EP1073735A1 - Host-encoded protein expressed on marek's disease virus (mdv)-infected cells and antibody thereto - Google Patents

Abstract

The invention relates to nucleic and amino acid sequences which encode an antigen which is associated with avian tumours, particularly avian tumours associated with Marek's disease virus. The invention also relates to the use of the sequences in medicine, particularly in the production of compositions useful in the treatment and prophylaxis of avian tumours.

Description

HOST-ENCODED PROTEIN EXPRESSED ON MAREK'S DISEASE (MDVHNFECTED CELLS AND ANTIBODY THERETO

The present invention relates to nucleic and amino acid sequences encoding an antigen which is associated with tumours, particularly avian 5 tumours and especially those avian tumours associated with oncogenic viruses such as Marek's disease virus. The invention also relates to expression vectors incorporating the above sequences and to the use of the vectors and antigen in medicine, especially in the treatment and prophylaxis of tumours.

10

Marek's disease virus (MDN) is a highly cell-associated avian alpha- herpesvirus that can cause a lymphoproliferative disease, Marek's disease (MD), manifested by neural lesions and/or lymphomas in the visceral tissues of susceptible birds [reviewed in Payne, L.Ν. (ed) Marek's Disease

15 (1985), Martinus Νijhoff Publishing, Boston]. MD is of major economic importance to poultry industries throughout the world. Strains of MDN have been classified into three serotypes. Type 1 viruses consist of oncogenic strains and their attenuated derivatives. The different Type 1 pathotypes range from those that are mildly oncogenic, causing MD only

20 in susceptible birds, to those that are very highly-oncogenic (very virulent +) and cause MD in birds that have been vaccinated. Type 2 MDN are naturally-occurring strains that are infectious but not oncogenic. Serotype 3 viruses consist of strains of the closely-related herpesvirus of turkeys (HVT) which are apathogenic in both turkeys and chickens. Currently

25 different vaccines based on all three serotypes of virus are commercially available and these are used singly or in various combinations. Almost all poultry used in commercial production are vaccinated but outbreaks of clinical disease continue to be reported and these have increased in incidence and severity as highly virulent pathotypes of MDN continue to emerge [Witter, R.L. (1997) Avian Dis. 41, 149-163]. In areas where outbreaks of MD are a particular problem poultry producers have responded by switching to more aggressive vaccines and resorting to using vaccines combinations (bivalent or trivalent) to improve protection. However the view is growing among vaccine manufacturers that it will be difficult to continue to control MD with conventional MD vaccines.

The present invention seeks to provide methods and agents which are useful in the identification, prophylaxis, or treatment of tumours.

In a first aspect the invention provides a purified polypeptide recognised by a monoclonal antibody secreted by the hybridoma AV37 deposited under the Budapest Treaty at the European Collection of Cell Cultures (ECACC), Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 OJG, UK on 3^rd March 1998, which has received the accession number 98030304.

As used herein, the term "purified" is intended to mean that the polypeptide is free from some and preferably most of the biological material in which it is found in nature, in other words, the polypeptide is not in the form in which it has previously existed. Thus, a polypeptide of the invention includes, for example, the polypeptide in a composition comprising a pharmaceutically acceptable carrier or exipient, or an adjuvant.

Preferably, the polypeptide is the AV37 polypeptide antigen having the sequence shown in Figure 5 (SEQ ID No. 2) and variants and fragments of that polypeptide: "Fragments" and "variants" are those which are useful to prepare antibodies which will specifically bind the said polypeptide or mutant forms thereof lacking the function of the native polypeptide. Such variants and fragments will usually include at least one region of at least five consecutive amino acids which has at least 90% homology with the most homologous five or more consecutive amino acids region of the said polypeptide. A fragment is less than 100% of the whole polypeptide. For example, as a fragment of the AV37 polypeptide shown in Figure 5 (SEQ ID No. 2) amino acids may be prepared as a synthetic peptide having the sequence NH₂-CDTLKNWFYDETLGRC-COOH (SEQ ID No. 3) of the AV37 polypeptide. It is believed that this sequence will be arranged on the outside of the protein structure and therefore be available to stimulate an antibody response.

Amino acids shown in Figure 5 (SEQ ID No. 2) may also be prepared as a synthetic peptide having the sequence NH₂-

DVMVPVEEEGKEFHHPTTATEK-COOH (SEQ ID No. 4) (C-terminal peptide) and NH₂-QPPFTSSHSCDTLKNWFYDETL-COOH (SEQ ID No. 5) (N-terminal peptide) of the AN37 polypeptide respectively. The latter sequences are from the C and Ν terminals and these peptides are known to be good candidates for making anti-peptide antibodies, ie. for use as peptide vaccines, as they tend to have more mobility (flexibility) than peptides that are fixed within the three dimensional structure of the protein. As a result of greater flexibility it is more likely that some of the peptide conformers will adopt a native conformation so that antibodies raised against them will also be more likely to recognise the native antigen.

It will be recognised by those skilled in the art that a polypeptide of the invention may be modified by known polypeptide modification techniques. These include the techniques disclosed in US Patent No 4,302,386 issued 24 November 1981 to Stevens, incorporated herein by reference. Such modifications may enhance the immunogenicity of the antigen, or they may have no effect on such immunogenicity. For example, a few amino acid residues may be changed. Alternatively, the antigen of the invention may contain one or more amino acid sequences that are not necessary to its immunogenicity. Unwanted sequences can be removed by techniques well known in the art. For example, the sequences can be removed via limited proteolytic digestion using enzymes such as trypsin or papain or related proteolytic enzymes.

