WO2003038440A2 - Chemokine binding molecules - Google Patents

Abstract

The present invention relates to a family of glycoproteins found in alphaherpesviruses (gG proteins) which bind chemokines and impair their biological function. Methods and means relating to the use of gG proteins in the treatment of diseases, in particular chemokine-mediated diseases, are provided.

Description

CHEMOKINE BINDING MOLECULES

The present invention concerns the modulation of the activity of chemokines. It relates in particular to a family of glycoproteins found in alphaherpesviruses which have been found to bind chemokines and impair their biological function. These glycoproteins have a variety of therapeutic applications.

Chemokines are small proteins that are secreted into the body and mediate diverse immunological responses including cellular migration, positioning and degranulation, angiogenesis and Thl/Th2 cytokine responses (Baggiolini, M. 1998. Nature 392:565-568,

Mackay, C. R. 2001. Nat Immunol. 2:95-101). Chemokines are divided into four families based on the number and configuration of conserved cysteine residues near the N- terminus : CC chemokines [including macrophage inflammatory protein (MIP) -l , RANTES (regulated on activation, normal T-cell expressed and secreted) and monocyte chemotactic protein (MCP) -1] , CXC chemokines [IL-8 and growth-related oncogene (GRO) -α] , the C chemokine [lymphotactin] and the CX₃C chemokine [fractalkine] . Chemokines interact both with specific receptors and cell, surface glycosaminoglycans (GAGs) via distinct binding sites (Schall, T. J., and K. B. Bacon. 1994. Curr. Opin. Immunol. 6:865-873).

Viruses which have co-evolved with the host immune system are known to express proteins which interact with crucial immune molecules to either modulate or inhibit their anti-viral role (Alcami, A., and U. H. Koszinowski. 2000. Immunol Today. 21:447-55, Tortorella, D. et al . 2000. Annu Rev Immunol. 18:861-926.) Examples of viral chemokine homologues, and viral proteins which mimic cellular chemokine receptors or which bind chemokines in solution are known (Alcami, A. et al 1998. J Immunol. 160:624-33, Lalani, A. S. et al 2000. Immunol Today.

21:100-6., Lalani, A. S. et al 1997. J Virol . 71:4356- 63, Parry, C. M. et al 2000. J Exp Med. 191:573-578).

Herpesviruses are grouped as alpha, beta and gamma based not only on comparison of their genome sequence and gene content, but also because of their biological properties. For example, ■ gammaherpesviruses establish latency in lymphoid cells whereas alphaherpesviruses establish latency in neurons .

Glycoprotein gG is encoded by a gene located in the unique short (US) sequences of the genome of alphaherpesviruses, a region that is variable among viruses in both its organisation and gene content. Although the predicted amino acid sequences of gG encoded by the different alphaherpesviruses share some similarity, there is also extensive sequence variation between them. Glycoprotein gG has been found to be expressed at the surface of virus particles and has also been observed to be proteolytically cleaved and secreted into the medium. The function of both membrane and secreted forms of gG remains unknown.

The US4 gene encoding glycoprotein gG was first characterised in HSV-2 as a 92 kDa glycoprotein that is also secreted from infected cells and has no known biological function (Marsden, H. S. et al (1984) J Virol. 50:547-54) . Processing involves the cleavage of a 104 kDa precursor to generate 72 kDa and 31 kDa intermediates. These intermediates are processed to generate the mature gG-2 and a secreted 34 kDa protein respectively. The 34 kDa secreted form is derived from the amino terminal portion of the 104 kDa precursor, while the 72 kDa is derived from the carboxy-terminus (Su, H. K. et al (1987) J Virol. 61: 1735-7, Su, H. K. et al (1993) J Virol. 67:2954-9) .

Corresponding gG glycoproteins have also been identified in HSV-1, bovine herpesvirus 1 (BHV-1) , BHV-5,

Rangiferine herpesvirus 1 (Ranifv-1) , Caprine herpesvirus 1 (CapHV-1) , Cervid herpesvirus 1 (CerHV-1) , equine herpesvirus 1 (EHV-1) , EHV-3, EHV-4, feline herpesvi'rus 1 (FeHV-1) , pseudorabies virus (PrV) and asnine herpesvirus 3 (AHV-1: Ficorilli et al . 1995. Arch. Virol. 140:1653- 1662) . Interestingly, varizella zoster virus (VZV) does not encode a gG homologue . Other alphaherpesviruses which comprise a gG glycoprotein suitable for use in accordance with the present invention are described in van Regenmortel, M. H. V., et al . , ed. , . 2000, Virus Taxonomy, Academic Press, San Diego.

HSV-1 gG was isolated from purified virions and infected cells, where it was determined to be a class I membrane protein of 59 kDa (Richman, D. D. et al (1986) J Virol. 57:647-55). Pathogenesis studies using an HSV-1 mutant showed no phenotype in vitro and only a mild attenuation in the mouse ear model (Balan, P. et al (1994) J Gen Virol. 75:1245-58). It has since been suggested that gG-1 is responsible for virus entry through apical surfaces of polarised epithelial cells in vi tro and in vivo in a mouse cornea model (Tran, L. C. et al . 2000. Proc Natl Acad Sci U S A. 97:1818-22) . The complete DNA sequence of EHV-1 and EHV -4 identified gG as a class I membrane protein with a predicted molecular mass of 45.2 kDa (Telford, E. A. et al 1992. Virology. 189:304-16, Telford, E. A. et al 1998 J Gen Virol. 79:1197-203). EHV-1 and EHV-4 gG were secreted into the medium of infected cells as a 120 kDa disulphide linked homodimer (Drummer, H. E. et al 1998. J Gen Virol. 79:1205-13). The gG coding sequence of EHV-3 has also been reported with a predicted molecular mass of 49 kDa (Hartley et al . 1999. Arch. Virol. 144:2023-2033)

gX from PrV was identified as a 498 amino acid protein that accumulates in the medium of infected cells (Rea, T. J et al 1985. J Virol. 54:21-9). It is released^" into the medium by a proteolytic cleavage, thus removing the transmembrane domain. The secreted form is 99 kDa in size and was cleaved from a mature 115 kDa form of gX (Bennett, L. M., et al 1986. Virology. 155:707-15). Its function in PrV pathogenesis remains unknown (Kimman, T. G. et al 1992. Vet Microbiol. 33:45-52, Thomsen, D. R. et al 1987. J Virol . 61:229-32).^'

BHV-1 and BHV-5 encode gG homologues that have 75% identity at the amino acid level. Antiserum raised against W -expressed BHV -1 gG recognised a 65 kDa polypeptide and a diffusely migrating species of proteins, ranging from 90 to greater than 240 kDa in supernatants and cell associated proteins of 61 and 70 kDa. The diffusely migrating species consists of a 65 kDa protein antigenically related to gG and linked to GAGs, namely chondroitin sulphate (Engelhardt, T., and G. M. Ken. 1996. Virology. 225: 126-35, Keil et al 1996 J. Virol. 70 3032-8) . BHV-1 gG has a role in prevention of apoptosis, and in efficient viral growth in rabbit kidney cells. It was also suggested that gG has a role in direct cell to cell transmission in Madin-Darby bovine kidney (MDBK) cells (Nakamichi, K. et al . 2001. Virology. 279:488-98. Nakamichi, K. et al 2000. Virus Res. 68: 175-81) .

The present inventors have observed that alphaherpesvirus gG glycoproteins bind chemokines . gG glycoproteins bind chemokines in both their soluble and membrane-bound forms . gG polypeptide is shown in the experiments described herein to block the interaction of chemokines with both cellular receptors and cell surface glycosaminoglycans and to inhibit the biological effects of chemokines in vi tro .

Although the genomes of many alphaherpesviruses have been sequenced, there was no indication, prior to the present findings, that these viruses encoded chemokine modulators .

This invention relates, in various aspects, to the use of alphaherpesvirus gG polypeptides or the active chemokine- binding domain (s) thereof in treatment of diseases and disorders where chemokines or analogues are implicated. Diseases mediated by chemokines include inflammatory disorders, including pulmonary inflammation (e.g. asthma and rhinitis) and skin inflammation (e.g. psoriasis), atherosclerosis, multiple sclerosis, arthritis, neoplasia, sepsis, bacterial and viral infections (including mycobacteria and HIV) and colitis (for review see Baggiolini M. (1998) Nature 392:565-568, Baggiolini, M. et al (1994) Advances in Immunology 55:97-179). Administration of gG polypeptides may be useful in neutralizing circulating chemokines by preventing binding to chemokine receptors and/or cell surface glycosaminoglycans and thereby preventing correct presentation of chemokines to cells, thus ameliorating chemokine-mediated responses. Similarly, nucleic acid encoding the amino acid sequence of a gG polypeptide may be inserted into vector systems for controlled cell or tissue expression in an organism.

Chemokines that are inhibited by gG that are associated with disease conditions include MlP-lα, involved for example in allergies, experimental autoimmune encephalomyelitis, anaemia, malarial infection and viral infection such as influenza and coxsackievirus B3 infection; IL-8, involved for example in psoriasis, atherosclerosis, pulmonary fibrosis and sepsis; RANTES, involved for example in asthma, rhinitis, polypsis, allergies and glomerulonephritis; MCP-1, involved for example in cardiovascular disease, lung fibrosis, and atherosclerosis; and eotaxin, involved for example in hypersensitivity and asthma.

gG polypeptides from alphaherpesviruses have the ability to bind chemokines, including CC, C, CXC and CX3C chemokines, and inhibit chemokine activity, for example by inhibiting chemokine interaction with receptors and/or glycosaminoglycans on the cell surface.

Furthermore, gG polypeptides are useful in vivo fox inhibition of chemokine activity in disorders mediated by chemokines. This provides for clinical uses of gG. Furthermore, in seeking treatments for chemokine mediated diseases, the interaction between gG and chemokines allows for screens and assays for molecules that modulate the interaction, either potentiating or inhibiting the binding .

In accordance with various aspects and embodiments of the present invention, gG polypeptide, preferably in an isolated or purified form, may be used in a variety of contexts for both assay methods and therapies.

One aspect of the present invention provides a method of inhibiting activity of a chemokine, the method comprising bringing the chemokine into contact with a gG polypeptide.

