IE84982B1

IE84982B1 - Viral Vaccines

Info

Publication number: IE84982B1
Application number: IE1999/0699A
Authority: IE
Inventors: Joseph Norman Ross Louis; David Scott Simon; Mckinley Binns Matthew
Original assignee: Merial
Filing date: 1989-09-12
Publication date: 2008-10-01

Abstract

Abstract Congress of Virology, 9-14 August, (1987) Edmonton. Canada; J. Virol. 62, 2373-2379). During the random sequencing studies 1988), we identified an M13 described earlier (Buckmaster gt al which came from the A antigen gene. This clone clone (No. 130) was then used to identify a 2.3 Kbp EggRl/PvuII fragment from IE990699 the RBIB strain of MDV containing the A antigen. This fragment was cloned into a SmaI/EcoR1 cleaved pUC13 vector by standard protocols. One plasmid (pMB419) was sequenced by the M13 dideoxynucleotide method. The sequence of the MDV RBJB A antigen and the predicted amino acid sequence of the protein are presented in Figure 6. The A antigen regions of MDV and HVT are non-essential genes and they can therefore be used as sites in MDV and HVT into which other genes can be inserted into the virus by homologous recombination. Several lines of evidence support this as outlined below. 1)During our study we isolated and sequenced another RBIB A antigen clone. This had one extra T residue in the string of T's 45 bases 3’ to the A antigen ATG codon. This extra T would cause a frameshift which would make it impossible for the gene to encode functional A antigen. As it is probable that this gene was cloned from a replicating MDV, the results suggest that the A antigen is non-essential to the virus. 2)0n conducting a similarity search it became clear that the MDV A antigen gene is the homologue of HSV gC and PRV gpIII glycoproteins. Both of these homologous genes are known to be non-essential [for the HSV homologue, see Rosenthal _g _i (1987) J. Virol. 61, 2438 — 2447]. 3)Strains of MDV lacking A antigen as judged. by agar gel diffusion tests [Churchill, A.E. _t ai (1969) J. gen. Virol. 4. IE990699 54] or producing low levels using the more sensitive 2D 557- D. _t_l (1982) Virology radio—immunoprecipitation (van Zaane. 121, 133-148] have been reported. Furthermore. in view of the fact that the A antigen is a major secreted glycoprotein. it may be a particularly suitable location for the presentation of foreign epitopes within the A antigen as soluble, secreted proteins. This may be achieved by cloning oligonucleotides encoding these epitopes in frame within the A antigen gene. STRATEGIES FOR INTRODUCING GENES INTO HVT VECTORS Two possibilities can be envisaged: 1) insertion into non- essential genes of the vector or 2) substitution of foreign gene for corresponding gene of the vector. This would be possible only in regions which already have substantial homology as may be the case between some genes of MDV and HVT. EXAMPLE 5: Insertion into non-essential genes of HVT or MDV la)Insertion at the TK locus of the vector. 1) HVT or MDV may be used as vectors for insertion and expression of avian herpesvirus genes. In particular gB, gH or gC of R818 HDV' may be inserted into HVT. One may use the promoter associated with the inserted gene or use heterologous IE990699 those of a different class of genes (for promoters. including example the immediate early promoter to optimise expression of gB). 2) HVT or MDV may be used as general vectors for the insertion and expression of genes unrelated to avian herpes viruses and likely to require manipulation of promoters for optimal expression. The procedure to be used for gene insertion is substantially as described previously for the insertion of hepatitis antigen in l. 1984 as above]. HSV [Shih Q; MDV and HVT DNA obtained as described above is infectious provided that precautions are taken'not to shear the DNA during extraction. Calcium phosphate precipitates of viral DNA prepared as described by Stow and Wilkie [(1976) J. gen. Virol. 33, 477] were added to sub—confluent monolayers of CEF. After absorption for 1h at 37°C, culture medium was added and cultures were incubated for 1 or 2 days until Confluent. Monolayers were then trypsinised, replated (1:1 or 1:2) in 199 containing 2 to 4% calf serum and incubated medium (wellcomel usually after 4 to 5 days. at 37°C until plaques developed, Approximately 200 plaques may be obtained per Ag of HVT DNA and approximately 50 per Ag of MDV DNA. IE990699 For homologous recombination and isolation of recombinant virus, genes of interest are inserted within non—essential genes such as TX or gC and co-transfected with wild-type viral DNA at molar ratios ranging from 10:1 to 2:1 as described above. Alternatively, intact wild—type virus may be used for co-infection. Restriction enzyme sites that could be used for the insertion of foreign antigens into the TK of HVT strain Fc—l26 include: BanII, Bsp1286, DraIII, EcoRI, HincII, Hgal, Nhel and NsgbII. RE sites that could. be used to produce defined TK deletion Ball. mutants hi MDV serotype I strain RBIB include; HaeII, insertion sites for foreign DNA that would NdeI and Sphl as and double digests of combinations of disrupt the TK gene. restriction enzymes IEcoK could also be used) to these four remove a portion of the TR gene. thus inactivating it. Some of these enzymes also have sites in the plasmid vector Thus in order into which the virus DNA fragments are cloned. to linearize the clone DNA without also cutting within the vector, partial_digests may be carried out. None of the above enzymes should cause any disruption to flanking genes, HSV—1 homologues of which are known to play an important role in virus multiplication. IE990699 Virus recombination may be detected by ‘plaque lifts‘ which involve transfer of infected Cells and released virus which have adhered to the agar overlay to nitrocellulose and hybridization of the denatured DNA released from the cells and virus to suitable probes as described by Villareal. L. _t _l (1977) Science 196, 183-185. Virus which hybridizes to the probe may be recovered from the monolayer. A similar procedure may be used to isolate recombinant virus which expressed epitopes of interest. In this instance the nitrocellulose "plaque lifts" are treated with antibody and the presence of bound antibody revealed using a suitable detection system such as labelled proteni A or phosphatase conjugated anti—globulin antibody. The gene of interest with appropriate promoters is first inserted within the cloned TH gene. The recombinant DNA is then co-transfected with infectious DNA of the vector in chick embryo fibroblasts or chicken kidney cells and TK- virus may be selected by growth in medium containing acyclovir [Ross, N. (1985) as above] or FMAU [Schat, K.A. _t _l (1984) Antiviral Research 4, 159-270]. Alternatively, or in addition, plaques are screened for the presence of the gene of interest using 'plaque lifts’ on nitrocellulose and hybridization to any relevant labelled probe. Plaques are also screened for expression of the epitopes of interest using monoclonal antibodies or antipeptide antibodies. \r—‘ IE990699 The main advantage of this strategy is that the selection procedure increases the chances of obtaining virus recombinants containing the gene of interest. It also offers the opportunity for optimum expression. Thus the of using different promoters use of an immediate early promoter may allow expression in latently infected cells. (b)Insertion at other non—essential sites of the vector. Since the A antigen (HVT and MDV homologues of HSV gC) is not essential for virus growth in givo and in vitro (see section on gC above) it is a potentially useful site for the insertion and expression of foreign genes. Moreover, since it is one of the most abundant antigens and is excreted, it may be particularly useful for enhancing the immunogenic properties of foreign proteins. The isolation of virus recombinants at this locus may be achieved by first inserting at least part of the gene of interest in frame within the gC gene and then co—transfecting with infectious viral DNA. Screening of virus plaques with sequence specific probes or with specific antibody allows the isolation of recombinants. An antigen~encoding sequence can also be inserted into the ribonucleotide reductase {large subunit) gene of HVT or of MDV - see Figures 8 and 9. IE990699 EXAMPLE 6: Substitution of MDV genes for their homologues in