Alternatively, smaller polypeptides corresponding to antigenic parts of the polypeptide may be chemically synthesised by methods well known in the art. These include the methods disclosed in US Patent No 4,290,944 issued 22 September 1981 to Goldberg, incorporated herein by reference.

Thus, a polypeptide of the invention includes a class of modified polypeptides, including synthetically derived polypeptides or fragments of the original polypeptide, having common elements of origin, structure, and immunogenicity that are within the scope of the present invention.

Peptides may also be synthesised by the Fmoc-polyamide mode of solid- phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxy carbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6- trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-teπninal residues, use is made of the 4,4'- dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl- acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4- hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/ 1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95 % trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.

Suitable peptide ligands of the invention that will bind to the AV37 monoclonal antibody may be identified using methods known in the art.

One method, disclosed by Scott and Smith (1990) Science 249, 386-390 and Cwirla et al (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382, involves the screening of a vast library of filamentous bacteriophages, such as M13 or fd, each member of the library having a different peptide fused to a protein on the surface of the bacteriophage. Those members of the library that bind to the AV37 monoclonal antibody are selected using an iterative binding protocol, and once the phages that bind most tightly have been purified, the sequence of the peptide ligands may be determined simply by sequencing the DNA encoding the surface fusion protein. Another method that can be used is the NovaTope (TM) system commercially available from Novagen, Inc., 597 Science Drive, Madison, WI 53711. The method is based on the creation of a library of bacterial clones, each of which stably expresses a small peptide derived from a candidate protein in which the ligand is believed to reside. The library is screened by standard lift methods using the antibody or other binding agent as a probe. Positive clones can be analysed directly by DNA sequencing to determine the precise amino acid sequence of the ligand.

Further methods using libraries of beads conjugated to individual species of peptides as disclosed by Lam et al (1991) Nature 354, 82-84 or synthetic peptide combinatorial libraries as disclosed by Houghten et al (1991) Nature 354, 84-86 or matrices of individual synthetic peptide sequences on a solid support as disclosed by Pirrung et al in US 5143854 may also be used to identify peptide ligands.

Monoclonal antibodies which will bind to the AN37 polypeptide and fragments and variations thereof can be prepared using standard monoclonal antibody technology. The antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques " , H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications" , J G R Hurrell (CRC Press, 1982).

Chimaeric antibodies are discussed by Νeuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799).

Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised, may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged. Such modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as

Ν-t-butoxy carbonyl. Such modifications may protect the peptide from in vivo metabolism. The peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the peptide to be formed as a loop, with the Ν- terminal and C-teraiinal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the peptide is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the peptide of the invention forms a loop.

According to current immunological theories, a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope. The peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin. More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser- Gly-Nal-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1. The latter compound may variously be regarded as a carrier or as an adjuvant or as both. Alternatively, several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking. Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(Ν- maleimidomethyl)cyclohexane-l-carboxylate, the latter agent exploiting the -SH group on the C-teiminal cysteine residue (if present).

If the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec" system is an example of such an arrangement. In a second aspect the invention relates to the use of a polypeptide of the invention in the manufacture of a composition for use in medicine. In particular, for use in the manufacture of a vaccine for the treatment or prophylaxis of tumours, preferably avian tumours and especially those associated with oncogenic avian viruses such as MDV, ALV and reticuloendotheliosis virus.

Active immunisation of the subject is preferred. In this approach, one or more AN37 polypeptides/proteins are prepared in an immunogenic formulation containing suitable adjuvants and carriers and administered to the subject in known ways. In avian species, in ovo injection of fertile eggs may be a convenient mode of administration. Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscoms" of EP 109 942, EP 180 564 and EP 231 039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a Registered Trade Mark.

It may be advantageous to use a AV37 polypeptide produced in a species other than the one being treated, in order to provide for a greater immunogenic effect. Another polypeptide can be used instead of the whole AV37 polypeptide in order to produce inhibitory antibodies in the patient. Such other polypeptides include fragments and analogues of the AN37 protein. Such polypeptides may be screened as above to ensure that they are capable of producing inhibitory antibodies in the subject.

A third aspect of the invention provides an isolated AN37 gene shown in Figure 5 (SEQ ID No. 1) and variations thereof: As used herein, the term "isolated" means that the gene is in isolation from at least most of the genome in which it is found, in other words the gene is not claimed in the form in which it has previously existed. Thus, for example, the gene of the invention includes the gene when that gene has been cloned into a bacterial vector, such as a plasmid, or into a viral vector, such as a bacteriophage, provided that such clones are in isolation from clones constituting a DNA library of the relevant genome.

The "gene" may comprise the promoter and/or other expression-regulating sequences which normally govern its expression and it may comprise introns, or it may consist of the coding sequence only, for example a cDNA sequence.

A "variation" of the gene is one which is (i) usable to produce a polypeptide or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said gene or (ii) an antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve its activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis," Science, 229: 193-1210 (1985), which is incorporated herein by reference. Since such modified genes can be obtained by the application of known techniques to the teachings contained herein, such modified genes are within the scope of the claimed invention. Moreover, it will be recognised by those skilled in the art that the gene sequence (or fragments thereof) of the invention can be used to obtain other DNA sequences that hybridise with it under conditions of high stringency. Such DNA includes any genomic DNA. Accordingly, the gene of the invention includes DNA that shows at least 55 per cent, preferably 60 per cent, and most preferably 70 per cent homology with the gene identified in the method of the invention, provided that such homologous DNA encodes a protein which is usable in the methods described below. In addition homologous genes in other species may be identified by demonstrating at least approximately 30% identity, preferably 35, 40, 50, 60 or greater % identity between the predicted amino acid sequences to those in the available protein databases.

DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1XSSC and 6XSSC and at temperatures of between 55 °C and 70 °C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By "high stringency" we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate.

"Variations" of the gene include genes in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 50% and preferably at least 90 or 95%) with equivalent stretches of the gene of the invention even though the overall homology between the two genes may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.

The invention also includes amino acid sequences which show at least 30% identity, preferably 35, 40, 50, 60 or greater % identity to the predicted amino acid sequences encoded by the gene of the invention.

Preferably a DNA sequence of the invention is expressed in a suitable host to produce a polypeptide of the invention. Thus, the DNA encoding a polypeptide of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.

The DNA encoding a polypeptide of the invention may be joined to a wide variety of other DNA sequences to form a vector for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

The vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.

Typical prokaryotic vector plasmids are pUClδ, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7/r99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.

An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.

Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps)

A variety of methods have been developed to operably link DNA to vectors via complementary cohesive terarini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify the DNA encoding a polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.

In a fourth aspect, the present invention relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.

In a fifth aspect, the present invention relates to the use of a polynucleotide vector construct of the invention in the manufacture of a composition for use in medicine. In particular, for use in the manufacture of a vaccine for the treatment or prophylaxis of tumours, preferably avian tumours and especially those tumours associated with oncogenic avian viruses such as MDN, ALN, Rous-associated virus and reticuloendotheliosis virus. In avian species, in ovo injection of fertile eggs with a solution of a polynucleotide vector construct of the invention may be a convenient mode of administration.

Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.

Electroporation is also useful for fransforming cells and is well known in the art for transforming yeast cell, bacterial cells and vertebrate cells.

For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25 μFD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182. Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described herein.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.

Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

Non-limiting examples embodying aspects of the invention will now be described with reference to Figures 1 to 7.

Figure 1: expression plasmid PI.18, having an EcoRV site into which the open reading frame (ORE) of the AV37 antigen can be inserted. Figure 2: Fowlpox virus transfer plasmid (pEFL929), having a Smal site into which the AV37 ORF can be inserted.

Figure 3: HVT transfer plasmid (pVECo4), having an EcoRV site into which the AV37 ORF can be inserted.

Figure 4: baculovirus transfer plasmid (pACYMl), having a Smal site within a muliple cloning site (mcs) into which the AV37 ORF can be inserted.

Figure 5(a): DNA sequence encoding the AV37 antigen together with amino acid sequence of mature AV37 antigen.

Figure 5(b): Amino acid sequence of mature AV37 antigen.

Figure 6: shows the results of DELFIA. Salmonella Ab was used as a positive control to show that the DELFIA worked. The reference numerals 1566, 1568 and 1571 are the ring numbers of virulent MDV infected birds with lymphomas (2); 391V and PK/2365 are birds which had been hyperimmunized with a virulent MDV(l).

Figure 7: shows the results of DELFIA following preabsorption with AV37 protein. Reference numerals 391V and 1559 indicate virulent MDV-infected birds with lymphomas. TABLE 1 - The Genetic Code and Single Letter Amino Acid

Designations (from Old & Primrose, Principles of Gene Manipulation, Third Edition, 1985, Appendix 7, page 346, Blackwell Scientific Publications, Oxford, UK.)

^aCodes for Met if in the initiator position.

Alanine A Leucine L

Arginine R Lysine K

Asparagine N Methionine M

Aspartic Acid D Phenylalanine F

Cysteine C Proline P

Cystine C Serine S

Glycine G Threonine T

Glutamic Acid E Tryptophan W

Glutamine Q Tyrosine Y

Histidine H Valine V

Isoleucine I Production of monoclonal antibody AV37 against a host antigen expressed on Marek's disease virus-transformed cell lines.

Production ofhybridoma secreting mAb AV37

Cells have been isolated from MD tumours and cultured in vitro to produce immortalised cell lines [Powell, P.C, Payne, L.N. , Frazier, J.A. and Rennie, M.C. (1975) Oncogenesis and Herpesviruses II, de-The, G. Epstein, M.A. and zur Hausen, H. (eds) I ARC Scientific Publications no. 11, Lyon]. 10⁷ cells from the MDV-transformed cell line HP9 [Payne, L.N. (1981) Int. J. Cancer 28, 757-766] were injected with Quil A into an adult BALB/c mouse by the intraperitoneal route. Four weeks later 10⁷ cells from the MDV-transformed cell line MDCC-HP89 [Payne, L.N. (1981) Int. J. Cancer 28, 757-766] were injected without adjuvant into the mouse by the same route. This was repeated 8 weeks later with 10⁷ MDCC-HP9 cells. Six weeks later the immune response of the mouse was boosted by injecting 10⁷ MDCC-HP9 cells by the intravenous route and a day later 10⁷ MDCC-HP9 cells by the intraperitoneal route. Three days later the mouse was killed, the spleen removed and splenocytes prepared. The splenocytes were fused with NSl myeloma cells using polyethylene glycol to produce hybridoma cells [Kohler, G. and Milstein, C. (1975) Nature 256, 495-497]. The hybridomas were cloned and cells from one well, 349(AD4a), secreted an antibody that gave a positive immunochemical reaction with at least 90% of MDCC-HP9 or -HP89 cell lines but recognised less than 10% thymocytes, when used in flowcytometric analysis. This clone was selected and taken through a further round of cloning and screening for a positive response with the

MDV-transformed cell line cells. The hybridoma selected gave a positive response with the HP9 and HP89 cell line cells but no response with lymphocytes from uninfected birds. This twice-cloned hybridoma was named AV37 and was deposited under the provisions of the Budapest Treaty at the ECACC on 3^rd March 1998. It has received the accession number 98030304.