Such a method may comprise administering the gG polypeptide to an individual, or it may take place in vi tro, for instance in cell culture. gG polypeptide may be provided to cells in vi tro ox in vivo by means of encoding nucleic acid, wherein the coding sequence is under control of appropriate regulatory sequences for production of the encoded gG polypeptide by expression from the nucleic acid.

A gG polypeptide may be a gG polypeptide of any alphaherpesvirus. Suitable alphaherpesvirus gG polypeptides are shown in Table 2 and include HSV-I US4, HSV-2 US4, EHV-I ORF70, EHV-3, EHV-4 gene 70, BHV-I US4, BHV-5 US4, FeHV-I gG, PrV gX and AHV-1. gG polypeptides are further described below.

gG polypeptide may bind and preferably inhibit a chemokine . Chemokines bound and inhibited by gG in accordance with the present invention include C, CC, CXC and CX3C chemokines. For example, such a gG polypeptide may bind to a CC chemokine such as MlP-lα and/or RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α and/or mouse KC and a C chemokine such as lymphotactin.

Chemokines are implicated in a number of diseases and disorders of the inflammatory system, including those mentioned above.

In accordance with a further aspect of the present invention, there is provided a method of treating a disease or disorder involving chemokine activity, the method comprising administering an gG polypeptide to an individual with such a disease or disorder. The disease or disorder may involve activity of a C, CC, CXC or CX₃C chemokine, for example a CC chemokine such as MlP-lα and RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α and mouse KC, or a C chemokine such as lymphotactin.

A disease or disorder involving chemokine activity is a disorder which is mediated by one or more chemokines or which is associated with increased chemokine activity.

Diseases and disorders that involve chemokine activity which may be treated in accordance with the present invention include inflammatory disorders, allergic diseases, for example asthma, rhinitis and anaphylaxis, chronic pulmonary diseases, psoriasis, atherosclerosis, multiple sclerosis, arthritis, neoplasia, sepsis, infectious diseases including for example malaria, tuberculosis and HIV, inflammatory bowel disease, diabetes, colitis and transplant rejection. gG polypeptides may also interact with the proteins from HIV and P. vivax that mediate attachment to chemokine receptors in the target cell. HIV binds to chemokine receptors such as CCR5 and CXCR4 in target cells and blockade of that interaction has been shown to prevent HIV infection. The malaria parasite P. vivax uses a chemokine receptor of unknown function (Duffy antigen) to enter and infect erythrocytes, and blockade of this interaction prevents infections by P. vivax.

gG polypeptides from alphaherpesviruses may be used as the basis for agents that can bind with higher affinity than gG proteins to the domains of HIV gpl20 that interact with the cellular chemokine receptors and as the basis for agents to block attachment of P. vivax to cne Duffy antigen on erythrocytes and initiation of malarial infection.

Further aspects of the present invention relate to the use of recombinant alphaherpesviruses with specific disruptions in the gene encoding gG that result in reduction or loss of function. Such mutant viruses are useful as vaccines because they have reduced virulence (i.e. they are attenuated: mutant virus has a reduced ability to replicate in vivo because of an enhanced antiviral immune response) and/or more immunogenic (increased recruitment of immune cells results in increased immunogenicity). Here 'recombinant' means transgenic i.e. genetically altered by human intervention, for example by techniques of genetic manipulation.

Recombinant alphaherpesvirus having reduced or abolished gG glycoprotein function, a recombinant alphaherpes virus having reduced or abolished gG glycoprotein function for use in a method of treatment of the human or animal body, a pharmaceutical composition comprising such a recombinant alphaherpesvirus and a pharmaceutically acceptable excipient and the use of such a recombinant ^' alphaherpes virus in the manufacture of a medicament for use in the treatment of alphaherpesvirus infection are all provided by aspects of the invention.

An alphaherpesvirus with reduced or abolished gG glycoprotein function may have a disrupted, inactivated or mutated gG gene, for example a gG gene that has been disrupted by insertion, deletion or frameshift, or a disrupted regulatory sequence for expressing the gG'gene. Suitable gG genes are discussed elsewhere herein. A disrupted gG gene may have no expression or express low levels of gG polypeptide, or may express mutant gG polypeptide with zero or low activity. Uses of such recombinant alphaherpesviruses are discussed further below.

In a further aspect, the present invention provides a gG polypeptide, a composition comprising a gG polypeptide (e.g. also comprising a pharmaceutically acceptable vehicle, diluent excipient or carrier) , a nucleic acid encoded a gG polypeptide or recombinant alphaherpesvirus as described above for use in a method of treatment of the human or animal body by therapy. The gG polypeptide, composition comprising the gG polypeptide or nucleic acid encoded a gG polypeptide may be for use in a method of treatment of any disease or disorder involving chemokine activity, e.g. as set out in the preceding paragraph. The disease or disorder may involve activity of a chemokine, for example a CC chemokine such as MlP-lα and RANTES, a CXC chemokine such as IL-8, uman BCA-1, human SDF-lα, mouse MIG, human GRO-α and mouse KC, a CX3C chemokine such as fractalkine and/or a C chemokine such as lymphotactin. gG polypeptides and encoding nucleic acids are further discussed below.

Further aspects of the present invention relate to the induction of antibodies against gG, for example by vaccination with recombinant protein. This neutralises the activity of gG after alphaherpesvirus infections and may enhance the anti-viral immune response (due to lack of gG activity that evades the immune system) and/or prevent infection (as gG expressed at the virus particle plays a role in virus attachment and cell tropism) .

Isolated antibody molecules that specifically bind a gG polypeptide and neutralise its activity, and therapeutic applications of such molecules as described below, are also provided by aspects of the invention. Suitable antibodies may be obtained and/or identified using assay methods as described herein.

Antibodies may be obtained using techniques that^* are standard in the art . Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , ^' 1992, Nature 357: 80-82). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal. As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naϊve, that is constructed from sequences obtained from an organism that has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism that has .been exposed to the antigen of interest .

Antibodies according to the present invention may be modified in a number of ways. Indeed, the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included. The reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. The mode of determining binding is not a feature of the present invention and those skilled in the art are able to Choose a suitable mode according to their preference and general knowledge.

Antibodies may also be used in purifying and/or isolating a polypeptide or peptide for use in the present methods, for instance following production of the polypeptide or peptide by expression from encoding nucleic acid therefor.

In a further aspect, the invention provides for use of a gG polypeptide, a nucleic acid encoded a gG polypeptide or an antibody molecule which specifically binds a gG polypeptide in the manufacture of a medicament for inhibiting chemokine activity, for example in the treatment of a disease or disorder which involves chemokine activity.

In a further aspect, the invention provides for use of an alphaherpesvirus having reduced or abolished gG polypeptide function in the manufacture of a medicament for treating a disease or disorder involving alphaherpesvirus infection.

A further aspect of the present invention provides a method of making a pharmaceutical composition comprising admixing a gG polypeptide, a nucleic acid encoded a gG polypeptide or a recombinant alphaherpesvirus having reduced or abolished gG polypeptide function or an antibody which specifically binds a gG polypeptide with a pharmaceutically acceptable excipient, vehicle, diluent or carrier, and optionally other ingredients.

As discussed further below, the present invention also encompasses substances that are able to affect the ability of an gG polypeptide to bind and/or inhibit activity of a chemokine, for instance to potentiate or increase binding and/or inhibition or inhibit or reduce binding and/or inhibition. Such substances may be useful in treatment of disease, especially alphaherpesvirus infection. A substance that potentiates the effect of a gG polypeptide may also be used to potentiate the effect of a gG polypeptide in therapy (for example in therapy of an inflammatory disorder), e.g. by combined, simultaneous or sequential administration. ^■

In various further aspects, the invention thus provides a pharmaceutical composition, medicament, drug or other composition for such a purpose, the composition comprising a gG polypeptide, a nucleic acid encoded a gG polypeptide, a recombinant alphaherpesvirus having reduced or abolished gG polypeptide function and/or a substance able to affect the ability of an gG polypeptide to bind and/or inhibit a chemokine, the use of such a polypeptide, nucleic acid, alphaherpesvirus and/or substance in a method of medical treatment, a method comprising administration of such a polypeptide, nucleic acid, virus and/or substance to a patient, e.g. for treatment (which may include preventative treatment) of a medical condition, e.g. a condition associated with one or more chemokine activities, use of such a polypeptide, nucleic acid, virus and/or substance in the manufacture of a composition, medicament or drug for administration for such a purpose, and a method of making a pharmaceutical composition comprising admixing such a polypeptide, nucleic acid, virus and/or substance with a pharmaceutically acceptable excipient, vehicle, diluent or carrier, and optionally other ingredients.

The polypeptide, nucleic acid, alphaherpesvirus ana/or substance may be used as sole active agents or in combination with one another or with any other active substance .

Whatever the polypeptide, nucleic acid, virus and/or substance used in a method of medical treatment of the present invention, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .

A polypeptide, nucleic acid, substance, alphaherpesvirus or composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient . The precise nature of the carrier or other material will depend on the route of administration; which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Administration may also be by aerosol for pulmonary delivery or by topical application to the skin.

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

The substance or composition may be administered in a localised manner to a desired site or may be delivered in a manner in which it targets cells.

Targeting therapies may be used to deliver the active substance more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

Instead of administering such substances directly, they may be produced in the target cells by expression from an encoding nucleic acid introduced into the cells, e.g. from a viral vector. The vector may be targeted to the specific cells to be treated, or it may contain regulatory elements that are switched on more or less selectively by the target cells.

Nucleic acid encoding a gG polypeptide or a peptide able to inhibit gG/chemokine binding may thus be used in methods of gene therapy, for instance in treatment of individuals, e.g. with the aim of preventing or curing (wholly or partially) a disorder.

Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired peptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see US Patent No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, and retroviruses . Many gene therapy protocols in the prior art have used disabled murine retroviruses.

As an alternative to the use of viral vectors in gene therapy other known methods of introducing nucleic acid into cells includes mechanical techniques such as microinjection, transfer mediated by liposomes and receptor-mediated DNA transfer.

Isolated or purified gG polypeptide is generally used in pharmaceutical contexts. A gG polypeptide suitable for use in accordance with the present invention may be obtained or derived from an alphaherpesvirus .