Substitution may be achieved by co—transfection of cloned MDV sequences and infectious HVT DNA as described in Example 5. Substitution of the g8 and gC genes derived from the RBIB strain of MDV for their counterparts in HVT may be effected as may substitution of the gH gene of MDV, other glycoproteins and immediate early genes. Recombinants expressing MDV sequences and epitopes may be detected using MDV-specific monoclonal antibodies or anti- peptide antibodies raised against unique MDV sequences as described above. The advantage of this procedure is that it is relatively simple and does not require manipulation of promoters. However. it may be limited to genes which share substantial homology. IE990699 EXAMPLE 7: Strategies for obtaining TK‘ mutants of MDV Deletion mutants. Deletions may be introduced within any suitable part of the gene. for example the domains of the gene that are required for nucleoside binding. This may be achieved by restriction enzyme double digestion. for example with HaeII and any of the following enzymes: Ball. NdeI. Sphl or EcoK. Appropriate fragments are then religated, followed by co» transfection with infectious viral DNA or transfection into virally—infected cells. Reference may be made to Figures 7 and 8, and to the section above relating to insertion of heterologous sequences, in choosing restriction enzymes and so on. TK- virus may be selected in the presence of’ acyclovir [Ross, N. (1985) as above] or FHAU [Schat, K.A. gt _l (1984) as above]. Plaque—purified clones may then be tested for the absence of the deleted portion of the TK gene by hybridization. The deletion mutants of MDV may be used themselves as attenuated viruses for vaccine preparation, or may have sequences for heterologous antigens inserted. A functional B—ga1actosidase gene under Insertional mutants. the control of a herpesvirus promoter or any other suitable sequence or a single base is first introduced in a domain of the TH gene which is essential for TK activity. The recombinant DNA is then co—transfected with infectious viral DNA or transfected into virally—infected cells to allow homologous IE990699 recombination to occur. Selection in the presence of acylovir or FMAU will yield TK' insertional mutants. If a B—ga1actos- idase gene is introduced. mutants can be detected by the production of blue plaques in the presence of X—gal. The TX gene and surrounding sequences may be subcloned into another suitable vector if necessary. l'VIj..-\n"H'l.lf 3'1 !:)'t~r'I I4-I. <>I ~."‘.I_l7‘/ ‘:2’!-‘.l'n 35-‘. L_;.,,.- -,;._1 . [|‘.v"i‘ The HVT TK gene is cloned in the plasmid vector pUC13 to generate a plasmid, which is termed pTK1B. This plasmid is linearised with, for example, the restriction endonuclease Rsr II which cleaves the plasmid only within the TK gene (nucleotide position 197 in Figure 5. enzyme recognition sequence CGGACCG). The "sticky" ends thus generated can be end repaired by standard techniques [see "Molecular Cloning: a Laboratory Manual", ed. Maniatis T.. Fritsch E.F., and Sambrook J. Cold Spring Harbor Laboratory 1982). The RBIB gB was originally cloned on two plasmids which may be termed RB1B—BamH1—I; and RB1B—§amH1—K;. (Note I; had lost one BamH1 site during cloning.) To generate a complete gB copy on one plasmid, both plasmids were cleaved with BamH1 and the fragments ligated. Recombinants containing the desired configuration were identified by restriction enzyme analysis of plasmid DNA’s. However. as described above. the complete gB IE990699 :5 :1 sequence was subsequently obtained on an EcoRI/Sal} fragment. Further information regarding the sequence encoding MDV g5 and its manipulation may be found in Ross _g __l J. gen. Virol (1989) [Q 1789-1804. The single recombinant plasmid of Ross gt al is then cleaved with ECORI and SalI, the ends are repaired and the plasmid is Alternatively, the MDV gB cloned into PTKIB prepared as above. open reading frame could be excised from plasmid MSB27 by digestion with HincII and NaeI and the products ligated to HVT TK plasmid pTKlB. cleaved partially with Hpal. Recombinant plasmids containing both TX and gB sequences could be identified by hybridisation and further characterised by Southern blotting. The recombinant plasmids are then introduced into cells containing HVT virus (viral DNA) and homologous recombination will introduce the gB gene into the TK gene. HVT viral recombinants can be selected with acyclovir or FMAU or alternatively detected with labelled gB probes. £"5.:'\f'x§‘l.i'. ‘|' :H H‘ x_j"' Blunt ended PTKIB is prepared as in Example 8. The RBIB gC is cleaved from the plasmid pMB419 (Example 4) with the restriction endonucleases EcoR1 and HindIII (site within the pUC13 polylinker). The sticky ends generated are again end- repaired by standard protocols. The end—repaired gC fragment is IE990699 then cloned into the linearized end-repaired pTKlB as in verified by analysis of the Example 8. (The cloning can be resulting clones with restriction enzymes, probing with radio- actively labelled fragments. or DNA sequencing, or any combination of these). The resulting plasmid with the RBIB gC gene cloned into the HVT TH gene can then be introduced into the HVT genome by transfecting the plasmid into HVT—infected cells using calcium phosphate precipitation or electro—poration. Homologous recombination, involving cross—overs either side of the gC gene, between the HVT virus and the flanking sequences of the HVT TK plasmid will carry the RBIB gC gene into the HVT viral genome. Viral recombinants can be selected for {as they are TK-) or identified (eg by probing) as described above. In analogous ways, the sequence information given above and in the Figures can be used to design cloning strategies for the insertion of these genes and others into the non—essential genes of the HVT described here or to generating combinations of antigen genes in HVT. EXAMPLE Lg [‘IIJV gl) ggw Figure 15 shows part of the sequence of the MDV gD gene. The sequence was obtained by sequencing random fragments of the Us IE990699 reg1on MDV DNA and comparing the sequence to the sequence of (see Buckmaster gt gl, Lgc. c1t.). The known herpesvirus genes sequence gave homology scores of 189 and 216 respectively with HSV gD and PRV gp50. The sequence information assists in the preparation of suitable probes to isolate and characterise the gene . IE990699 CIJKIMS 1. A vaccine against Marek’s Disease, comprising a vector which contains a DNA fragment encoding the gD gene from MDV serotype 1 or 2 or 3. 2. A vaccine according to claim 1, characterized in that the vector is a viral vector and the said DNA fragment is inserted into a non~essential site if said viral vector. 3. A vaccine according to claim 1 or 2, characterized in that the DNA fragment comprises the coding portion of the nucleotide sequence appearing on Figure 15. 4. A vaccine according to claim 1 or 2, characterized in that the DNA fragment comprises the coding portion and at least a part of the 5' and/or 3' non—coding portions of the gD gene. S. A vaccine according to claim 1, 2 or 3, characterized in that the vector‘ comprises further‘ a promoter‘ which is heterologous to the said DNA fragment. 6- A vaccine according to any one of claims 2 to 5, characterized in that the viral vector is MDV. 7- A vaccine according to claim 6, characterized in that the MDV vector is HVT. 8. A vaccine according to claim 5 or 7, characterized in that it comprises MDV—susceptible cells and said viral vector. 9. A vaccine according to claim 2. 3 or 5, characterized in that the viral vector is a Poxvirus, in particular fowlpox virus. 10. DNA fragment comprising the nucleotide sequence appearing on Figure 15. 11. A vector comprising a DNA fragment according to claim 12. A plasmid vector comprising a DNA fragment according to claim 10, which is suitable for transfection of an MDV—or HVT—susceptibLc cell. ANNE RYAN & CO. AGENTS FOR THE APPLICANTS IE990699 Abstract Viral Vaccirgs A vaccine effective against Marek’s disease virus (MDV) comprises (a) an MDV attenuated by virtue of being TK- or (b) a host expressing an MDV antigen, namely the respective MDV homologues of the HSV gB, gC, gD or gH glycoproteins (or antigenic parts thereof) or the respective MDV homologues of the HSV-1 immediate early genes IE-68 or IE- l75. 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Description