Use ofmAb AV37 to identify cells expressing AV37 antigen

The monoclonal antibody (mAb AV37) secreted by the AV37 clone recognised an antigen present on virtually all cells of cell lines derived from MD tumours apart from the cell line MDCC-MSB-1 [Yamada M., Matsuda, H. and Nii, S. (1983) GANN, 74, 502-508]. The AV37 antigen did not appear to be expressed on the lymphoid cells from uninfected birds (spleen, thymus, bursa of Fabricius or bone marrow). However the monoclonal antibody appeared to give a weak positive response on a very small number of peripheral blood lymphocytes ( <0.1 % of the population). Stimulation of lymphocytes with the plant lectin concanavalin A together with the addition of a conditioned medium from activated chicken lymphoblasts increased the proportion (up to a maximum of 6%) of cells expressing the AV37 antigen. After infection with MDV the antigen was detected on up to 2% of peripheral blood lymphocytes at 3 and 7 days after infection. It was also detectable on splenocytes at 5 days after infection and on thymocytes at 12 days after infection.

The AV37 antigen was present on a variable proportion (2-70%) of the cells in different MD tumours. These AV37⁺ cells were dispersed throughout the tumour tissue but tended to aggregate in discrete areas. Flow cytometric analysis showed that most AV37⁺ tumour cells were CD4⁺ and expressed high levels of the MDV gene product meq, a basic leucine zipper protein with transcription factor activity. pp38 and gB, viral proteins involved in lytic infection, were not present. A high proportion of CD4⁺ AV37⁺ cells were shown to be in the S-G₂M phase of the cell cycle and only a small proportion had sub-diploid DNA indicating this population of cells is actively proliferating but not undergoing activation- induced-programmed cell death, meq has been associated with blocking programmed cell death in vitro, suggesting that the AV37⁺ cells in tumours are immortalised and therefore likely to be the neoplastically- transformed cells. A further important finding was that the antigen recognised by the AV37 monoclonal antibody is expressed on cell lines transformed by other avian oncogenic viruses, including retroviruses such as avian leukosis virus and reticuloendotheliosis (see Table 2). This indicates that the AV37 antigen is an avian tumour-specific antigen.

TABLE 2 - Reactivity OfmAbs AV37 To MATS As With Avian Cell Lines

Key to Table 2

1 ALV = avian leukosis virus; MDV = Marek's disease virus; MNNG - methyl nitronitrosoguanidine; RAV = Rous-associated virus; REV = reticuloendotheliosis virus.

2 HL SC = Hyline Poultry Farms, Dallas Centre, IA, USA; RIR - Rhode Island Red; RPRL - Regional Poultry Research Laboratory, East Lansing, MI, USA.

3 Oghura, H. et al. (1984) GANN, 75, 410-414;

Hihara, H. et al.(1974) Natl. Inst. Animal Health Quart. U, 163-173;

Baba, T.W. (1985) Virol., 144, 139-151;

Nazerian, K. et al. (1977) Av. Dis., 21, 69-76; Okazaki,W. et al. (1980) Av. Path. 9, 311-329;

Powell, P.C. et al. (1974) Nature, 251 , 79-80;

Payne, L.N. et al. (1981) Int. J. Cancer, 28, 757-766;

Nazerian, K. (1987) v. Path., 16, 527-544;

Akiyama, Y. and Kato, S. (1974) Biken, 17, 105-116; Shaw, I. (1997) PhD thesis, University of London.

23b CHARACTERISATION OF AV37 ANTIGEN

Purification of AV37 antigen

The AV37 antigen was purified from HP9 cells whose surface proteins had been labelled with Na¹²⁵I using the lactoperoxidase method as described in Vainio, 0., Riwar, B., Brown, M.H. and Lassila, 0. (1991) [J. Immunol, 147. 1593-1599]. The cell surface proteins were solubilised with 2% NP40 lysis buffer containing protease inhibitors. Radiolabelled antigen that formed a soluble immune complex with the AV37 antibody was immunoprecipitated using rabbit anti-mouse immunoglobulin antibodies that had been bound to Streptococcus protein G. The immunoprecipitates were dissociated with a buffer, with and without mercaptoethanol [Laemmli, U.K. (1970) Nature, 227, 680-685]. When analysed by SDS polyacrylamide gel electrophoresis the radiolabelled antigen was shown to have a relative molecular weight of 75kDa under both reducing and non-reducing conditions.

Cloning and Sequencing of AV37 gene

(i) The gene expressing the antigen recognised by the AV37 monoclonal antibody was cloned from a complementary DNA (cDNA) library prepared from cells of a MDV-transformed cell line. Messenger RNA was prepared from HP9 cells by the method of Chirgwin et al. [Chirgwin, J.M., Przbyla, A.E. MacDonald, R.J. and Rutter, W.J. (1979) Biochem. 18, 5294-5299] and purified by oligo(dT) chromatography. Double-stranded cDNA was ligated to non self-complementary BsiXl adapters, size fractionated by agarose gel electrophoresis and inserted into the plasmid pCDM8 [Seed, B. (1987) Nature, 3 29, 840-842]. The ligated DNA was electroporated into Escherichia coli MC1061/p3 [Dower, W.J., Miller, J.F. and