A nucleic acid sequence encoding the amino acid sequence of a gG polypeptide may be readily identified from its position in the alphaherpesvirus genome at US4 or ORF70 (for example the gG gene in HSV-1 is the US4 ORF which is located between US3 and US5 in the HSV-1 genome) .

Alphaherpesvirus gG polypeptides show chemokine binding activity and share little or no sequence identity or similarity with other known chemokine binding molecules.

As described above, gG polypeptides may be membrane bound or soluble and may include gG polypeptides of alphaherpesviruses, such as equine herpesvirus 1 (EHV-1), EHV-3,^' EHV-4, bovine herpesvirus 1 (BHV-1), BHV-5, Rangiferine herpesvirus 1 (RanHV-1) , Caprine herpesvirus 1 (CapHV-1) , Cervid herpesvirus 1 (CerHV-1) , feline herpesvirus 1 (FeHVl) , pseudorabies virus (PrV) , herpes simplex virus 1 (HSV-1) , HSV-2 and Asnine herpesvirus 1 (AHV-1) as well as fragments and alleles of such polypeptides as discussed further below. In some embodiments, a gG polypeptide from EHV-1, EHV-3, BHV-1,

BHV-5, RanHV-1, CapHV-1, CerHV-1 or FeHVl may be used, in particular a gG polypeptide from EHV-1, EHV-3, BHV-1 or BHV-5.

In some embodiments, a gG polypeptide may bind a C chemokine, a CC chemokine and/or a CXC chemokine, but not a CX3C chemokine. For example, a gG polypeptide according to such an embodiment may bind one of a C chemokine, a CC chemokine or a CXC chemokine, or may bind both C and CC chemokines, both C and CXC chemokines, or both CXC and C chemokines or may bind C, CC and CXC chemokines. Examples of C, CC, CXC, and CX3C chemokines are well known in the art .

The primary sequence of the gG polypeptide may be substantially similar to that of a known alphaherpesvirus gG protein, for example HSV-I US4, HSV-2 US4, EHV-I ORF70, EHV-3, EHV-4 gene 70, BHV-I US4 , BHV-5 US4 , FeHV-I gG, PrV gX or AHV-1 as shown in table 2 and may be determined by routine techniques available to those of skill in the art. In essence, such techniques may include using polynucleotides derived from known gG?gene sequences on public databases (as shown in Table 2) as probes to recover and to determine the sequence of the gG gene in other alphaherpesvirus species .

A wide variety of techniques are available for this, for example PCR amplification and cloning of the gene using a suitable source of mRNA, or by methods including obtaining a cDNA library from an alphaherpesvirus- infected cell,- probing said library with a polynucleotide as described under stringent conditions, and recovering a cDNA encoding all or part of the gG polypeptide of that alphaherpesvirus. Where a partial cDNA is obtained, the full length coding sequence may be determined by primer extension techniques.

An "active portion" of a protein means a sequence which is less than said full length sequence, but which retains its essential biological activity. In particular, the active portion retains the ability to bind to and preferably inhibit activity of a chemokine, e.g. a CC chemokine such as MlP-lα and RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α and mouse KC, a C chemokine such as lymphotactin and/or a CX3C chemokine such as fractalkine . Fragments consisting of or comprising active portions of gG proteins are useful as gG polypeptides in accordance with the present invention.

A gG polypeptide as described herein may include heterologous amino acids, such as an identifiable sequence or domain of another protein, or a histidine tag or other tag sequence, and the invention includes a polypeptide consisting essentially of a portion of a gG polypeptide able to bind and preferably inhibit a chemokine .

Isolated gG polypeptides will be those as defined herein in isolated form, free or substantially free of material with which it is naturally associated such as other polypeptides with which it is found in the cell. The polypeptides may of course be formulated with diluents or excipients and still for practical purposes be isolated - for example the polypeptides may be mixed with a carrier if used^' to coat microtitre plates for use in immunoassays, and may be mixed with a pharmaceutically acceptable vehicle, excipient, diluent or carrier when employed in a method of treatment as discussed.

Preferably, gG polypeptides are glycosylated, either naturally or by systems of heterologous eukaryotic cells. In other embodiments (for example if produced by expression in a prokaryotic cell) , gG polypeptides are unglycosylated or lack native glycosylation. The term "lacking native glycosylation" may be used with reference to a polypeptide which either has no glycosylation (e.g. following production in a prokaryotic cell) or has a pattern of glycosylation that is not the native pattern, e.g. a pattern which is conferred by expression in a particular host cell type, such as baculovirus infected insect cells. gG polypeptides which are unglycosylated or lack native glycosylation retain the ability to bind chemokines as described.

gG polypeptides may be modified for example by the addition of a signal sequence to promote their secretion from a cell or of histidine residues to assist their purification. Fusion proteins may be generated that incorporate (e.g.) six histidine residues at either the N-terminus or C-terminus of the recombinant protein. Such a histidine tag may be used for purification of the protein by using commercially available columns which contain a metal ion, either nickel or cobalt (Clontech, Palo Alto, CA, USA) . These tags also serve for detecting the protein using commercially available monoclonal antibodies directed against the six histidine residues (Clontech, Palo Alto, CA, USA) .

gG polypeptides which are amino acid sequence variants, alleles, derivatives or mutants may also be used in accordance with the present invention, such forms having at least 40% sequence identity, for example at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to an alphaherpesvirus gG protein such as HSV-1 US4 , HSV-2 ^' US4, EHV-1 ORF70) , EHV-3, EHV-4 gene 70, BHV-1 US4, BHV-5 US4, FeHV-1 gG, PrV gX or AHV-1 as shown in Table 2. Such a form may have at least 50% sequence similarity, for example at least 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to an alphaherpesvirus gG protein as shown in Table 2. A gG polypeptide which is a variant, allele, derivative or mutant may have an amino acid sequence which differs from a sequence described herein by one or more of addition, substitution, deletion and insertion of one or more (such as from 1 to 20, for example 2, 3, 4, or 5 to 10) amino acids.

Such variants, alleles, derivatives or mutants retain the ability of the wild-type sequence to bind chemokine.

Amino acid similarity and identity are generally defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps . Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al .

(1990) J. Mol . Biol . 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444- 2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J^". Mol Biol . 147 : 195-197), generally employing default parameters .

Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. A gG polypeptide may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid. gG polypeptides may also be generated wholly or partly by chemical synthesis, for example in a step-wise manner. The isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, such as a diluent.

A gG polypeptide may be used in methods of identifying and/or obtaining molecules that affect or modulate the ability of a gG polypeptide to bind a chemokine and/or inhibit chemokine activity or function. Such molecules may be useful in a therapeutic (which may include prophylactic) context. This is discussed in detail below.

A polypeptide may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin.

As noted, a preferred way of producing a gG polypeptide is to employ encoding nucleic acid in a suitable expression system to produce the polypeptide recombinantly.

Nucleic acids suitable for use in accordance with aspects of the present invention may include nucleic acids that comprise a gG nucleic acid sequence set out in Table 2.

Nucleic acids useful in the invention further include nucleic acids which comprise a sequence having at least 50%, 60% or 70% identity, more preferably at least 80%, such as at least 90%, 95%, 98% or 99% sequence identity to the one of nucleic acid sequences of HSV-I US4, HSV-2 US4, EHV-I ORF70, EHV-3, EHV-4 gene 70, BHV-I US4 , BHV-5 US4, FeHV-I gG, PrV gX or AHV-1 or its complement as shown in Table 2.

Preferred nucleic acid for use in accordance with the invention may encode the amino acid sequence of a wild type alphaherpesvirus gG glycoprotein, in which case it may include a nucleic acid sequence as shown in Table 2 or a different nucleotide sequence, as permitted by degeneracy of the genetic code.

A nucleic acid sequence as described above may be provided as a component of a nucleic acid construct, for instance where the coding sequence is placed under regulatory control of a heterologous sequence, such as a promoter. A stop codon may immediately follow the gG coding sequence, e.g. TAA, as occurs naturally in the cloned sequence, or TAG or TGA, or additional coding sequence encoding a peptide tag, protein domain or other heterologous polypeptide sequence may follow, providing a nucleotide sequence encoding a fusion protein.

Nucleic acid sequences encoding all or part of a gG gene can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992) . These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences. Modifications to the wild type sequences described herein can be made, e.g. using site directed mutagenesis, to lead to the expression of modified polypeptides or to take account of codon preference in the host cells used to express the nucleic acid.

In general, short sequences for use as primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art .

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-50 nucleotides) based on the sequence information available on public databases as referenced herein, to a region of the mRNA or genomic sequence encoding the mRNA which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a cell infected with alphaherpesvirus, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Such techniques may be used to obtain all or part of the sequences described herein.

Polynucleotides which are not 100% homologous to the sequences referenced herein, for example gG coding sequences from other alphaherpesviruses, may be used in accordance with the invention and can be obtained in a number of ways .

Variants (for example allelic forms) of the gG polypeptide and nucleic acid sequences described herein may be obtained from other alphaherpesvirus for example by probing DNA libraries with probes including all or part of a nucleic acid encoding a gG polypeptide as described herein under conditions of medium to high stringency (for example for hybridization on a solid support (filter) overnight incubation at 42 °C in a solution containing 50% formamide, 5 x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulphate and 20 μg/ml salmon sperm DNA, followed by washing in 0.03 M sodium chloride and 0.03 M sodium citrate (i.e. 0.2 x SSC) at from about 50 °C to about 60 °C) .

An isolated nucleic acid may be employed which hybridizes to a gG nucleotide sequence as described above under the abovementioned hybridization and washing conditions. Such a nucleic acid is suitable for use as a probe for detecting a gG gene, for example in Southern blots.

Databases may also be screened using conventional in silico methods to identify alphaherpesvirus gG coding sequences . Alternatively, such polynucleotides may be obtained by site directed mutagenesis of a gG nucleotide sequence as set out above or an allelic variant thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Further changes may be desirable to represent particular coding changes that are required to provide, for example, conservative substitutions.

In the context of cloning, it may be necessary for one or more gene fragments to be ligated to generate a full- length coding sequence. Also, where a full-length encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be sub- cloned into expression vectors and activity assayed by transfection into suitable host cells, e.g. with a reporter plasmid.