VIRAL VACCINES The present invention relates to viral vaccines which may be used to provide immunity against disease and to nucleotide sequences for inclusion in the viruses of such vaccines.

Background and Description of prior art Herpesviruses are large double stranded DNA viruses consisting of an icosahedral capsid surrounded by an envelope. The group has been classified as alpha. beta and gammaherpesviruses on the basis of genome structure and biological properties [Roizman. B _; ._l (1981) Inter—virology 16. 201-217]. Avian herpes viruses include Marek's Disease Virus (MDV) ta gammaherpesvirus) which causes a lymphomatous disease of considerable economic importance in chickens [reviewed in Payne. L.N. (ed) Marek’s Disease (1985), Martinus Nijhoff Publishing, Boston] and Infectious Laryngotracheitis Virus (ILTV) (an alphaherpesvirus) which Causes an acute upper respiratory tract infection in chickens resulting in mortality and loss of egg production.

A recent unexpected finding in our laboratory is that there is sufficient amino acid homology between MDV. ILTV and mammalian herpesviruses. particularly varicella zoster (VZV) and Herpes Simplex Virus (HSV) to allow identification of numerous conserved genes. These include the MDV and Herpesvirus of - 84982 Turkeys (HVT) homologues of glycoproteins gB, gC and gﬂ of HSV; the ILTV. MDV and HVT homologues of TH and ribonucleotide reductase genes and the ILTV homologue of gB and genes 34 and of VZV [Buckmaster. A t al (1988) J. gen. Virol, 69. 2033- .

Strains of MDV have been classified into three serotypes. Type 1 comprises pathogenic strains and their attenuated derivatives. Type 2 are a group of naturally-occurring non- pathogenic strains and type 3 is HVT. For more than a decade, vaccination with HVT has been remarkably effective in controlling Marek’s disease. However. in -recent years. new strains of MDV have been isolated which cause disease despite vaccination with HVT. Losses due to these ’very virulent’ strains have occurred in parts of the U(S.A., Europe and the Middle East. Although the degree of protection can be improved by using a mixture of HVT, type 2 MDV and attenuated derivatives of very virulent strains for vaccination. the results have been erratic. These observations and the fact that there are MDV type—specific epitopes that are not shared by HVT or type 2 HDV have led us to the conclusion that improved vaccines might be constructed which are antigenically more related to MDV than existing vaccines. [Reviewed by Ross and Biggs in Goldman J.M. and Epstein M.A. (eds) Leukaemia and Lymphoma Research, Vaccine Intervention against Virus—Induced Tumour, p 13-31. Macmillan. 1986].

A number of herpesvirus antigens have been shown to confer protective immunity when expressed in a recombinant vaccinia virus. These include the gB gene of HSV [Cantin E.M. gt _l (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5908—5912]. gD of HSV [Paoletti. E. _t _l (1984) Proc. Natl. Acad. Sci. U.S.A. 8], 193—197] and gp5O of pseudorabies virus (PRV), a homologue of HSV gD [Marchioli. C.C. _t _l (1987) J. Virol. 61, 3977-3981].

Because of the absolute requirement of gB for virus penetration and infectivity and because it is conserved among herpes- viruses, g3 and its homologues are important immunogens.

Moreover, the presence of gB at the surface of infected cells has been shown to be an important target for humoral and cell- mediated immune responses [Blacklaws, B.A. t 1 J.gen. Virol. . 1103-1114 (1987); McLaughlin~Taylor, E. _t _l_ (1988) J. gen. Virol. 69. 1731-1734]. The recently described glycoprotein gH of HSV is also essential for infectivity and may also be an important immunogen [Desai. P.J. _t _l (1988) J. gen. Virol. 69, 1147-1156]. It has also been shown that gIII of pseudorabies virus (PRV), a homologue of gC, is a major target for neutralizing antibody and for cytotoxic T cells although it is a non-essential protein. Also of interest is the unexpected participation of immediate early proteins in 1‘ cell mediated cytotoxic reactions in cells infected with cytomegalovirus (CMV) [Kozinowski U.H. _t _l (1987) J. Virol. 61, 2054-2058].

Similar antigens could play an important role in the rejection of latently infected and transformed lymphocytes in Marek’s disease since immediate early RNA have been transcripts detected in lymphoblastoid cell lines established from Marek’s disease tumours.

Although many recombinant vaccines have been constructed using the poxvirus vaccinia as a vector, there are also reports of the use of herpesviruses as vectors for the expression of foreign genes. Thus hepatitis antigen has been expressed in HSV [Shih. M.F. _; _l (1984) Proc. Natl. Acad. Sci. U.S.A. 81, -5870] and human tissue plasminozen activator has been D.R. et al expressed in PRV [Thomsen. (1987) Gene 57. 261-265.