24 Ragsdale, CW. (1988) Nucl. Acids Res. 16, 6127-6145]. Plasmid was prepared from pooled transformed colonies by standard methods [Maniatis, T. Fritsch, E.F. and Sambrook, J (I 982) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, New York]. COS-7 cell transfections were conducted in suspension or on adherent cells [Metzelaar, M, Winjgaard, P.L.J., Peters, P., Sixma, J.J. Nieuwenhuis, H.K. and Clevers, H.C (1991) J.Biol. Chem. 266,3239-3245]. Screening of acetone-fixed transfected cells with the monoclonal antibody AV37 and recovery of the plasmid from positively stained cells were carried out as described by Horst [Horst, M. Wijngaard, P.L.J. , Metzelaar, M., Bast, E.J.E.G. and Clevers, H.C. (1991) Nucl. Acids Res. 19, 4556]. This AV37⁺ clone was given the name pMATl. A control monoclonal antibody AV7 did not detectably stain cells that were transfected with pMATl, though these transfected cells stained strongly with the monoclonal antibody AV37. The pMATl clones were sequenced using the PRISM™ Ready Reaction DyeDeoxy™ Terminator cycle sequencing kit (Applied Biosy stems). The complete sequence of the clones was determined on each strand. Sequence data were analysed with the Wisconsin Package software (Genetics Computer Group) [Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucl. Acids Res. 12, 387-395]. The complete nucleic and amino acid sequence is shown in Figure 5(a). The UW-GCG software package [Devereux, J., Haeberli, P. and Smithies, O, (1984) Nucl. Acids Res. 12, 387-395] was used to search various DNA databases.

Unexpectedly, the gene encoding the AV37 antigen was not detected on MDV DNA using Southern blot analysis, indicating that it is not a viral gene. It was, however, amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) from total RNA extracted from uninfected chicken lymphocytes and from cells obtained from chicken embryos from 5 days of incubation. This suggests that AV37 is an oncofoetal antigen similar to those described for mammals [reviewed in: Boon, T

25 and Old, L.J. Current Opinion in Immunology (1997), 9, 681-683; Van den Eyde, B. and vand der Bruggen, P. Current Opinion in Immunology (1997), 9, 684-693]..

The AV37 monoclonal antibody also recognised lymphoblastoid cell lines that have been produced by transformation with other oncogenic viruses. Positive responses were obtained with cell lines, as described in Table 2 (above). This indicates that the AV37 antigen is a marker of transformation and is present on the cells of tumours that have been caused by other oncogenic viruses.

(ii). The gene expressing the antigen recognised by the AV37 monoclonal antibody was cloned from a complementary DNA (cDNA) library made from the Marek's disease virus-transformed lymphoblastoid cell line, MDCC-HP9. All MDCC-HP9 cells express very high levels of the AV37 antigen. Messenger RNA was prepared from HP9 cells by the method of Chirgwin et al. [Chirgwin, J.M., Przbyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochem. 18, 5294-5299] and purified by oligo(dT) chromatography. Double-stranded cDNA was ligated to non self-complementary BsXl adapters, size-fractionated by agarose gel electrophoresis and inserted into the plasmid vector pCI-nx, a gift from Dr J. Young at IAH, Compton. pCI-nx is a variation on the commercially available pCI- neo (Promega, Madison, Wisconsin). The library was transfected into electrocompetent Escherichia coli MCI 061 [Dower ,W.J., Miller, J.F. and Ragsdale,CW. (1988) Nucl. Acids Res. 16,6127-6145]. Plasmid was prepared from pooled transformed colonies by standard methods [Mamatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, Cold Spring, Harbor, New York]. COS-7 cells were transfected in suspension [Metzelaar, M., Winjgaard, P.L.J., Peters, P., Sixma, J.J. Niewenhuis, H.K. and Clevers, H.C. (1991) J. Biol. Chem. 266, 3239-3245. Dynabeads^® M280 coated with sheep anti- mouse immunoglobulin (Dynal, Oslo, Norway) were incubated with AV37 antibody, washed and incubated with COS-7 cells transfected with the HP9 library. These

26 were then magnetically sorted as described by O'Regan et al. [O'Regan, M.N., Parsons, K.R., Tregaskes, CA. and Young, J.R. (1999) Immunogenetics, 49, 68- 71]. There were three rounds of this selection and enrichment. Twenty four positive clones were isolated. The clone that was sequenced expressed the AV37 antigen when transfected into either COS-7 cells or chicken embryo fibroblasts. The AV37 gene was sequenced by MWG-Bicotech (Milton Keynes, UK) using a LI- COR 4200 system. The complete sequence of the clone was determined on each strand.

The UW-GCG software package [Devereux, J., Haberli, P. and Smithies, O. (1984) Nucl. Acids Res. 12, 387-395] was used to search various DNA databases. These searches indicated that AV37 is a member of the tumour necrosis factor receptor super-family.

The mature AV37 antigen is a type 1 membrane protein of 446aa which has most similarity to (1) human and mouse CD30, overall amino acid identity with human and mouse CD30 is approximately 32% .

Amino acid sequence of AV37 antigen

To determine the amino acid sequence directly, the AV37 antigen was isolated from the MDCC HP9 cell line. The AV37 monoclonal antibody reacts with a 70 kD band after immunoblotting (Western) a lysate of membrane proteins from the HP9 cell line. The AV37 monoclonal was produced by growing hybridoma cells in medium containing immunoglobulin-free serum. The AV37 immunoglobulin was purified using protein A (Pharmacia) and then coupled to CNBr-activated Sepharose (Pharmacia) using standard techniques [Coligan, J.E., Kruisbeek, A.M., Marulies, D.H., Shevach, E.M. and Strober, W (1999) Current Protocols In Immunology, vol. 2 sect 8, suppl. 29, John Wiley and Sons Inc.]. A lysate of membrane

27 proteins was prepared from 10¹⁰ HP9 cells and fractions containing the AV37 antigen eluted by passing the lysate down the AV37-Sepharose column. Those fractions containing the AV37 antigen were identified using by immunoblotting samples of the fractions. (The AV37 monoclonal antibody reacts with a 70kD band after immunoblotting (Western) a lysate of membrane proteins from the MDCC-HP9 cell line.) Positive fractions were pooled, precipitated with acetone and prepared for N-terminal sequencing using Edman degradation [See Niall, HD (1973) Methods In Enzymol. vol.2, pp.942-1010] . The N-terminal seven amino acids were sequenced and these confirmed exactly the predicted start site (amino acid 22) of the mature AV37 protein.