Preferably, a polynucleotide encoding a gG polypeptide in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, phagemid or baculoviral, cosmids, YACs, BACs, or PACs as appropriate.

The vectors may be provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vi tro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in methods of gene therapy. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.

For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press . Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds. John Wiley & Sons, 1992.

Vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the invention which includes cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding ^{■ •} sequence encoding the polypeptides, and recovering the expressed polypeptides. Polypeptides may also be expressed in in vi tro systems, such as reticulocyte lysate.

Following production of a gG polypeptide, it may be tested for gG activity, e.g. by determination of ability to bind a chemokine for example a CC chemokine such as MlP-lα or RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α or mouse KC, a C chemokine such as lymphotactin or a CX3C chemokine such as fractalkine, or inhibit chemokine activity in an assay, such as in an assay substantially as described in the experimental section herein. Additionally, or alternatively, the polypeptide produced may be tested for immunological characteristics of gG polypeptide, e.g. ability to bind one or more antibody molecules that recognise gG polypeptide. In various further aspects the present invention relates to screening and assay methods and means, and substances identified thereby, especially substances that affect ability of gG polypeptide to bind and/or inhibit chemokine activity.

Thus, further aspects of the present invention provide the use of gG polypeptide (e.g. a polypeptide or polypeptide fragment as described herein that binds a chemokine, and/or encoding nucleic acid therefor) , in screening or searching for and/or obtaining/identifying a substance, e.g. peptide or chemical compound, which interacts and/or binds with a gG polypeptide and interferes with its chemokine binding activity.

In various aspects the present invention is concerned with provision of methods for identifying and/or obtaining a compound that interacts with or binds a gG polypeptide and modulates its chemokine binding activity, and thereby modulates chemokine activity.

Methods may include identifying a compound as an agent that interacts with a gG polypeptide and modulates its chemokine binding activity. Such an agent or substance may be isolated, and/or purified, synthesised, manufactured and/or used to modulate gG polypeptide activity as discussed.

Determination of the ability of a test compound to interact and/or bind with a gG polypeptide may be used to identify that test compound as a candidate for a modulator of ability of gG polypeptide to bind and/or inhibit a chemokine, e.g. a CC chemokine such as MlP-lα or RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α or mouse KC, a C chemokine such as lymphotactin or a CX3C chemokine such as fractalkine.

Identification of ability of a test compound or substance to bind a gG polypeptide may be followed by one or more further assay steps involving determination of whether or not the test compound is able to inhibit gG binding to a chemokine and/or affect gG activity (such activity being ability to bind and/or inhibit activity of a chemokine) .

Inhibition of chemokine activity may include inhibiting the binding of a chemokine to a chemokine receptor or inhibiting the binding of a chemokine to a cell surface glycosaminoglycan.

For example, methods of the invention may further comprise contacting a chemokine, a gG polypeptide and a chemokine receptor or glycosaminoglycan in the presence of a test compound under conditions in which the gG polypeptide inhibits the binding of the chemokine to the receptor or glycosaminoglycan.

An increase or reduction in the amount of inhibition in the presence relative to the absence of test compound being indicative that the test compound is a modulator of gG polypeptide activity.

Naturally, assays involving determination of ability of a test substance to modulate gG activity may be performed where there is no knowledge about whether the test substance can bind or interact with gG polypeptide, but a prior binding/interaction assay may be used as a "coarse" screen to test a large number of substances, reducing the number of candidates to a more manageable level for a functional assay involving determination of ability to modulate gG polypeptide activity.

A method of identifying and/or obtaining a compound which modulates chemokine activity may comprise:

(a) bringing into contact a gG polypeptide, a chemokine polypeptide; and a test compound, under conditions in which in the absence of the test compound being an inhibitor, the gG polypeptide and the chemokine polypeptide interact or bind; .

(b) determining interaction or binding between the gG polypeptide and the chemokine polypeptide.

A difference in interaction or binding in the presence and absence of the test compound is indicative of a modulating (i.e. an inhibitory) effect of the relevant test compound.

A method may include identifying the test compound as an inhibitor of binding between gG polypeptide and chemokine .

Suitable gG polypeptides for use in such methods are described above and may be soluble or membrane bound. A suitable chemokine polypeptide may be any chemokine which binds to the gG polypeptide, for example, a CC chemokine such as MlP-lα or RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α or mouse KC, a C chemokine such as lymphotactin or a CX3C chemokine such as fractalkine . Other suitable chemokines are set out in A. Zlotnik and 0. Yoshie. (2000) Immunity- 12:121-127. A quantitative method, that is a method in which the degree of binding can be measured, whether increased or decrease, allows for identification of test compounds that are able to potentiate or inhibit gG polypeptide binding to chemokine polypeptide.

A method may further include determining the ability of the test compound to modulate gG polypeptide activity as described above.

It is not necessary to use the entire full-length proteins for assays of the invention that test for gG polypeptide activity (see below) . Fragments may be generated and used in any suitable way known to those of skill in the art. Suitable ways of generating fragments include, but are not limited to, recombinant expression of a fragment from encoding DNA. Such fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small fragments (e.g. up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods that are well known in the art .

A method of identifying and/or obtaining a compound which modulates chemokine activity may comprise:

(a) incubating an isolated gG polypeptide and a test compound in the presence of a chemokine under conditions in which chemokine activity is inhibited by the gG polypeptide; and (b) determining chemokine activity.

An inhibitor or potentiator (i.e. a modulator) of gG polypeptide activity may be identified (or a candidate substance suspected of being a gG polypeptide inhibitor or potentiator may be confirmed as such) by determination of chemokine activity compared with a control experiment in which the test compound is not applied. In other words, a difference in chemokine activity in the presence and absence of the test compound is indicative of a modulating effect of the relevant test compound.

A method may include identifying the test compound as an modulator chemokine activity. Such a compound may modulate (i.e. inhibit) binding between the gG polypeptide and the chemokine.

A suitable chemokine binds to an alphaherpesvirus gG polypeptide as described herein and may be a CC chemokine such as MlP-lα or RANTES, a CXC chemokine such as IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α or mouse KC, a C chemokine such as lymphotactin or a CX3C chemokine such as fractalkine .

Chemokine activity may be determined by any standard method, for example by determining the transient calcium flux or cellular migration induced by the chemokine, as described herein. Chemokine activity may also be determined by measuring the binding of the chemokine to a chemokine receptor or a cell surface glycosaminoglycan.

An assay according to the present invention may also take the form of an in vivo assay. The in vivo assay may be performed in a cell line in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell. A preferred assay of the invention includes determining the ability of a test compound to modulate gG polypeptide activity of an isolated or purified gG polypeptide which may be a full- length gG glycoprotein or an active portion thereof.

The precise format of the assay of the invention may be varied by those of skill in the art using routine skill and knowledge. For example, the interaction between the polypeptides may be studied in vi tro by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support. Suitable detectable labels include ³⁵S-methionine- which may be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.

The protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se . A preferred in vi tro interaction may utilise a fusion protein including glutathione-S- transferase (GST) . This may be immobilized on glutathione agarose beads. In an in vi tro assay format of the type described above a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the beads may be rinsed to remove unbound protein and the amount of bound protein can be determined by counting the amount of label present in, for example, a suitable scintillation counter.

Combinatorial library technology (Schultz, JS (1996)

Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide. The amount of test substance or compound, which may be added to an assay of the invention, will normally be determined by trial and error depending upon the type of compound used. Compounds that may be used may be natural or synthetic chemical compounds used' in drug screening programmes . Extracts of plants that contain several characterised or uncharacterised components may also be used. Other candidate inhibitor compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.

Following identification of a substance or compound that modulates or affects the binding of a gG polypeptide to a chemokine, the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, as already discussed.

A substance identified as a modulator of gG polypeptide activity may be peptide or non-peptide in nature. Non- peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. -These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process . In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be ^• screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo ox clinical testing.

Mimetics of substances identified as having ability to modulate polypeptide activity using a screening method as disclosed herein are included within the scope of the present invention. A polypeptide, peptide or substance able to modulate binding of a gG polypeptide and a chemokine according to the present invention may be provided in a kit, e.g. sealed in a suitable container that protects its contents from the external environment. Such a kit may include instructions for use.

Methods of the invention may also be used to identify the level or amount of chemokine in a sample . A method of determining the amount or level of chemokine in a sample may comprise, contacting the sample containing the chemokine with a gG polypeptide; and, determining binding of chemokine to said gG polypeptide, the amount of binding being indicative of the amount of chemokine in said sample.

The sample may be contacted with the gG polypeptide under conditions in which the gG polypeptide binds to a chemokine, if present in the sample.

A suitable sample may be a tissue, blood, serum or plasma sample obtained from an individual. The individual may be suspected of suffering from a disease or disorder described herein.

Using routine skill and knowledge, those of skill in the art may vary the precise format of such methods. For example, the interaction between the polypeptides may be studied by bringing the sample into contact with the gG polypeptide that has been immobilised on a solid support as described above . The binding of the chemokine may then be detected by conventional techniques, for example using a labelled chemokine specific antibody.

The gG polypeptide may be immobilized using an antibody against that protein bound to a solid support or via other technologies, which are known per se . In an in vi tro assay format of the type described above, the level of chemokine in a sample can be assayed by determining^' its ability to diminish the amount of labelled chemokine which binds to the immobilized GST-fusion polypeptide for example, by SDS-polyacrylamide gel electrophoresis.

Alternatively, the beads may be rinsed to remove unbound protein and the amount of protein that has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter .

Further aspects and embodiments of the present invention will be apparent to those skilled in the art. The following experiments provide support for and exemplification by way of illustration of aspects and embodiments of the invention.

All documents mentioned in this specification are incorporated by reference .

Figure 1 shows a Kyte and Doolittle hydropathy plot of EHV -I ORF70 (gG) using the GCG programme PEPPLOT.

Figure 2 shows the inhibition of binding of chemokines to U937 cells by gG from EHV-1 and BHV-I. Supernatants from insect cells infected with AcBHV-IFL (a,c) or Acorf70FL, or purified secreted EHV-1 ORF70 (e) were tested for inhibition of IL-8 and/or MlP-lα binding to U937 cells.