In both cases, foreign genes were inserted in cloned fragments of non-essential herpes genes which were then introduced into virus vector by homologous recombination. The hepatitis virus gene was fused to a herpesvirus promoter and the recombinant DNA was inserted within the TK gene 0f 35V- Homologous recombination co—transfection of the following recombinant DNA and wild-type HSV DNA resulted iJ1 TK‘ Virus clones that expressed the hepatitis antigen.

In the case of PRV. the gX gene mapping in U: was used as the site for insertion of the The strategy Used foreign gene. involved insertion of the ‘TH gene of HSV in the gx gene of a PRV mutant that had a defect in its TX gene resulting in a T3 positive virus. The human tissue plasminogen activator gene “as then inserted within a cloned fragement of HSV TR and the recombinant was introduced into the PRV mutant by homologous recombination. TH- virus was selected which expressed the human gene (Thomsen gt Q; as above). Similarly, VZV has been used as a vector [Lowe gt al (1987) Proc. Natl. Acad. Sci. U.S.A. 84, -3900]. Several herpesvirus genes have also been shown to be associated with virulence and to be non-essential for growth in vitro. These include the TK genes of HSV [Jamieson. al (1974) J. gen. Virol. 24, 465-480; Field, H. and wildy, P., (1987) J. Hygiene (Cambridge) 81, 267-277] and of PRV. Indeed it has long been known that PRV is readily attenuated by deletion of TK activity [Tatarov, G. (1968) Zentralbl. Vet. Med B, 848-853]. Furthermore, attenuation of the Bartha strain of PRV has been attributed to a defect in gI, a non-essential structural glycoprotein mapping in Us [Mettenleiter, T. _g _l (1987) J. Virol. 61, 4030-4032].

Genes of HSV mapping in the internal repeat region (TRS) flanking the long unique sequence have also been associated with pathogenicity [Rosen, A. _g _l (1986) Virus Research 5, 157-175; Thompson, R.L. _t _l (1983) Virology 131, 180-192L Several additional genes of HSV have been shown to be non- essential for growth in vitro although it is not known whether they are associated with virulence. These include UL24 (Sanders, P.G., (1982), J. gen. Virol. 63, 277-295, large subunit of ribonucleotide reductase (Goldstein D.J. and Weller, S.K. (1988) J. Virol. 62. 196-205). SC (Draper K.G. t l A.T. Q (1984) J. Virol. 51, 578-585), dUTPase (Fisher. F.B. & Preston.

V.G. (1986) Virology 148. 190-197). and Ln 55 and UL 56 (MacLean, A.R. & Brown, S.M. (1987) J. gen. Virol. 68, 1339- ). Moreover there is evidence that several genes of HSV mapping in Us are also non—essential for growth in [weber, P.C. t 1 (1987) Science 236, 576-579}.

W0 88/07088 (published only on 22 September 1988) Qisclosed hybrid viral vectors based on HVT or MDV and including a gene of interest in a non-essential site, such as the TK region or the region encoding protein A. Protein A. in this context, appears to be the same as gC. disclosed by Velicer and Coussens.

Summary of the invention One aspect of the present invention provides a nucleotide sequence substantially free of the sequences which would adjoin it in the wild-type virus associated with the sequence, the sequence being selected from the group consisting of: la) the MDV homologue of the HSV gB gene, (b) the MDV homologue of the HSV gH gene. (c) the TR gene of MDV, (d) the MDV homologue of the immediate early gene IE—17S Of HS‘/pl vitro Ie) the MDV homologue of the immediate early gene IE—68 of HSV—I (f) the MDV homologue of the HSV gD gene and minor variations thereof.

In addition. the TK sequence of HVT, referred to hereinafter sometimes as sequence (x). and the MDV analogue of HSV gC, referred to hereinafter sometimes as sequence (Y).and. minor variations of either may be used as insertion sites for certain heterologous sequences or as deletion sites to obtain less virulent viruses but are not novel per gg.

Each of sequences (a) to (f), (x) and (y) may be associated with further elements such as suitable stop and start signals and other 5’ and 3' non—coding sequences, including promoters, enabling expression of the sequence. Such further elements may associated with the in its naturally- be those sequence occurring state or may be heterologous to that sequence.

In particular the promoter may be one associated with one of the sequences (d) and (f) above.

The term "minor variations thereof" is intended to include changes in the nucleotide sequence which do not affect its essential nature, for example minor substitutions of nucleotides for one another. In the case of sequences which are intended for insertion into a vector to encode an antigen. the "essential nature of the sequence refers to the protein or glycoprotein encoded. Conservative changes in the nucleotide sequence which give rise to the same antigen will clearly be included. as will changes which cause conservative alterations in the amino acid sequence which do not affect adversely the antigenic nature of the antigen. in particular. antigenic portions of the antigen sequences may be used alone, for example, the regions corresponding to nucleotides 816-863, 1377-1595, 1377-1630 or 1824—198b of MDV gB, or nucleotides 483-633, 843-933 or 1203-1278 or MDV gC, and minor variations thereof. These sequences and the peptides encoded thereby form further aspects of the invention. In the case of a sequence which is an insertion site, it is necessary only that the sequence should be non-essential for the infectivity and replication of the virus and have sufficient homology with the defined sequence to enable recombination to occur. Thus an insertion of the nucleotide into the sequence could completely change the reading frame from then on in a downstream direction. In the case of an antigen—encoding sequence this the amino acid sequence undesirably would usually alter (depending on where the frameshift occurred), but in the case of an insertion site, the degree of homology would be almost the same. thereby allowing recombination to take place with almost the same ease.

Generally speaking. in an insertion site, if a nucleotide homology of at least 75% is present, the sequence is regarded as a "minor variation". Preferably. the sequence is at least , 85. 90. 95 or 99% homologous. It will be appreciated that such degrees of homology relate to substantially the entire portion of each sequence (a) to (f) and (X) defined above.

Shorter sequences may be used as probes in the identification or isolation of such longer sequences.but in this case the degree of homology will in general need to be greater in order to ensure accurate hybridisation.

Thus, a further aspect of the invention provides sub—sequences of at least 13 nucleotides having at least 90% (preferably 95%, % or 100%) homology to at least one portion of any of the said sequences (a) to (f), (X) and (y) above.

In the above list, sequences (a), (b) and (d) to (f) are useful as antigen—expressing sequences and sequence (y) is useful as an insertion site for heterologous sequences. Sequence (C) is useful for deletion to provide TX’ mutants.

The sequences may readily be isolated from naturally-occurring HVT and MDV viruses, using the sequence information given herein and standard techniques, for example involving the preparation of oligonucleotide probes and use thereof to hybridise to the naturally—occurr1ng DNA.

The isolated polypeptides encoded by sequences (a). (b) and (f) above are novel and form a further aspect of the invention, together with minor variations thereof and any glycosylated forms thereof which result from expression of the said sequences in MDV~susceptible cells.