Use of nucleic acid encoding AV37 antigen in vector constructions and in vaccination methods and compositions

1 Source of AV37 ORF

The open reading frame (ORF) encoding the AV37 antigen can be obtained by PCR amplification of a cDNA clone (pMATl .AV37) using the following primers.

Examples of primers pairs suitable for obtaining ORF of the AV37 gene:

Forward

5' TGG AAA GGA ACT GGA GTG 3' (SEQ ID No. 6)

Reverse

5^* GCA AGC TGT GAA TTA GCC 3' (SEQ ID No. 7)

Forward

5' CTG CTG CTT CTC CAG GAC ATT C 3' (SEQ ID No. 8)

Reverse

28 5' ATT CCT TTC CCT CCT CTT CCA C 3' (SEQ ID No. 9)

Forward

5' AGA CTT CAG GAG GAG CAA C 3' (SEQ ID No. 10)

Reverse

5' GCT TCA CAC ACC TTC TCA G 3' (SEQ ID No. 11)

These primers have been designed to amplify the ORF of AV37 flanked by EcoRV sites.

It will be appreciated that each primer sequence may also be useful on its own as a hybridisation probe (eg a radiolabelled probe) for variations of the AV37 nucleotide sequence of Figure 5(a) (SEQ ID No. 1) in accordance with standard techniques.

2. Construction of expression plasmid

The PCR product obtained above can be digested with EcoRV and the fragment cloned into the EcoRV site of the expression plasmid PI. 18 (Fig 1). The PI.18 plasmid was obtained from Dr J.S. Robertson, National Institute of Biological Standards and Control, South Mimms, EN6 3QG, UK. It is derived from pUC plasmid [Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] and is non-mobilisable. It has been engineered to express foreign genes in mammalian and avian cells. Expression is driven by the HCMV immediate-early promoter and has been optimised by including an intron between the promoter and the cloning site. Transcription is terminated by the CMV polyA signal. The orientation of the AV37 fragment can be tested by restriction enzyme analysis and the correct orientation confirmed by sequencing. The recombinant plasmid obtained (PI.18-AV37) can be transfected into chick embryo fibroblasts and expression of the AV37 antigen under the CMV

29 I.E. promoter tested by immunofluorescence using mAb AV37. This plasmid can be used directly for vaccination of chickens. Preferably, it is first purified on Qiagen™ anion exchange resin.

Vaccination methodology

Chicks can be vaccinated with various amounts of the recombinant plasmid DNA (30-100 μg) in multiple sites in the leg and pectoral muscle. Boosts can be given after suitable intervals (eg. 15 days) and the chicks challenged with a virulent strain (RB1B) of MDV. Chicks immunized with the PI. 18 plasmid alone serve as controls.

3. Construction offowlpox virus recombinant

In this example, a known avian vaccine is modified by the insertion of an AV37 encoding nucleotide sequence of the invention. The resulting agent thus comprises a combination of a nucleotide sequence/polypeptide of the invention and a known avian vaccine.

The EcoRV fragment containing the AV37 ORF can be obtained from PI. 18-AV37 by digestion with EcoRV and will be cloned into the Smal site of the fowlpox virus transfer plasmid pEFL29 (Fig 2). The correct orientation of the AV37 gene can be checked as above. The construct can be transfected into chick embryo fibroblasts previously infected with the FP9 strain of fowlpox virus as described by Yu et al (1994) [Vaccine, 12, 227-237] and Li et al (1994) [J. Virol. Methods, 50, 185-196]. Recombinant virus progeny can be identified by the formation of blue plaques in the presence of Bluogal (Sigma), a substrate for β-galactosidase. A number of recombinant virus can be plaque purified and tested for expression of the AV37 antigen by immunofluorescence using the monoclonal antibody AV37.

30 Vaccination methodology

Chickens can be vaccinated i/m or intra-dermally, usually by wing web scarification, with varying doses (eg. 10⁶-10⁷ plaque-forming units) of the recombinant. They may be boosted by a second immunisation with the same dose and by the same route after 14 days. Vaccinated chickens and controls immunised with FP9 vector can be challenged 6 days later with RBIB virus.

4. Construction of herpesvirus of Turkeys (HVT) recombinant

In this example, a known avian vaccine is modified by the insertion of an AV37 encoding nucleotide sequence of the invention. The resulting agent thus comprises a combination of a nucleotide sequence/polypeptide of the invention and a known avian vaccine.

The EcoRV fragment containing the AV37 ORF obtained as above can be cloned into the EcoRV site of the HVT transfer plasmid pVECo4 (Fig 3) [Sondermeijer et al (1993) Vaccine, 11, 349-358. The correct orientation of the AV37 gene can be checked as above. The construct and infectious HVT DNA can be co-transfected into CEF as described (Morgan et al (1992) Avian Diseases, 36, 858-870; Ross et al (1993) J. Gen. Virol. 74, 371-377]. A number of virus plaques can be picked and recombinant virus identified by screening infected CEF for expression of AV37 by immunofluorescence as above.

Vaccination methodology

Chicks can be vaccinated i/m with 1000 to 5000 plaque forming units (p.f.u.) of recombinant HVT or parental HVT and can be challenged 6 days later with RBIB

31 virus.