Increasing concentrations of culture supernatants, expressed as cell equivalents, or purified EHV-1 ORF70 were incubated in duplicate with ¹²⁵I-IL-8 or ^{1 Ξ}I-MIP-Iα prior to addition of U937 cells. Binding of ¹²⁵1-chemokine to cells was measured and expressed as the mean+/-SD. To control for binding specificity to U937 cells, ¹²⁵i- chemokines were pre-incubated with medium alone (no SN) , purified M3 or an excess of unlabelled chemokine. Figure 3 shows the inhibition of transient calcium flux induced by chemokines. HeLa cells transfected with CCR5 and CXCR4 were pre-labelled with Fura-2 AM and activated with 150 ng MlP-lα preincubated for lh with medium alone or the indicated doses of purified secreted BHV-I gG or control protein (IgG Fc) . Changes in fluorescence were monitored and calculated as described in below.

Figure 4 shows the inhibition of cell migration in response to chemokines. The activity of human GRO-α was measured in a transwell migration assay using freshly isolated human neutrophils. Three μg of purified secreted BHV-I gG were preincubated (a) or not (b) with the indicated amounts of human GRO-α before addition to the assay. The percentage of cells migrating in response to the chemokine is represented (mean+/-SD) .

Figures 5 to 7 show affinity constants of gG binding to chemokines. Nickel chelate FlashPlates pre-coated overnight with purified gG.His were incubated in triplicate with the indicated doses of ^{1 5}I-GRO-α. Bound radioactivity was determined and analysed using the

Ligand programme .

Figure 5 show saturation curve and Scatchard analysis of

¹²⁵I-GRO-α binding to gG.His from EHV-1.

Figure 6 shows saturation curve and Scatchard analysis of ¹²⁵I-GRO-α binding to gG.His from BHV-1.

Figure 7 shows saturation curve and Scatchard analysis of ¹²⁵I-GRO-α binding to gG.His from BHV-5. Figures 8 to 10 show the binding specificity of gG encoded by EHV-1, BHV-1 and BHV-5. Nickel chelate FlashPlates were coated with gG.His from EHV-1, BHV-1 or BHV-5. Three hundred pM of iodinated human IL-8 (EHV-1) or GRO-α (BHV-1 and BHV-5) was added in the absence or presence of 500-fold excess of unlabeled competitor CC, CXC, C or CX₃C chemokines of human (h) or mouse (m) origin, or vMIP. Bars represent the mean percentage inhibition of binding +/- SD of triplicate samples.

Figure 8a shows the binding specificity of EHV-1 gG with human chemokines .

Figure 8b shows the binding specificity of EHV-1 gG''with murine or viral chemokines .

Figure 9a shows the binding specificity of BHV-1 gG with human chemokines .

Figure 9b shows the binding specificity of BHV-1 gG with murine or viral chemokines .

Figure 10a shows the binding specificity of BHV-5 gG with human chemokines .

Figure 10b shows the binding specificity of BHV-5 gG with murine or viral chemokines .

Figures 11 to 15 show the inhibition of chemokine-heparin interactions by purified gG.

Figure 11 shows results obtained from FlashPlates pre- coated with heparin-BSA and incubated with ¹²⁵I-IL-8 in the absence of gG but with increasing doses (10, 50 and 150 ng per well) of murine TNF (mTNF) , human IgG, His- tagged Fas ligand (FasL) or His-tagged glucocorticoid- induced TNFR superfamily-related protein ligand (GITRL) . FlashPlates coated with BSA alone (BSA) were also tested to determine background binding.

Figure 12 shows results obtained with FlashPlates pre- coated with heparin-BSA and incubated with ^12SI-IL-8 in the presence of purified gG from EHV-1 (EHV-lgG.His) , BHV-1 (BHV-lgG.His) or BHV-5 (BHV-5gG.His) . The amount of bound radioactivity (mean ±SD of duplicate samples) is shown.

Figure 13 shows results obtained with FlashPlates pre- coated with heparin-BSA were pre-incubated with ^12SI-GRO-α Purified recombinant EHV-1 gG (300 ng per well) or medium was added to the wells and bound radiolabeled chemokine (mean ±SD of triplicate samples) determined at the indicated times . Background binding to BSA alone was subtracted.

Figure 14 shows results obtained with FlashPlates pre- coated with heparin-BSA were pre-incubated with ¹²⁵I-MCP- lα. Purified recombinant EHV-1 gG (300 ng per well) or medium was added to the wells and bound radiolabeled chemokine (mean ±SD of triplicate samples) determined at the indicated times. Background binding to BSA alone was subtracted.

Figure 15 shows chemokine binding activity expressed at the surface of recombinant baculovirus-infected insect cells. Sf21 insect cells were infected with AcEHV-lgG or AcEHV-lgGs for 24 h, incubated for 30 min at room temperature with the indicated chemokines in suspension and bound chemokine determined. The binding difference between AcEHV-lgG and AcEHV-lgGs was observed three times for IL-8 and twice for GRO-α. Table 1 shows the amplification primers used to generate gG coding sequence in the experiments described herein.

Table 2 shows the database accession numbers of examples of alphaherpesvirus gG glycoproteins .

EXPERIMENTAL

Materials and Methods Reagents

Radioiodinated recombinant human IL-8, GRO-α and MCP-1 (2200 Ci/mmol) were from Perkin Elmer Life Sciences (Boston, MA) . Recombinant human MlP-lα, IL-8, MCP-1, GRO- αand SDF-lα, and murine BCA-1, IP-10, eotaxin, fractalkine, lymphotactin and TNF were from R&D Systems (Minneapolis, MN) . Recombinant human BCA-1, lymphotactin fractalkine, 1-309, eotaxin, TARC, MIP-3, ENA-78, IP-10,

RANTES and I-TAC, vMIP-2, and murine SDF-1, MIP-2, JE,

MIP-3α, C-TAC, RANTES, MIG, KC and Exodus-2 were from PeproTech (Rocky Hill, NJ) . Recombinant, histidine-tagged human GITRL and FasL were from R&D Systems (Minneapolis,

MN) . Human IgG was from Sigma.

Viruses and cells The EHV I strains AB4 , V592, RacH and Army 183, and EHV-3 were grown on equine embryonic lung (EEL) cells. EHV-4 strain MD and EHV-2 were grown on equine embryonic kidney (EEK) cells. RanHV-1, CapHV-1, CerHV-1, BHV-1, BHV-4, BHV-5, FeHV-1 and PrV were grown on MDBK cells. Varicella-Zoster virus (VZV) was grown on MRC-5 cells.

HSV-1 strain SC16, HSV-2 strain HVD and MHV-68 were grown on baby hamster kidney (BHK) 21 cells. W strain Lister and VV Lister Δ35K were grown on BSC-1 cells. AcNPV was propagated in Spodopterafrugiperda 21 (St2I) or Hi5 insect cells. Polymorphonuclear (PMN) cell separation Enriched populations of human neutrophils were isolated from blood using known methods (Baly et al (1997) Methods Enzymol. 287:69-88). Briefly, venous blood samples were collected from healthy donors onto citrate-phosphate- dextrose solution and mixed with an equal volume of 2% dextran T500 for 30 min at room temperature. Cell suspension was carefully layered over a Lymphoprep gradient (FLOBIO) and centrifuged for 40 min at 600 x g at room temperature. The gradient fraction containing PMN was collected. After hypotonic lysis of contaminating erythrocytes, purified PMN cells were washed twice in RPMI 1640 with 0.1% fetal calf serum (FCS) .

Binding assay

Cells were infected with herpesviruses, W or baculovirus at a multiplicity of infection of 10 plaque forming units (pfu) per cell and supernatants were harvested 1-3 days post-infection, prepared and concentrated as described (Alcami et al (1998) supra, Symons , J. A. et al (1995) Cell. 81:551-60). Infectious virus in the supernatants was inactivated with 4, 5 ' , 8-trimethylpsoralen and exposure to UV light. Binding medium was RPMI 1640 containing 20 mM Hepes (pH 7.4) and 0.1% bovine serum albumin (BSA) . Cross-linking experiments with bis (sulfosuccinimidyl) suberate (BS³) (PIERCE) (5 mM) or ethylene glycolbis succinamidyl succinate (EGS) (SIGMA) to ¹²⁵i-chemokines (0.4 nM) were performed in 25 μl as described (Alcami et al (1998) supra) . The amount of medium used was equivalent to 5-7 x 10⁴ cells. Samples were analysed by 12% acrylamide SDS-PAGE. In the competition assays with U937 cells, supernatants were pre-incubated with 100 pM ¹²⁵I -chemokine in 100 μl for Ih at 4°C. Subsequently, 2.5 x 10⁶ U937 cells were added in 50 μl and incubated for 2 h at 4°C. Bound ¹²⁵1-chemokine was determined by phthalate oil centrifugation as described (Alcami et al (1998) supra) .

Proximity assay for determination of the affinity constant

FlashPlates™ (Perkin Elmer) were used for determination of the affinity constant. Nickel-coated FlashPlates were incubated overnight with purified His-tagged protein in 0.1 % BSA in phosphate buffered saline, washed, and incubated at room temperature with increasing concentrations of radiolabelled chemokine. Bound radiolabelled chemokine was determined by scintillation counting in a 96-well plate counter. Non-specific binding, determined in the presence of 100-fold excess unlabeled chemokine was subtracted from the total counts . Binding data were analysed by the Ligand program (Munson, P. J., and D. Rodbard. 1980 Anal. Biochem. 107:220-239).

Construction of Recombinant Baculoviruses

The gG ORFs were PCR-amplified from infected cell DNA or supernatants with Pfu polymerase using oligonucleotides specific for each virus (Table 1) , which contained unique restriction endonuclease sites at either end of the gene.

The DNA products were cloned into pBAC-I (Novagen) . The nucleotide sequence of the cloned ORF was confirmed by sequencing before the recombinant baculoviruses were constructed as described (Alcami et al (1998) supra) . Recombinant naturally cleaved and secreted forms of gG containing a C-terminal 6xHis tag were produced in baculovirus-infected Sf21 insect cells. Recombinant baculoviruses were grown in Hi5 insect cells in the absence of serum for protein analysis and purification of recombinant protein.