A second aspect of the invention provides MDV mutants which are insertional or deletional mutants in the TX gene.

The mutation may be in the coding or non—coding sequences of the region identified.

An MDV antigen—expressing gene may be isolated from a virulent strain of MDV and inserted into the TK region of a less virulent strain of MDV; this insertion would result in a novel "virus" if it did not result in a naturally-occurring virus.

Other heterologous antigen-encoding sequences may be included, as well as an MDV antigen-encoding sequence. for example.

The heterologous sequence may alternatively be one coding for an antigen associated with any one of the following diseases: avian encephalomyelitis (epidemic tremor). avian influenza (fowl plague), avian leukosis, avian paramyxoviruses other than Newcastle disease (PMV2 to PMV7), avian reovirus diseases (enteric disease, tenosynovitis). chicken anaemia (caused by chicken anaemia agent). coccidiosis, egg drop syndrome (EDSTS). fowl pox, infectious bronchitis, infectious bursal disease (Gumboro). inclusion body hepatitis (adenovirus). lymphoproliferative disease of turkeys, Newcastle disease, reticuloendotheliosis in chickens, reticuloendotheliosis in turkeys, rotavirus enteritis. turkey haemorrhagic enteritis and turkey rhinotracheitis. The sequence may alternatively encode paramyosin (a muscle protein common to all invertebrate parasites) or an antigenic part thereof, somatostatin or a grouth—promoting part thereof or an immune regulator.

The vectors in accordance with the invention will then provide multivalent vaccine protection.

The mutant viruses are potentially useful in vaccines as attenuated viruses, without necessarily having a heterologous sequence inserted.

A convenient process for preparing the deletional or insertional mutants of the second aspect of the invention comprises simply introducing into a suitable cell, for example by co-transfection, a deletional or insertional mutant version of the TH region and either whole viral DNA or a whole virus virus). The naked DNA of such (for example the wild—type viruses has been found to be infectious. provided that it has not been sheared. A calcium phosphate precipitate of the DNA is generally advantageous. Suitable cells include chicken embryo fibroblasts. chicken kidney cells and duck embryo fibroblasts. all preferably grown in sub—confluent monolayers in Petri dishes. The transfected DNA and the whole viral DNA will then recombine with one another in the infected cells by homologous recombination and the desired recombinants can be screened for, for example by the detection of hybridisation to suitable probes or by an immunoassay using suitable antibodies to the gene product of the region in question.

For homologous recombination to take place, the viral DNA must replicate. At present, no cell—free replication system for MDV is known. However. if such a system becomes available. then the process of the invention could be operated therein. The environment in which the replication and recombination occur is not critical.

Regions (3), (b) and (d) to (f), which were identified above as being responsible for encoding immunologically useful viral antigens, can be inserted into suitable vectors, for example into HVT or other vectors such as foulpox-virus. bacteria OF fungi. In the case of viral vectors, especially herpesvirus vectors and poxvirus vectors, such insertion can be achieved by recombination between the antigen—encoding sequence, flanked by suitable non—essential sequences. and the vector's genome in a suitable host cell as described above. When HVT is the vector. the will usually be an HVT MDV vector. When promoter 0? fowlpox-virus or other virus is the vector, the promoter will Po J1 usually be a promoter which is endogenous to the vector. In the case of bacteria and fungi. the antigen-encoding sequence may be inserted using known or yet-to—be-discovered techniques of DNA manipulation. A non-pathogenic strain of Salmonella may be used as such a host. The heterologous sequence may be inserted into the host’s genome or be carried on an independently-replicating plasmid. A promoter which is endogenous to the host will usually be used to control expression of the heterologous (viral antigen—encoding) sequence.

The flanking sequences which are used may comprise all. virtually all or less of the region into which the heterologous sequence is to be inserted. If all the region is employed, then the sequence of that region will clearly still be present in the resulting virus, but the function of that region will have been deleted. If less than the whole region is used as flanking sequences, then the result will be a structural as well as functional deletion. Either approach may be used.

Thus, three strategies can be envisaged for the construction of improved Marek’s disease vaccines: (1) Construction of HVT that selected MDV genes; (2) recombinant express Construction of deletional or insertional mutants of highly virulent strains of MDV, which are attenuated and hence vaccines; (3) Construction of recombinant suitable for use in viruses that express MDV proteins in other vectors such as foul pox virus.

To prepare a vaccine in which HVT CH‘ MDV jg the virus or vector. the virus is grown in suitable cells such as chick embryo fibroblasts in a standard culture medium which as 199 medium (Wellcome or Flow Laboratories) for 3 to 4 days at about 370C. The cells are harvested by trypsinisation and suspended in medium containing 10% dimethyl sulphoxide and 4% calf serum before storage in liquid nitrogen in sealed ampoules.

For vaccination. typically, day—old chicks are injected intramuscularly with about 1,000 plaque-forming units. Immunity follows within a few days.

It should be noted that MDV and HVT are cell-associated viruses and are infectious only when present in cells. Thus, a vaccine infected based on such viruses will always include suitable cells.

The vaccines of the invention may be used to protect any fowl susceptible to MDV, including commercially—reared poultry such as chickens, turkeys, ducks and quail.

Preferred aspects of the invention will now be described by way of example and with reference to the accompanying drawings. in which: Figure 1 is a map of the HDV genome showing in part the ﬁgml site distribution and the location of the gB and TK genes.

Figure 2 (On 18 sheets) shows the nucleotide sequence of the gB gene of the R818 strain of HDV, with the numbering referring to the MDV nucleotides. the sequence of part of the HVT gB gene shown under the line, homologies indicated by vertical bars, and amino acid differences between MDV gB and HVT gB shown above the line; Figure 3 is a map of the HVT genome showing the positions of the gH (hatched). TK (solid black) and major capsid protein (MCP. dotted) genes, with HindIII sites shown as "H"; Figure 4 (on 3 sheets) shows the nucleotide sequence of most of the HVT gH gene, with the corresponding amino acid sequence shown above the line; Figure 5 (on 10 sheets) shows the nucleotide sequence of the HVT TK gene, with the numbering referring to the HVT nucleotides, the sequence of part of the MDV TK gene shown under the line, homologies indicated by vertical bars and amino acid differences between MDV TK and HVT TK shown above the line; Figure 6 (on 6 sheets) shows the nucleotide sequence of the gC gene of the RBIB strain of MDV. With corresponding amino acids shown above the line; Figure 7 shows part of the nucleotide sequence of the HVT homologue of the VZV62/HSV—1 IE 175 gene with corresponding amino acids shown above the line; Figure 8 shows part of the nucleotide sequence of the HVT ribonucleotide reductase (large subunit) gene with corresponding amino acids shown above the line; Fjgllre 9 (on 2 sheets) shows part of the nucleotide sequence of the MDV ribonucleotide reductase (large subunit) gene with corresponding amino acids shown above the line; Figure 10 shows part of the nucleotide sequence of the HDV ribonucleotide reductase (small subunit) gene with corresponding amino acids shown above the line; Figure 11 shows part of the nucleotide sequence of the MDV homologue of the HSV-1 IE—175 gene with corresponding amino acids shown above the line; Figure 12 shows part of the MDV homologue of the HSV-1 IE—68 gene with corresponding amino acids shown above the line; Figure 13 is a schematic representation of homologous recombination at a non—essential region of a viral genome and a homologous region of DNA cloned within a plasmid vector; Figure 14 (on 27 sheets) supplements Figures 4 and S. and shows the nucleotide and predicted amino acid sequences from the region containing the MDV and HVT TH and gH and flanking amino acid those bracketed MDV sequences are genes. The potentially encoded by this region «of nucleotide sequence if the upstream ATG triplet were the true gene initiation site.