5. Construction of baculovirus recombinant

The EcoRV fragment containing the AV37 ORF can be cloned into the Smal site within the multiple cloning site (mcs) of the baculovirus transfer plasmid PACYM I (Fig 4) [Matsuura, Y. et al. (1987) J. Gen. Virol.68, 1233-1250]. The recombinant plasmid and linearised baculovirus DNA can be co-tranfected into SF9 insect cells as described by Matsuura, Y. et al. (1987). Virus progeny can be plaque-purified and tested for their capacity to express the AV37 antigen in SF9 cells by immunofluorescence as above.

Vaccination methodology

Extracts of the infected SF9 cells or AV37 antigen purified by affinity chromatography can be used for immunization. For the first inoculation, the antigen can be emulsified in Freund's complete adjuvant. Further inoculations with incomplete adjuvant and a final inoculation without adjuvant can be given after suitable intervals (eg. 3 weeks). Vaccinated chicks will be challenged with RBIB virus one week after the last inoculation. Chicks immunised with extracts of insect cells infected with wild type baculovirus serve as controls.

In all vaccination strategies the degree of protection conferred can be determined by observing the recipient birds for clinical signs of MD and tumour formation. Blood samples may be collected after suitable intervals and screened for the presence of antibodies against the AV37 antigen. All recipient birds can be sacrificed eg. after 15-26 weeks and a post mortem examination undertaken for signs of tumours.

32 6. Further vaccination methodology

Chickens can be vaccinated by the in ovo route. Fertile incubated eggs on about the eighteenth day of incubation will have their shells drilled and up to 0.1ml inoculum containing either the recombinant AV37 antigen in the HVT vector, fowlpox vector or baculovirus vector or as plasmid DNA can be injected into the allantoic or amniotic cavity of the developing chick embryo. Useful automatic egg injection apparatus is available from Embrex Inc. , USA.

Further inoculations without adjuvant can be given to the hatched chicks by the appropriate routes after suitable intervals (eg. 2 - 3 weeks). Vaccinated chicks will be challenged with RBIB virus one week after the last inoculation. Chicks immunised, with HVT, fowl pox, or extracts of insect cells infected with wild type baculovirus serve as respective controls.

7. A significant immune response to AV37 is induced in MDV-infected chickens

Sera were obtained from: (1) genetically-resistant chickens that had been hyperimmunised with a virulent MDV and (2) susceptible chickens that had been infected with virulent MDV and had developed lymphomas. These sera reacted positively to the purified AV37 antigen in an immmunosorbent assay (Figure 6). In contrast, control sera from disease-free chickens did not react to the purified AV37 antigen in an immmunosorbent assay. This suggests that the MDV-infected and immunised chickens produced antibodies against the AV37 antigen. In addition when these sera were pre-incubated with the purified AV37 antigen the antibodies were depleted from the sera and the positive readings abolished (Figure 7).

Brief description of the experiment:

33 Line 6 chickens that are genetically resistant to MD were hyperimmunised by repeated injections of an oncogenic strain of MDV (HPRS-16 strain). Sera were obtained and stored at -20°C pending analysis. In addition, Rhode Island Red chickens that are susceptible to MD were infected with MDV (HPRS-16 strain) and sera were obtained when Marek's disease lymphomas developed. Control sera were obtained from the same strains of chickens that were kept in disease-free conditions.

The AV37 monoclonal antibody was produced by growing AV37 hybridoma cells in medium containing immunoglobulin-free serum. The immunoglobulin was purified using protein A (Pharmacia) and then coupled to CNBr-activated Sepharose (Pharmacia) using standard techniques [Coligan, J.E., Kruisbeek, A.M., Marulies, D.H., Shevach, E.M. and Strober, W (1999) Current Protocols in Immunology, vol. 2 sect 8, suppl. 29, John Wiley and Sons Inc.]. A lysate of membrane proteins was prepared from 10¹⁰ HP9 cells and fractions containing the AV37 antigen eluted by passing the lysate down the AV37-Sepharose column. Those fractions containing the AV37 antigen were identified using by immunoblotting samples of the fractions.

The AV37 antigen was coated onto wells of a plastic microtitre plate by incubating the lysate fraction containing purified AV37 antigen overnight at 4°C The plates were washed and blocked with a blocking buffer containing bovine serum albumin as the blocking agent. The hyperimmune sera from MDV-immunised chickens, sera from MD-infected chickens with tumours or control sera from uninfected chickens were diluted and incubated in the AV37-coated wells. The plate was washed 3 times and then incubated with anti-chicken immunoglobulin that had been conjugated to Europium and used in a dissociation enhanced lanthanide fluorescence assay (DELFIA) [ Wood, P. & Barnard, G. (1997) Principles and Practice of Immunoassay. Edit Price David C.P., chap 17, Newman Macmillan, London]. Hyperimmune sera and sera from MDV-infected chickens with tumours reacted positively (higher lanthanide fluorescence) than sera from uninfected chickens (see

34 Figure 6).

To test that the response was specific, the same sera were pre-incubated in wells that had been coated with either the purified AV37 antigen or with horse serum. The samples were then used in the DELFIA. The hyperimmune sera and sera from MDV-infected chickens with tumours AV37 failed to react in the DELFIA (Figure 7) indicating that the AV37-specific antibodies in the sera had been removed.

35

Claims

1. A purified polypeptide recognised by a monoclonal antibody secreted by the hybridoma AV37 deposited under the Budapest Treaty at the European Collection of Cell Cultures (ECACC), UK on 3rd March 1998, which has received the accession number 98030304.

2. A polypeptide as claimed in Claim 1 wherein the polypeptide is expressed on cell lines transformed by an oncogenic avian virus.