Protein Purification The gG-6xHis fusion proteins were purified from supernatants of Hi5 insect cells infected with recombinant baculoviruses on a nickel-Sepharose column (Pharmacia) following standard protocols.

Measurement of Calcium Mobilisation in HeLa cells Stably Transfected with CCRS and CXCR4

Changes in intracellular calcium flux were determined in HeLa cells transfected with CCR5 and CXCR4 following standard protocols (Pozzan, T. et al 1983. Science. 221:1413-5). Briefly, cells were re-suspended at a concentration of 5 x 10^s cells/ml in Hanks balanced salt solution (HBSS) containing 20mM HEPES, 2% dialysed FCS, 2.5ml 10% F-127 detergent and 5μM Fura-2 AM. After 45 min at 37°C, shaking and in the dark, cells were washed, re- suspended at 2.5 x 10^s cells in 1.5 ml in the above HBSS. Changes in fluorescence were monitored at 37°C using a spectrometer (LS50, Perkin-Elmer Corp) at an excitation wavelength of 345nm and emission wavelength of 495nm. Maximal fluorescence (Fmax) was determined by the addition of 0.1% Triton X-100 (final concentration) and minimal background fluorescence (Fmin) was determined by the addition of 25mM EGTA. Free calcium was calculated using the following equation: [Ca2+]i, nM=224 (F- Fmin) / (Fmax-F) .

Chemotaxis assays

Cell chemotaxis was evaluated in 24 well transwell plates (Costar) . A 600μl aliquot of chemokine diluted in RPMI 1640 with 0.1% FCS was placed in the lower compartment and lOOμl of cell suspension (5 x 10⁵ cells/ml) was placed in the upper chamber, separated by a polycarbonate filter (3μm pore size) .

After incubation at 37°C for 3 h the transwell insert was removed and the cells in the lower chamber were removed, concentrated 6-fold and counted on a Neubauer counting chamber .

Chemokine binding assays

Radioiodinated recombinant human IL-8, GRO-α and MCP-1 (2200 Ci/mmol) were from Perkin Elmer Life Sciences (Boston, MA) . Recombinant human MlP-lα, IL-8, MCP-l; GRO- α-and SDF-lα, and murine BCA-1, IP-10, eotaxin, fractalkine, lymphotactin and TNF were from R&D Systems' (Minneapolis, MN) . Recombinant human BCA-1, lymphotactin, fractalkine, 1-309, eotaxin, TARC, MIP-3α, ENA-78, IP-10, RANTES and I-TAC, vMIP-2, and murine SDF-1, MIP-2, JE, MIP-3α, C-TAC, RANTES, MIG, KC and Exodus-2 were from PeproTech (Rocky Hill, NJ) . Recombinant, histidine-tagged human GITRL and FasL were from R&D Systems (Minneapolis, MN) . Human IgG was from Sigma.

Recombinant gG.His fusion proteins were purified on nickel-sepharose columns (Pharmacia) from supernatants of Hi5 insect cells grown in EX-Cell 405 (JRH Biosciences) and infected with recombinant baculoviruses. Purified His-tagged proteins were visualized by SDS-PAGE and Coomassie staining and were quantified by protein assay (BioRad) .

Nickel chelate-coated FlashPlates (Perkin Elmer Life Sciences) were used in accordance with the manufacturer's instructions. Purified His-tagged protein (1-10 ng/well) was incubated overnight in 0.1% BSA in PBS, washed and incubated at room temperature for 1-2 h with increasing doses of ^{1 5}i-chemokine in the absence or presence of excess unlabeled chemokine. Bound ¹²⁵I-chemokine was determined in a Packard TopCount Microplate Scintillation Counter. Non-specific binding, determined in the presence of 100-fold excess unlabeled chemokine, was subtracted and binding data were analyzed by the Ligand programme .

Heparin binding Assays

Basic Flashplates were coated with 2.5 μg per well heparin-BSA (Sigma) in 10 mM phosphate buffer pH 7.0. Wells were washed, blocked for 2 h with phosphate buffer containing 2% BSA and washed again. Iodinated chemokine (400 pM) , preincubated for 1 h at room temperature with purified gG or control proteins, was then added to the well. Plates were read after overnight incubation at 4°C. Time course experiments were conducted as above except that ¹²⁵1-chemokine was bound to immobilised heparin-BSA overnight before the addition of purified proteins.

Background binding was measured by ^12ΞI-chemokine binding to wells treated only with 2% BSA in binding buffer.

Identification of soluble chemokine binding activity in cultures infected with alphaherperviruses

A chemokine cross-linking assay was used to identify expression of soluble vCKBPs in supernatants from cells infected with different alphaherpesviruses.

Media from cultures uninfected (mock) or infected with the indicated viruses were incubated with ¹²⁵IMIP-Iα or ¹²⁵I-IL-8 and cross-linked with EGS . Samples were analysed by SDS-PAGE in a 12% polyacrylamide gel and. autoradiography. ¹²⁵I-MIP-lα-vCKBP complexes were observed at levels well above background for BHV-1, BHV5, RanHV-1, CapHV-1, CerHV-1 and FeHV-1. The size of the ligand-vCKBP complex ranged from 54 to 80 kDa suggesting a vCKBP size of 49- 72 kDa after subtraction of the 8 kDa monomeric MlP-lα. With BHV-1, BHV-5, RanHV-1 and CerHV-1, ligand binding to a very diffusely migrating species of proteins with an apparent molecular mass of between 60 kDa and greater than 175 kDa was observed.

Chemokine binding activity expressed by MHV-68 and W were used as positive controls (Alcami et al (1998) supra, Parry et al (2000) J. Exp. Med. 191 573-578)

The specificity of the ligand-vCKBP interaction was demonstrated by the absence of binding in mock-infected cells or cells infected with W Lister A35K, a mutant in which the vCKBP of 35 kDa has been inactivated (Alcami et al (1998) supra) . The gammaherpesvirus BHV-4 did not express a chemokine binding activity that can be detected in this assay. ¹²⁵I-IL-8vCKBP complexes of 50 to 80 kDa were observed with EHV-I and EHV-3 supernatants. EHV-4 did not show chemokine binding activity in this assay.

^12BI-IL-8 cross-linking to produce a ligand-vCKBP complex was also observed for BHV-I, BHV-5, RanHV-I and FeHV-I, but not for CapHV-I and CerHV-I which showed MlP-lα binding activity. Variation in the complex size may reflect the degree of glycosylation or different polypeptide lengths of the vCKBP encoded by different viruses . No ¹²⁵I-fractalkine-vCKBP complex was observed for any of the supernatants tested using EGS or BS³ as a cross- linker, although a complex was apparent in the positive control consisting of MHV68 supernatants containing M3.

Chemokine Binding Specificity of the Alphaherpesvirus CKBP

Cross-linking assays were carried out as before with the addition of 2000-fold excess of unlabelled chemokine in order to determine the binding specificity of the vCKBPs identified in the supernatants. Samples were analysed by SDS-PAGE in a 12% polyacrylamide gel and autoradiography.

BHV-I supernatant was incubated with 0.4nM ¹²⁵I-MTP-lα in the presence of a 2, 000-fold excess of human BCA-1, human SDF-lα, murine MIG, human GROα, human IL-8, murine KC, human MCP-1, human RANTES, human lymphotactin or human fractalkine.

Addition of excess unlabelled human IL-8 did not compete in the cross-linking assays, although a vCKBP-1251-IL-8 complex was formed in the direct cross-linking assay. This provides indication that the affinity of the BHV -I vCKBP is much higher for MlP-lα than it is for IL-8.

The vCKBP from BHV-1 was able to bind with high affinity at least one CXC non-ELR chemokine, mouse MIG, but binding to the CXC non-ELR chemokine B-cell attracting chemokine I (BCA-I) was not observed. Human growth- related oncogene α (GRO-α) , a CXC ELR chemokine (like IL- 8) , was able to totally compete the binding of MlP-lα, indicating that BHV-1 vCKBP has a higher affinity for GRO-α than IL-8. Partial competition is also seen for murine KC. Human human monocyte chemoattractant protein I (MCP-I) , a CC chemokine, was able to compete the binding of MlP-lα.

Human lymphotactin, the only member of the C class of chemokines, also competed with MlP-lα binding.

Fractalkine, the only member of the CX3C class of chemokines was not observed to inhibit the binding of MlP-lα to the vCKBP, confirming the direct cross-linking results .

The addition of excess unlabelled MlP-lα in the cross- linking assay resulted in the formation of a ladder in the gel, as well as inhibition of the ¹²⁵I-MIP-lα-vCKBP complex. The ladder probably results from the radiolabelled MlPlα forming multimers with the unlabelled MlP-lα, which are subsequently cross-linked by EGS .

Chemokines interact with glycosaminoglycans (GAGs) through their carboxyl terminus. ¹²⁵I-MIP-lα was preincubated with an excess of heparin or heparan sulphate before incubation with the supernatant, to determine whether the vCKBPs bind to the GAG binding site.

Cross-linked complexes appeared uniform across the GAG dose range, indicating the GAGs do not interfere with chemokine binding.

A similar analysis of the chemokine binding specificity was performed with EHV-I. EHV-I supernatant was incubated with ¹²⁵I-IL-8 in the presence of 2,000fold excess of cold (i.e. non- radioactive) chemokine.

The CXC chemokines human BCA-I, human SDF-Ia, mouse MIG, human GRO-α and mouse KC were all observed to inhibit the binding of radiolabelled IL-8 to the vCKBP . The C chemokine family member lymphotactin was able to completely block binding of radiolabelled IL-8 to the vCKBP. Of the CC chemokines tested, human RANTES and human MlP-lα were able to compete the binding of IL-8, but human MCP-I was not. Addition of an excess of GAGs did not interfere with the chemokine binding activity encoded by EHV-I, indicating that the chemokines' do not bind through the GAG binding site.