Asterisks denote stop codons. Spaces have been inserted into the sequences in order to optimize alignments. Colons between the MDV znui HVT DNA sequences indicate nucleotides conserved between the two viruses. MDV amino acids are only shown in positions where they differ from that in HVT; and Figure 15 shows the partial nucleotide sequence of the MDV homologue of HSVgD. the predicted amino acids being shown above the MDV nucleotide sequence and residues in bold type being conserved between the MDV and HSV-1 gD regions.

EXAMPLES: General Approaches Selected short sequences of the avian herpesviruses cloned in the bacteriophage vector M13 were used as probes to identify longer fragments that might contain the entire genes of interest. This was achieved by Southern blot hybridization of restriction fragments. Full details are given below. yjrug Strains. The ‘highly oncogenic' strain RBIB of NDV [Schat, H.A. t I (1982) Avian Pathol. 11, 593-605] was obtained from Professor B. Calnek, Cornell University. Ithaca, U.S.A. The virus received has been plaque purified in chicken kidney vells in tissue culture. It was passaged twice in SPF RIR chickens and 4 times in chick embryo fibroblasts (CEF). Its ’highly oncogenic' nature was demonstrated by a high incidence of gross tumours when inoculated in genetically resistant N- line chickens.

The FC126 strain of HVT [witter, R.L. _t _l (1970) Am. J. Vet.

Res. 31, 525-538], obtained from the Wellcome Research Laboratories. Beckenham, Kent, had been passaged 14 times in CEP. It was subsequently grown in duck embryo fibroblasts (DEF) and CEF in our laboratory. It was then plaque-purified and grown further in CEF. Viral DNA used for cloning in the present work was extracted from virus that had been passed 29 times since the original isolation.

Tissue culture. CEF were grown in roller bottles in 199 medium lwellcome), supplemented with penicillin, streptomycin, Fungizone (Regd. T.M.) and calf serum as described previously {Ross, L.J.N. t 1 (1975) J. gen. Virol. 28. 37-47].

CKC were grown in 10 cm Petri dishes [Churchill. A.E. and Biggs P.M., (1967) Nature, 215, 528-530].

Isolation of MDV DNA. Cell associated R818 was inoculated onto confluent monolayers of CEF in roller bottles at a multiplicity of infection of approximately 0.001 plaque—form1ng units (pfu) per cell, and the cultures were incubated at 37°C. After 3 days. the medium was discarded and replaced with fresh 199 medium containing 2% calf serum. Cells were harvested for virus purification after 2 to 3 days when cytopathjc effect was extensive. Virus was obtained by rate zonal centrifugation of the cytoplasmic fraction of infected cells (1980) J. gen. Virol. 51, 245-253]. Viral DNA was extracted by treating purified virus with sarcosyl. proteinase K and Tris buffer pH 9 overnight at 37°C and purified by rate zonal centrifugation in glycerol gradients as described previously (Lee egg al, 1980). High weight viral DNA was molecular precipitated with ethanol and resuspended in 10 mM Tris pH 7.5 im 1mM EDTA (TE).

Cloning of MDV DNA. One pg of MDV DNA was cut with the restriction enzyme BamH1 and ligated to BamH1—cut, dephosphorylated pUC13 DNA (Pharmacia). Competent E.coli strain transformed according to standard procedures T01 cells were [Hanahan, D. (1983) J. Mol. Biol. 166, 557-580] and were grown in the presence of ampicillin and X-gal. White colonies were picked and tested for the presence or MDV inserts by hybridization to nick-translated MDV DNA [Grunstein M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3961].

Positive colonies were cultured in small volume and plasmid DNA isolated by the procedure of Holmes, D.S. and Quigley, M.

[Lee, Y.S. gt Q; [(1981) Anal. Biochem. 114. 193—297]. The size of the inserts was determined by electrophoresis of BamH] digests of the recombinant DNA in agarose gels. Plasmids containing MDV inserts ranging from less than 1 to 18 Kbp were obtained.

Random sequencing of viral DNA. Sonicated fragments of viral DNA were cloned into Smal-cut, dephosphorylated M13.mp10 (Amersham International PLC) and plaques containing MDV inserts identified by hybridization to MDV DNA. The sequence was were determined by the dideoxy method (Sanger, F. et al (1977) Proc.

Natl. Acad. Sci. U.S.A. 74. 5463-5467] using 358 dATP).

The same procedure was used to sequence cloned fragments of MDV DNA except that plaques were identified by hybridization to labelled insert so as to avoid colonies containing pUC13 fragments.

EXAMPLE 1: gB gene of MDV An M13 clone of HVT homologous to the gB gene of VZV and HSV hybridized to BamH1 fragment I3 of MDV (see Figure IL Sequencing of this fragment obtained from as BamH1 library of the RBIB strain of MDV showed that two thirds of the gene, starting with the NH; terminus, was contained within 13. The remainder of the gene was identified in the adjacent restriction fragment K3. Figure 1 shows the map position of the gene which is 2.6Kbp long. Its mRNA has been estimated to be approximately 2.8 Kb. The translated protein is 865 amino acids long (Figure 2). This includes approximately 20 amino acids which may be part of a signal sequence domain. The primary translated sequence of MDV gB has a few features in common with gB of other herpes viruses such as the alignment of cysteine residues and the presence of hydrophobic sequences which are presumably capable of spanning a lipid bilayer [Pellet, P.E. gt _l (1985), J. Virol. 53, 243-253]. However. MDV gB has only 48% amino acid similarity with gB of HSV and has many unique features such as the insertion of 23 amino acids (residues -1920, Figure 2) and the presence of extra sites with glycosylation potential. Comparison of the sequence of MDV gB with limited sequence data (702 bases) available for HVT gB (Figure 2) has shown 76.9% nucleic acid similarity and 87.1% amino acid similarity between these two glycoproteins. Amino acid substitutions in HVT gB compared to MDV gB were particularly marked in a region (residues 1323 — 1433) equivalent to a domain of HSV gB associated with virus neutralization [Pellet P.E. _t _l (1985) as above]. Amino acid substitutions between MDV and HVT gB were also noted in other regions of unknown function.