3. A polypeptide as claimed in Claim 2 wherein the oncogenic avian virus is selected from Marek's disease virus, avian leukosis virus, Rous-associated virus, or reticuloendotheliosis virus.

4. A polypeptide as claimed in Claim 3 wherein the virus is Marek's disease virus.

5. A polypeptide, preferably as claimed in any one of Claims 1 to 4, wherein the polypeptide comprises all or a fragment or variation of the following amino acid sequence of Figure 5(b) (SEQ ID No. 2):

6. A polypeptide fragment as claimed in Claim 5 having an amino acid sequences selected from:

(a) NH2-QPPFTSSHSCDTLKNWFYDETL-COOH (SEQ ID No. 5); and

(b) NH₂-DVMVPVEEEGKEFHHPTTATEK-COOH (SEQ ID No. 4); and/or

(c) NH₂-CDTLKNWFYDETLGRC-COOH (SEQ ID No. 3) of the AV37 polypeptide

36

7. A monoclonal antibody raised against a polypeptide as claimed in any one of Claims 1 to 6.

8. A purified polypeptide recognised by a monoclonal antibody as claimed in Claim 7.

9. An isolated nucleotide sequence or variation thereof which encodes a polypeptide as claimed in any of Claims 1 to 6 or 8.

10. An isolated DNA sequence or variation thereof which encodes a polypeptide as claimed in any of Claims 1 to 6 or 8.

11. A DNA sequence as claimed in Claim 10 comprising all or part of the nucleotide sequence shown in Figure 5(a) (SEQ ID No. 1).

12. A polynucleotide vector construct comprising a nucleotide sequence as claimed in any one of Claims 9 to 11.

13. An expression vector comprising a DNA sequence as claimed in Claim 10 or 11 wherein the DNA sequence is arranged in the vector to permit expression of the DNA sequence in a suitable host cell.

14. An expression vector as claimed in Claim 13 wherein the vector comprises a plasmid.

15. An expression vector as claimed in Claim 13 wherein the vector comprises viral DNA.

16. An expression vector as claimed in Claim 15 wherein the viral DNA is from

37 fowlpox virus, herpesvirus of turkeys and/or baculovirus.

17. Use of a vector as claimed in any one of Claims 12 to 16, or a polypeptide as claimed in any one of Claims 1 to 6 or 8 in the manufacture of a composition for use in medicine.

18. Use of a vector as claimed in any one of Claims 12 to 16, or a polypeptide as claimed in any one of Claims 1 to 6 or 8 in the manufacture of a composition for use as a vaccine.

19. Use as claimed in Claim 17 or 18, in the manufacture of a composition for use in the treatment or prophylaxis of avian tumours.

20. Use as claimed in Claim 18, wherein the vaccine is against one or more oncogenic avian viruses.

21. Use as claimed in Claim 20 wherein the avian oncogenic viruses are selected from one or more of Marek's disease virus, avian leukosis virus, Rous-associated virus and reticuloendotheliosis virus.

22. Use as claimed in any one of Claims 17 to 21 in combination with another vaccine.

23. Use as claimed in Claim 22 wherein the other vaccine is selected from HVT and fowlpox.

24. A composition comprising a polypeptide as claimed in any one of Claims 1 to 6 or 8 together with a pharmaceutically acceptable exipient.

38

25. A composition comprising a polypeptide as claimed in any one of Claims 1 to 6 or 8 together with an adjuvant.

26. A method of making a polypeptide comprising expressing a nucleotide sequence or variation thereof as claimed in any one of Claims 8 to 10 in a suitable host cell and purifying the polypeptide.

27. The hybridoma AV37 deposited under the Budapest Treaty at ECACC, UK on 3 March 1998 which has received accession number 98030304.

28. A monoclonal antibody secreted by the hybridoma of Claim 27.

29. Use of a monoclonal antibody as claimed in Claim 7 or 28 in a method of identifying a polypeptide or a fragment thereof.

30. A host cell transformed with a polynucleotide vector construct as claimed in any one of Claims 12 to 16.

31. A nucleotide sequence selected from

5' TGG AAA GGA ACT GGA GTG 3' (SEQ ID No.6)

5' GCA AGC TGT GAA TTA GCC 3' (SEQ ID No.7)

5' CTG CTG CTT CTC CAG GAC ATT C 3' (SEQ ID No.8)

5^* ATT CCT TTC CCT CCT CTT CCA C 3' (SEQ ID No.9)

5' AGA CTT CAG GAG GAG CAA C 3' (SEQ ID No.10)

5' GCT TCA CAC ACC TTC TCA G 3' (SEQ ID No.11)

32. Use of one or more of the nucleotide sequences of Claim 31 as a primer in a

39 nucleic acid amplification reaction.

33. Use of one or more of the nucleotide sequences of Claim 31 as a hybridisation probe for variants of the nucleotide sequence of any one of Claims 9 to 11.

34. A method of making an antibody comprising providing a polypeptide or fragment or variant thereof as claimed in any one of Claims 1 to 6 or 8; administering the polypeptide or fragment or variant thereof to a mammal to raise an antibody response; and obtaining the antibody from the mammal.

35. A method of making an hybridoma capable of producing a monoclonal antibody comprising providing a polypeptide or fragment or variant thereof as claimed in any one of Claims 1 to 6 or 8; administering the polypeptide or fragment or variant thereof to a mammal to raise an antibody response; obtaining an antibody- producing cell from the mammal and fusing it with an immortal cell to form an hybridoma capable of secreting a monoclonal antibody.

36. An antibody obtained by the method of Claim 34.

37. A monoclonal antibody produced by an hybridoma as claimed in Claim 35.

40