Expression of EHV-I chemokine binding activity in infected cells

A cross-linking assay was performed of 0.4 nM ¹²⁵I-IL-8 to supernatants from cells infected with EHV-I strain AB4 and harvested at 4 , 20 or 24 h post-infection. Infections were also performed- in the presence of phosphonoacetic acid (PAA) or tunicamycin (Tm) and harvested 24 h post- infection. Infected cell lysates were also tested and used as a positive control . Samples were analysed by SDS PAGE in a 12% polyacrylamide gel and autoradiography.

These experiments indicated that the chemokine binding activity is produced at late times of infection (20 and 24 h) but is not detected at 4 h post-infection. Cultures infected with EHV-1 in the presence of phosphonoacetic acid (PAA), an inhibitor of viral DNA synthesis, did not produce the chemokine binding activity. This confirmed that synthesis of the EHV-1 vCKBP occurs after viral DNA replication.

Cells infected with EHV-1 in the presence of tunicamycin (Tm) , an inhibitor of N-glycosylation, did not secrete to the medium the chemokine binding activity. This suggested that the vCKBP is N-glycosylated and that the lack of carbohydrate impaired protein secretion or resulted in loss of chemokine binding activity.

Medium and cell lysates of cultures infected with EHV-1 strain AB4 in the absence or presence of Tm were also tested in the crosslinking assay. Samples were analysed by SDS-PAGE in a 12% polyacrylamide gel and autoradiography.

No chemokine binding activity was detected in cell extracts from Tm-treated cultures, indicating that the lack of activity is not due to an inhibition of protein secretion alone. For example, polypeptide may be synthesized within the cell with reduced or no carbohydrate modification, resulting in an unfolded product .

Expression of vCKBP by EHV-1 natural isolates

To determine whether the chemokine binding activity is widespread throughout EHV-1 natural isolates, a collection of supernatants were prepared from viruses isolated in the field. Supernatants from the wild isolates were identified by a unique number (50483,

82449, 10492, 32281, 84047, 82644, 82244, 32202, 10013, 62062, 60966, 82529) and tested in a cross-linking assay alongside the EHV-1 strains AB4 , Army 183 (highly virulent) and RacH (apathogenic vaccine strain) . ¹²⁵I-IL8-vCKBP complexes of 55-60 kDa were formed in all the supernatants tested. This confirmed that the vCKBP protein is highly conserved in the wild population of EHV-I. The specificity of the ligand-vCKBP binding was demonstrated by the absence of binding in the mock- infected cultures, and with the positive control of EHV-1 AB4 supernatant .

Identification of glycoprotein gG of alphaherpesviruses as the secreted chemokine binding activity Open reading frames (ORFs) were identified from the EHV-1 genome sequence, based on the presence of a predicted signal peptide and a size consistent with the results from the cross-linking experiments. EHV-1 ORF8, ORF26, ' ORF59, ORF60 and ORF70, which all have unknown function, were selected and expressed in the baculovirus system, as described above. Two different recombinant baculovirus clones were selected for the initial screening to control for possible mutations introduced in the PCR reactions, which might have led to a loss of binding activity.

Cross-linking assays were performed of ¹²⁵I-IL-8 with EGS to supernatants from insect cell cultures infected with the recombinant baculovirus clones . Samples were analysed by SDS-PAGE in a 12% polyacrylamide gel and autoradiography. Supernatants from cultures infected with EHV-1 strain AB4 were used as a positive control.

A ¹²⁵I-IL-8-vCKBP complex of 50-55 kDa was only produced by the recombinant baculovirus expressing ORF70. EHV-1 ORF70 is known to encode glycoprotein gG, which is anchored through the C-terminal transmembrane domain in the virus particle envelope. EHV-1 gG has also been shown to be proteolytically cleaved and released in large amounts to the medium. The binding activity detected in the cross-linking experiments to supernatants from infections with EHV-1 and the recombinant baculovirus AcORF70 is likely to be due to the secreted, cleaved form of gG.

The finding that gG encodes the chemokine activity is consistent with the results observed with BHV-1 supernatants. BHV-1 gG has been shown to consist of a glycoproteoglycan of high molecular mass (Keil (1996) supra) , and we found that with BHV -I supernatants chemokines cross-linked to a very diffusely migrating species of proteins with an apparent molecular mass of between 60 kDa and greater than 175 kDa.

Genes encoding the glycoprotein gG have been identified in most alphaherpesviruses . To determine whether gG homologues encoded by BHV -I, BHV -5, HSV-I and HSV-2 encode chemokine binding activity, full length proteins and truncated versions lacking the transmembrane domain were expressed in the baculovirus system (Table I) . The viral genes were PCR-amplified and their DNA sequence compared to that found in public databases.

The full length EHV-1 gG had 100% sequence identity with the published sequence. A secreted form of EHV-1 gG without the transmembrane domain and with a C-terminal 6xHis tag had one substitution at residue 208 (Valine to Alanine) . The BHV-1 full length and secreted versions showed one substitution when compared to the published sequence at residue 331 (Glutamate to Lysine) . The BHV-5 full length gG had five amino acid substitutions when compared to the published sequence at residues 157 (Valine to Leucine) , 220 (Glutamate to Aspartate) , 221 (Isoleucine to Alanine) , 266 (Alanine to Glycine) and 392 (Alanine to Threonine) . The genes encoding gG from HSV-1 and HSV -2 were both expressed without their transmembrane domains and had 100% identity to the previously published sequence.

The viral genes encoding full length and truncated versions of gG were cloned into baculovirus transfer vectors and recombinant baculoviruses were isolated.

Supernatants from insect cell cultures infected with the different recombinant baculoviruses were tested for the presence of secreted chemokine binding activity in cross- linking assays .

MlP-lα binding activity was detected in all the protein samples tested apart from HSV-1 gG and HSV-2 gG, or the control supernatant from a mock-infected culture. Recombinant EHV-1 gG bound MlP-lα whereas supernatants from EHV-1-infected cultures did not bind this chemokine. This difference may reflect a low binding affinity for MlP-lα and the requirement of much more protein, produced in the baculovirus system, for detection.

IL-8 binding activity was detected in all samples apart from HSV-I, HSV-2 and the negative control. This experiment confirmed that gG from BHV-1 and BHV-5 are vCKBPs. The recombinant form of BHV-1 and BHV-5 gG does not include the diffusely migrating species seen in viral infections of mammalian cells. This may be due to a deficient protein glycosylation in insect cells.

Cross linking assays of ¹²⁵I-MIP-lα or ¹²⁵I-IL-8 with BS³ to uninfected insect cell cultures or insect cell cultures infected with recombinant baculovirus demonstrated that expression of truncated versions of gG lacking the transmembrane domain in the baculovirus system produced secreted chemokine binding activity. Secreted versions of gG expressed in the baculovirus system were encoded by EHV-1, BHV-I and BHV-5.

Inhibition of chemokine binding to cells To determine whether the interaction of gG with chemokines prevented their binding to specific chemokine receptors, binding assays of radiolabelled chemokines to the human monocytic cell line U937 were performed (figure 2) .

Expression of full-length gG from BHV-1 or EHV-1 from insect cells infected with recombinant baculoviruses produced a secreted from of the protein that inhibited the binding of ¹⁵I-IL-8 or ¹²⁵I-MIP-lα to the surface of U937 cells. Specificity of the ligand-gG interaction was demonstrated by the absence of inhibition of binding in the supernatants from cells infected with wild type baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV) .

The positive controls of recombinant MHV-68 M3 and 500- fold molar excess of cold chemokine reduced almost completely the binding of the chemokine to cells. The recombinant secreted version of EHV-1 gG, expressed in the baculovirus system with a C-terminal 6xHis tag, was purified by affinity chromatography as described above. Figure 2e shows that purified soluble gG also blocks the binding of ¹²⁵I-IL-8 to U937 cells. Inhibition of Transient Calcium flux in response to Chemokines

The inhibition of chemokine binding to cellular receptors indicates that gG blocks the- biological effects of chemokines. A well established index of chemokine receptor activation is the induction of transient increases in cytoplasmic calcium concentrations. Purified recombinant soluble gG from BHV-1 efficiently blocked signal transduction induced by MlP-lα, a ligand of CCR5, in HeLa cells expressing CCR5 and CXCR4 (Fig. 3) .

Inhibition of chemokine-induced cell migration

The ability of chemokines to induce infiltration of ''cell into tissues can be studied in vi tro by measuring cell migration along chemotactic gradients. The migration of human neutrophils in response to GRO-α was very efficiently inhibited by purified recombinant gG from BHV-I expressed as a secreted version in the baculovirus system (Fig. 4) .

Chemokine binding affinity and specificity of purified recombinant gG encoded by EHV-1, BHV-1 and BHV-5 Recombinant secreted forms of gG fused to a C-terminal 6xHis tag and expressed in the baculovirus system were purified in nickel chelate columns (gG.His) , and chemokine binding activity with human ¹²⁵I-GRO-α was demonstrated by cross-linking. Chemokine binding affinity was determined with human ¹²⁵I-GRO-α using a scintillation proximity assay. Nickel chelate FlashPlates (PerkinElmer Life Sciences) , containing a thin layer of scintillant in the interior of each well, were coated with purified gG.His. Increasing doses of ^12SI-GRO-α were added and bound chemokine determined in a scintillation counter. No free ¹²⁵I-GR0-α was detected. The affinity constants were determined by Scatchard analysis of the saturation curves, with K_D values of 42 ± 3.1 pM, 36 ± 2.5 pM and 113 ± 33 pM for gG.His from EHV-1, BHV-1 and BHV-5, respectively (Figs 5 to 7) .