EXAMPLE 2: gH gene of HVT and gH gene of MDV An M13 clone of HVT containing sequences homologous to HSV gH was isolated during our earlier work on gene identification and mapping (Buckmaster gt Q; (1988) as above). This clone, when used as a probe, hybridized to a 6Kbp ﬁigdlll fragment of HVT (Figure 3). Sequencing revealed that this fragment contained approximately one quarter of the gH gene including the carboxy terminus. The adjacent ﬂigdlll fragment (3.2 Kbp) containing the remainder of the gH gene was identified by hybridization using a cloned ﬂpgl fragment of HVT which overlapped the ﬁindlll site. Figure 4 shows the sequence of the coding region of the gH gene of HVT (2.3 Kbp) and flanking sequences. The % V of HVT and its amino acid identity between the gH gene HSV1, VZV and EBV was only 20, 24 and 20 homologue in respectively (estimated from maximised amino acid overlaps of , 644 and 153 respectively).

EXAMPLE 3: TK gene of HVT and TK gene of MDV The whole coding region of the TK gene of HVT (1053 bp) was contained within the 3.2 Kbp ﬂigdlll fragment described above (Figure 3). The sequence of the entire gene and flanking regions is shown in Figure 5. Similarly the whole of the MDV TX gene is contained within the 3.6 Kbp Egmﬂl K2 fragment of HDV (Figure 1). The complete sequence of MDV TK gene is shown in Figure 14. Comparison of the MDV and HVT TK sequences shows P.) Rf, that the two genes have 60% amino acid identity. By contrast, the % amino acid identities between the TR gene of HVT and the TR genes of HSV 1. VZV and EBV are only 30. 27 and 24 respectively {estimated from amino acid overlaps of 320, 332 and 193 respectively). The predicted amino acid sequences of HVT and MDV TK show characteristic ATP and/or CTP binding site motifs described for a number of virus and eukaryotic proteins that are associated with phosphorylation (Gentry, G.A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6815-6819). These conserved sequences are examples of useful sites for insertion and expression of foreign genes and for producing TX" deletion mutants.

EXAMPLE 4: A antigen gene of MDV (gPS7—65) (gC homologue) The A antigen gene is of interest in vaccine development both as an immunogen (it encodes a major glycopolypeptide product) and also because we have identified it as the homologue of HSV gC, a potential non—essential region. The A antigen gene was mapped within the ﬂgmﬂl B fragment of MDV (Isfort gt al 1987). and the nucleotide sequence determined for the GA strain of MDV (Coussens and 4Velicer, Abstract OP18.51, VII International Congress of Virology, 9-14 August, (1987) Edmonton, Canada; J.

Virol. 62, 2373-2379). During the random sequencing studies described earlier (Buckmaster et al 1988), we identified an M13 clone (No. 130) which came from the A antigen gene. This clone was then used to identify a 2.3 Kbp ECORI/Eyull fragment from the R818 strain of MDV containing the A antigen. This fragment was cloned into a Smal/EcoR] cleaved pUC13 vector by standard protocols. One plasmid (pMB419) was sequenced by the M13 dideoxynucleotide method. The sequence of the MDV RBIB A antigen and the predicted amino acid sequence of the protein are presented in Figure 6. The A antigen regions of MDV and HVT recombination. Several lines of evidence support this as outlined below.

)During our study we isolated and sequenced another RBIB A antigen clone. This had one extra T residue in the string of T's 45 bases 3’ to the A antigen ATG codon. This extra T would cause a frameshift which would make it impossible for the gene to encode functional A antigen. As it is probable that this gene was cloned from a replicating MDV, the results suggest )On conducting a similarity search it became clear that the MDV A antigen gene is the homologue of HSV gC and PRV gpIII glycoproteins. Both of these homologous genes are known to be non—essential [for the HSV homologue, see Rosenthal gt 1; (1987) J. Virol. 61, 2433 — 24471.

)Strains of MDV lacking A antigen as. judged by 893? 991 diffusion tests [Churchill, A.E. t al (1969) J. gen. ViFO1- W - 64] or producing low levels using the more sensitive 2D radio—immunoprecipitation (van Zaane, D. gt al (1982) Virology ], 133-146] have been reported.

Furthermore. in view of the fact that the A antigen is a major secreted glycoprotein, it may be a particularly suitable location for the presentation of foreign epitopes within the A antigen as soluble, secreted proteins. This may be achieved by cloning oligonucleotides encoding these epitopes in frame within the A antigen gene.

STRATEGIES FOR INTRODUCING GENES INTO HVT VECTORS Two possibilities can be envisaged: 1) insertion into non- essential genes of the vector or 2) substituticni of foreign gene for corresponding gene of the vector. This would be possible only in regions which already have substantial homology as may be the case between some genes of NDV and HVT.

EXAMPLE 5: Insertion into non—essential genes of HVT or MDV (a)Insertion at the TK locus of the vector.

) HVT or MDV may be used as vectors for insertion and expression of avian herpesvirus genes. In particular gB, gH or gC of R818 HDV may be inserted into HVT. One may use the promoter associated with the inserted gene or use heterologous promoters, including those of a different class of genes (for example the immediate early promoter to optimise expression of SB).

) HVT or MDV may be used as general vectors for the insertion and expression of genes unrelated to avian herpes viruses and likely to require manipulation of promoters for optimal expression.

The procedure to be used for gene insertion is substantially as described previously for the insertion of hepatitis antigen in HSV [Shih gt al, 1984 as above].

MDV and HVT DNA obtained as described above is infectious provided that precautions are taken not to shear the DNA during extraction. Calcium phosphate precipitates of viral DNA prepared as described by Stow and Wilkie [(1976) J. gen. Virol. , 477] were added to sub-confluent monolayers of CEF. After absorption for 1h at 37°C, culture medium was added and cultures were incubated for 1 or 2 days until confluent.

Monolayers were then trypsinised, replated (121 or 1:2) in 199 medium (Wellcome) containing 2 to 4% calf serum and incubated at 37°C until plaques developed, usually after 4 to 5 days.

Approximately 200 plaques may be obtained per yg of HVT DNA and approximately 50 per Ag of MDV DNA.

For homologous recombination and isolation of recombinant virus, genes of interest are inserted within non—essential genes such as TX or gC and co-transfected with uild—type viral DNA at W013? F3ti0S ranging from 10:1 to 2:1 as described above. Alternatively, intact wild-type virus may be used for co—infection.

