16); SANLNFFSPDFGLYTPNASA (SEQ ID NO: 17); SKLLYNYGACRTGCYMAGR (SEQ ID NO: 18); TELSLFCDSHGLGLSPYRQC (SEQ ID NO: 19); TPNTVRDFYYNVSLPSYMLI (SEQ ID NO: VMDECVFSSISVLFCNHMLH (SEQ ID NO: 21); and YTDSSMAVTLMKFASNFLF (SEQ ID NO: 22). The mimotope according to any one of claims 1-7, wherein the mimotope comprises a retro, inverso, or retro-inverso sequence of at least one of SEQ ID NOs 3, 4 and 6-22.

11. The mimotope according to any one of claims 1-10, wherein the mimotope is a peptide further comprising a heterologous amino acid sequence.

12. The mimotope according to any one of claims 1-11, wherein the mimotope is stabilized by a covalent modification.

13. The mimotope according to claim 12, wherein the modification is a cyclization and/or incorporation of gamma-lactam or other bridge.

14. The mimotope according to any one of claims 1-13, wherein the mimotope is a structural mimic of either a linear or conformational EBV epitope.

15. The mimotope according to any one of claims 1-14, wherein the mimotope is conjugated to a linker group.

16. The mimotope according to claim 15, wherein the linker group comprises at least one glycine or at least one serine residue.

17. The mimotope according to any one of claims 1-16, wherein the mimotope is conjugated to an adhesive compound.

18. The mimotope according to claim 17, wherein the adhesive compound is an adhesive peptide.

19. The mimotope according to any one of claims 1-18, wherein the mimotope is conjugated to a carrier compound. The mimotope according to any one of claims 1-19, wherein the mimotope is conjugated to at least one fusion partner.

21. An isolated nucleic acid molecule encoding a mimotope according to any one of claims 1 20, wherein the mimotope is a peptide mimotope.

22. The nucleic acid molecule according to claim 21 prepared by cloning from a library of sequences or from an organism.

23. The nucleic acid molecule according to claim 22, wherein the organism is a phage or a bacterium.

24. A diagnostic reagent comprising at least one mimotope according to any one of claims 1-20. The diagnostic reagent according to claim 24, comprising two or more mimotopes selected from the group consisting of: a. a mimotope comprising the sequence DNYWSFSDSTYWTLRYSSG (SEQ ID NO: 8); b. a mimotope comprising the sequence ELISSCLVWSARGCLFGGGI (SEQ ID NO: c. a mimotope comprising the sequence GGWYSFDSPYLMSITEMRLR (SEQ ID NO: 12); and d. a mimotope comprising the sequence YTDSSMAVTLMKFASNFLF (SEQ ID NO: 22).

26. The diagnostic reagent according to either claim 24 or claim 25, wherein the mimotope(s) is/are conjugated to a single carrier compound or other scaffold.

27. The diagnostic reagent according to any one of claims 24-26, wherein at least one mimotope is conjugated to a label.

28. The diagnostic reagent according claim 27, wherein the label is selected from the group consisting of a radioactive compound, a chemiluminescent compound, an electroactive compound, a fluorescent compound, and a direct particulate compound.

29. The diagnostic reagent according to claim 27, wherein the label is an indirect label. The diagnostic reagent according to claim 29, wherein the indirect label is alkaline phosphatase or horseradish peroxidase.

31. An assay device comprising at least one mimotope according to any one of claims 1-20.

32. The assay device according to claim 31, wherein the assay device is selected from the group consisting of a direct enzyme-linked immunosorbent assay device, an indirect enzyme-linked immunosorbent assay. device, a direct sandwich enzyme- linked immunosorbent assay device, an indirect sandwich enzyme-linked immunosorbent assay device, a competitive enzyme-linked immunosorbent assay device, biacore device, SAW device, a lateral flow assay device, and a combination of at least two of the above-mentioned devices.

33. The assay device according to either claim 31 or claim 32, wherein the at least one mimotope is releasably immobilised on the solid support.

34. The assay device according to any one of claims 31-33, wherein the at least one mimotope is non-releasably immobilised on the solid support. A method for screening for a mimotope that selectively binds to an antibody to an EBV epitope comprising contacting one or more candidate compound(s) from a randomised library, wherein a compound that selectively binds to the antibody is identified as a mimotope.

36. The method according to claim35, wherein the EBV epitope is a conformational epitope.

37. The method according to either claim 35 or claim 36, wherein the method comprises phage display technology.

38. A method for selecting a mimotope according to any one of claims 1-20, which method comprises panning a phage display library encoding a plurality of randomised peptides with an antibody that binds specifically to EBV.

39. The method according to claim 38, wherein the antibody that binds specifically with EBV is a monoclonal antibody. The method according to claim 39, wherein the monoclonal antibody is an antibody directed again gp125, an antibody is directed against p18, MAb Fl produced by a hybridoma cell line deposited under deposit number PTA-6833, MAb A2 produced by a hybridoma cell line deposited under deposit number PTA- 6832, or MAb A3 produced by a hybridoma cell line deposited under deposit number PTA-6834.

41. A method for isolating an anti-mimotope molecule that binds to the mimotope according to any one of claims 1-20, the method comprising contacting a biological sample with the mimotope, and isolating any complexes formed between the mimotope and anti-mimotope molecules present in the sample.

42. An anti-mimotope isolated according to the method of claim 41.

43. A pharmaceutical composition comprising the anti-mimotope according to claim 42.

44. Use of the anti-mimotope according to claim 42 in the manufacture of the pharmaceutical composition for use in the treatment of EBV infection. A method of diagnosing EBV infection in a subject which comprises: a. contacting a biological sample from the subject with at least one mimotope according to any one of claims 1-20; and b. assaying for the presence or absence of a mimotope-antibody complex, wherein the presence of the mimotope-antibody complex is indicative of EBV infection.

46. The method according to claim 45, wherein the biological sample is selected from the group consisting whole blood, serum, plasma and saliva.

47. A diagnostic kit comprising at least one mimotope according to any one of claims 1-20.

48. The diagnostic kit according to claim 47, further comprising at least one additional such as one or more suitable reagents for performing an immunoassay, a control, or instructions for use of the kit.

49. The diagnostic kit according to either claim 47 or claim 48, wherein the kit comprises at least one mimotope selected from the group consisting of: a. a peptide comprising the sequence DNYWSFSDSTYWTLRYSSG (SEQ ID NO: 8); b. a peptide comprising the sequence ELISSCLVWSARGCLFGGGI (SEQ ID NO: c. a peptide comprising the sequence GGWYSFDSPYLMSITEMRLR (SEQ ID NO: 12); and d. a peptide comprising the sequence YTDSSMAVTLMKFASNFLF (SEQ ID NO: 22). A vaccine composition comprising at least one mimotope according to any one of claims 1-20.

51. The vaccine composition according to claim 50, further comprising at least one of a pharmaceutically acceptable carrier, a diluent a wetting or emulsifying agent, a pH buffering agent, and adjuvant.

52. The vaccine composition according to either claim 50 or claim 51, wherein the vaccine composition is formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration. Dated this eighth day of August 2006 Diatech Pty Ltd Patent Attorneys for the Applicant: F B RICE CO