Binding of ¹²⁵I-chemokines to purified gG.His in the presence of 500-fold excess unlabeled chemokines was determined in the FlashPlate assay. This provided a broad spectrum of binding specificity for 30 chemokines of human (Figures 8a, 9a and 10a) , mouse or viral origin (Figs 8b, 9b and 10b) . Purified gG.His from BHV-1 and BHV-5 bound to most human and mouse CC chemokines and to some human and mouse CXC chemokines. By contrast, EHV-1 gG.His showed a narrower binding specificity for" both human and mouse CC and CXC chemokines. EHV-1 gG.His bound to the human and mouse C chemokine lymphotactin, whereas BHV-1 gG.His bound only to human lymphotactin and BHV-5 gG.His did not bind to the C chemokine. None of the recombinant proteins bound the CX₃C chemokine fractalkine . BHV-5 gG.His, and to a lesser extent BHV-1 gG.His, interacted with vMIP-2 encoded by Kaposi's sarcoma- associated herpesvirus.

gG can alter the heparin binding characteristics of chemokines

Pre-incubating purified gG.His from EHV-1, BHV-1 and BHV- 5 with ¹²⁵I-IL-8 prior to addition to FlashPlates. precoated with heparin-BSA greatly reduced binding of the chemokine to heparin (Fig 12) . No effect was observed with several control proteins, some expressing a His-tag (Fig.11) . Moreover, gG also disrupted pre-established chemokine-GAG interactions. FlashPlates coated with heparin-BSA were pre-incubated with ¹²⁵I-GRO-α, and addition of EHV-1 gG.His was observed to efficiently detach ¹²⁵I-GRO-α from heparin-BSA over time (Fig. 13) . By contrast, no effect was observed on the binding of ¹²⁵I- MCP-1, a chemokine not recognized by EHV-1 gG.His, to heparin-BSA (Fig. 14) .

These results demonstrate that gG blocks the interaction of chemokines with heparin. Chemokines are presented to leukocytes on a solid phase after retention at the surface of endothelial cells by interacting with GAGs. This interaction is critical for transcytosis and correct chemokine presentation to the^' passing leukocyte. The chemokine domains that interact with receptors or GAGs may be different and have been well defined at the molecular level for some chemokines . Thus the binding of alphaherpesvirus vCKBP to chemokines masks both receptor and GAG binding sites in the chemokine. The ability of gG to inhibit chemokine function is enhanced by this blocking of both receptor and GAG. gG is also observed to displace chemokine once it has bound to heparin, providing indication that gG disrupts established chemokine gradients .

gG encodes chemokine binding activity at the cell surface gG encoded by several alphaherpesviruses is anchored in membranes through a C-terminal transmembrane domain and proteolytic cleavage leads to secretion. To determine whether the membrane form of gG also binds chemokines, insect cells infected with recombinant baculoviruses expressing the full-length gG (AcEHV-lgG) or a truncated form of gG lacking the C-terminal transmembrane domain of EHV-1 gG (AcEHV-lgGs) were tested.

2 x 10⁶ infected Sf21 cells were scrapped and incubated in suspension with 200 pM ¹²⁵I-chemokines for 30 min at room temperature and bound chemokine determined by phthalate oil centrifugation.

Infection with AcEHV-lgG was observed to clearly increase the ability of cells to bind human GRO-α and IL-8 at the cell surface (figure 15) , as compared to AcEHV-lgGs- infected cells. As a control, no difference was observed in the binding to MCP-1, a chemokine not recognized by EHV-1 gG. Chemokine binding activity was observed in media from cells infected with either recombinant.

Chemokine binding activity is therefore evident in recombinant EHV-1 gG when expressed at the surface of insect cells

VIRUS ACC. NO. 6XHIS TAG PROTEIN FORMf PRIMERS

EHV-1 M86664 No Full length, 411 aa 5'-CGCGAATTCATGTTGACτGTCTTAGCAGC-3•

C-terminal Secreted form, 355 aa 5^CGCGAATTCATGTTGACTGTCrTAGCAGC-3' 5'-CGCAAGCTTGTGTGTACTGTCATCGCTGTT-3^•

BHV-1 AJ004801 No Full length, 444 aa 5'-ACCGGATCCATGCCTGCCGCCCGGACGG-3'

(AcBHV-lF ) 5'-TCCAAGCTTrCAGACGCTGAGCATCGGCT-3'

C-terminal Secreted form, 391 aa S'-ACCGGATCCATGCCTGCCGCCCGGACGG-S¹

(AcBHV-1's') 5^,-CCCiGCGGCCGCGCCAAAGAGCCCAAACTCCG-3^•

BHV-5 x99755 Full length, 440 aa 5'-CATGGATCCATGCCCGCCGCCGCTCAAGC-3^•

(AcBHV-5FL) 5'-TCCAAGCTrCTAGACGCGGAGCATGGGCτ-3^•

Secreted form, 396 aa 5'-CATGGATCCATGCCCC<:CGCCGCTCAAGC-3'

(AcBHV-SV) 5'-CTCGCGGCCGCGCGCCGCGTGGCGGGATCAG-3^• xl4112 No Secreted form, 167 aa 5'-AC∞AAπCATGTCGCAGGGCGCCATGCG-3'

(AcHSV-lV) 5^•-C CAAGCTτCTATCGTCCCTrrGAσGTGAG'^C-3^•

Z86099 No Secreted form, 457 aa S'-CTCGAATTCATGCACGCCATCGCTCCCAGO'

(AcHSV-2Y) 5'-CCTAAGCTTCTACTGCGGAGGCGCTGCTGGTG-3'

t The name of the recombinant baculovirus is indicated in parenthesis

Table 1

Table 2

Claims

Claims :

1. A method for obtaining a modulator of chemokine activity comprising: (a) bringing into contact a gG polypeptide, a chemokine polypeptide; and a test compound, under conditions in which in the absence of the test compound being an inhibitor, the gG polypeptide and the chemokine polypeptide interact or bind; (b) determining interaction or binding between the gG polypeptide and the chemokine polypeptide.

2. A method for obtaining a modulator of chemokine activity comprising: (a) incubating an isolated gG polypeptide and a test compound in the presence of a chemokine under conditions in which chemokine activity is inhibited by the gG polypeptide; and

(b) determining chemokine activity..

3. A method according to claim 1 or claim 2 wherein the gG polypeptide is an alphaherpesvirus gG polypeptide

4. A method according to claim 3 wherein the gG polypeptide is an alphaherpesvirus gG polypeptide shown in Table 2.

5. A method according to any one of the preceding claims wherein the chemokine is a C, CC, CXC chemokine.

6. A method according to claim 5 wherein said chemokine is MlP-lα, RANTES, IL-8, human BCA-1, human SDF-lα, mouse MIG, human GRO-α, mouse KC or lymphotactin.

7. A method according to any one of the preceding claims comprising identifying the test compound as a modulator of chemokine activity.

8. A method according to claim 7 comprising isolating and/or synthesising a test compound identified as a modulator of chemokine activity.

9. A method according to claim 8 comprising formulating said test compound into a composition with a pharmaceutically acceptable excipient.

10. A compound which modulates the binding of a gG polypeptide to a chemokine, the compound being obtained using a method according to any one of claims 1 to 8.

11. A compound according to claim 10 which is an antibody specific for a gG polypeptide or a peptide fragment of a gG polypeptide.

12. A method of producing a pharmaceutical composition comprising; identifying a compound which modulates the activity of a chemokine using a method according to any one of claims 1 to 7; and, admixing the compound identified thereby with a pharmaceutically acceptable carrier.

13'. A method according to claim 12 comprising the step of modifying the compound to optimise the pharmaceutical properties thereof .

14. A method for preparing a pharmaceutical composition for treating a condition involving chemokine activity. comprising; i) identifying a compound which is an agonist/antagonist of a gG polypeptide, ii) synthesising the identified compound/ and; iii) incorporating the compound into a pharmaceutical composition.

15. A recombinant alphaherpesvirus having reduced or abolished gG glycoprotein function.

16. A recombinant alphaherpesvirus according to claim 15 selected from the group consisting of HSV-I, HSV-2, EHV- I, EHV-3, EHV-4, BHV-I, BHV-5, FeHV-I, PrV and AHV-1.

17. A recombinant alphaherpes virus having reduced or abolished gG glycoprotein function for use in a method of treatment of the human or animal body.

18. Use of such a recombinant alphaherpesvirus according to claim 15 in the manufacture of a medicament for use in the treatment of alphaherpesvirus infection

19. A method of making an attenuated alphaherpes virus comprising; providing an alphaherpesvirus; and, disrupting the gG glycoprotein gene of said virus.

20. An antibody molecule which specifically binds a gG polypeptide and neutralises the chemokine binding activity of said gG polypeptide.

21. A gG polypeptide or a nucleic acid encoded a gG polypeptide, or a fragment thereof, or an antibody molecule according to claim 20 for use in a method of treatment of the human or animal body.

22. Use of an gG polypeptide, a nucleic acid encoded a gG polypeptide or a fragment thereof, or an antibody molecule according to claim 20 in the manufacture of a medicament for treating a disease or disorder involving chemokine activity.

23. Use according to claim 22 wherein said disease is selected from the group consisting of inflammatory diseases, allergic diseases, ^"chronic pulmonary diseases, psoriasis, atherosclerosis, multiple sclerosis, arthritis, neoplasia, sepsis, infectious diseases, inflammatory bowel disease, diabetes, colitis and transplant rejection.

24. Use according to claim 23 infectious diseases include malaria, tuberculosis and HIV.

25. A method of making a pharmaceutical composition comprising admixing a gG polypeptide, a nucleic acid encoded a gG polypeptide, recombinant alphaherpesvirus according to claim 15 or an antibody according to claim 20 with a pharmaceutically acceptable excipient, vehicle, diluent or carrier, and optionally other ingredients.

26. A pharmaceutical composition comprising a gG polypeptide, a nucleic acid encoded a gG polypeptide, recombinant alphavirus according to claim 15 or an antibody according to claim 20 and a pharmaceutically acceptable excipient, vehicle, diluent or carrier.

27. A method of inhibiting activity of a chemokine comprising bringing the chemokine into contact with a gG polypeptide.

28. A method according to claim 27 wherein the gG polypeptide is provided to cells in vi tro by means of encoding nucleic acid.

29. A method of treating a disorder involving chemokine activity, the method comprising administering an gG polypeptide or a nucleic acid encoding gG polypeptide to an individual with the disorder.

30. A method according to claim 29 wherein the disorder is selected from the group consisting of inflammatory diseases, allergic diseases, chronic pulmonary diseases, psoriasis, atherosclerosis, multiple sclerosis, arthritis, neoplasia, sepsis, infectious diseases, inflammatory bowel disease, diabetes, colitis and transplant rejection.

31. A method of determining the amount of chemokine in a sample comprising, contacting the sample containing the chemokine with a gG polypeptide; and determining binding of chemokine to said gG polypeptide, the amount of binding being indicative of the amount of chemokine in said sample .