Restriction enzyme sites that could be used for the insertion of foreign antigens into the TK of HVT strain Fc-126 include: Nhel and Nspbll.

BanII, Bsp1286, DraIII, EcoRI, incII. Hgal, RE sites that could be used to produce defined TK deletion include; Ball, mutants nu MDV serotype I strain RBIB ﬁagil, insertion sites for foreign DNA that would NdeI and Sphl as disrupt the TK gene, and double digests of combinations of these four restriction enzymes ( coK could also be used) to remove a portion of the TK gene, thus inactivating it.

Some of these enzymes also have sites in the plasmid vector Thus in order into which the virus DNA fragments are cloned. to linearize the clone DNA without also cutting within the vector, partial digests may be carried out.

None of the above enzymes should cause any disruption to HSV-1 homologues of which are known to play an flanking genes, important role in virus multiplication.

Virus recombination may be detected by ’plaque lifts‘ which involve transfer of infected cells and released virus which have adhered to the agar overlay to nitrocellulose and hybridization of the denatured DNA released from the cells and virus to suitable probes as described by Villareal, L. t 1 (1977) Science 196, 183-185. Virus which hybridizes to the probe may be recovered from the monolayer.

A similar procedure may be used to isolate recombinant virus which expressed epitopes of interest. In this instance the nitrocellulose "plaque lifts" are treated with antibody and the presence of bound antibody revealed using a suitable detection system such as labelled protein A or phosphatase conjugated anti—globulin antibody.

The gene of interest with appropriate promoters is first inserted within the cloned TK gene. The recombinant DNA is then co-transfected with infectious DNA of the vector in chick embryo fibroblasts or chicken kidney cells and TK' virus may be selected by growth in medium containing acyclovir [Ross, N. (1985) as above] or FMAU [Schat, K.A. _g _l (1984) Antiviral Research 4, 159-270]. Alternatively, or in addition, plaques are screened for the presence of the gene of interest using ‘plaque lifts’ on nitrocellulose and hybridization to any relevant labelled probe. Plaques are also screened for expression of the epitopes of interest using monoclonal antibodies or antipeptide antibodies.

The main advantage of this strategy is that the selection procedure increases the chances of obtaining virus recombinants containing the gene of interest. It also offers the opportunity of using different promoters for optimum expression. Thus the use of an immediate early promoter may allow expression in latently infected cells. (b)Insertion at other non—essential sites of the vector.

Since the A antigen (HVT and MDV homologues of HSV gC) is not essential for virus growth in vivo and in vitro (see section on gC above) it is a potentially useful site for the insertion and expression of foreign genes. Moreover, since it is one of the most abundant antigens and is excreted. it may be particularly useful for enhancing the immunogenic properties of foreign proteins. The isolation of virus recombinants at this locus may be achieved by first inserting at least part of the gene of interest in frame within the gC gene and then co-transfecting with infectious viral DNA. Screening of virus plaques with sequence specific probes or with specific antibody allows the isolation of recombinants. can also be inserted into the An antigen—encoding sequence ribonucleotide reductase (large subunit) gene of HVT or of MDV - see Figures 8 and 9.

EXAMPLE 52 Substitution of NDV genes for their hornologueg in ~__._...___.____________, Substitution may be achieved by co-transfection of cloned MDV HVT DNA as described in Example 5. sequences and infectious Substitution of the gB and gC genes derived from the RBIB strain of MDV for their counterparts in HVT may be effected as may substitution of the gH gene of MDV, other glycoproteins and immediate early genes.

Recombinants expressing MDV sequences and epitopes may be detected using MDV-specific monoclonal antibodies or anti- peptide antibodies raised against unique MDV sequences as described above.

The advantage of this procedure is that it is relatively simple and does not require manipulation of promoters. However. it maY be limited to genes which share substantial homology.

EXAMPLE 7: Strategies for obtaining TK‘ mutants of MDV Deletion mutants. Deletions may be introduced within any suitable part of the gene. for example the domains of the gene that are required for nucleoside binding. This may be achieved by restriction enzyme double digestion. for example with Haell and any of the following enzymes: Ball. Ndel, Sphl or EcoK.

Appropriate fragments are then religated, followed by co- transfection with infectious viral DNA or transfection into vira1ly—infected cells. Reference may be made to Figures 7 and , and to the section above relating to insertion of heterologous sequences. in choosing restriction enzymes and so on. TK- virus may be selected in the presence of acyclovir [Ross, N. (1985) as above] or FMAU [Schat, K.A. gt al (1984) as above]. Plaque-purified clones may then be tested for the absence of the deleted portion of the TR gene by hybridization.

The deletion mutants of MDV may be used themselves as attenuated viruses for vaccine preparation. or may have sequences for heterologous antigens inserted.

Insertional mutants. A functional B—galactosidase gene under the control of a herpesvirus promoter or any other suitable sequence or a single base is first introduced in a domain of the TH gene which is essential for TK activity. The recombinant DNA is then co—transfected with infectious viral DNA or trnnsfected into virally-infected cells to allow homologous recombination to occur. Selection in the presence of acylovir or FMAU will yield TK‘ insertional mutants. If a B—galactos- idase gene is introduced, mutants can be detected by the production of blue plaques in the presence of X—gal.

The TK gene and surrounding sequences may be subcloned into another suitable vector if necessary.

EXAMPLE 8: Insertion of MDV RBIB gB gene into HVT The HVT TK gene is cloned in the plasmid vector pUC13 to generate a plasmid, which is termed pTK1B. This plasmid is linearised with, for example, the restriction endonuclease Rsr II which cleaves the plasmid only within the TK gene Figure 5, enzyme recognition (nucleotide position 197 in sequence CGGACCG). The "sticky" ends thus generated can be end repaired by standard techniques (see "Molecular Cloning: a Laboratory Manual", ed. Maniatis T., Fritsch E.F., and Sambrook J. Cold Spring Harbor Laboratory 1982).

The RBIB gB was originally cloned on two plasmids which may be termed RBIB-BamH1—Ia and RB1B~BamH1—K:. (Note I: had lost one BamH1 site during cloning.) To generate a complete gB copy on one plasmid, both plasmids were cleaved with BamH] and the fragments ligated. Recombinants containing the desired configuration were identified by restriction enzyme analysis of plasmid DNA's. However, as described above, the complete gB I‘) 3 sequence was subsequently obtained on an EcoRI/Sal] fragment.

Further information regarding the sequence encoding MDV gB and may’ be found in Ross gt 1 J. gen. Virol its manipulation (1939) 7__0_ 1789-1804.

The single recombinant plasmid of Ross gt Q; is then cleaved with EcoRI and Sall. the ends are repaired and the plasmid is cloned into PTK1B prepared as above. Alternatively, the MDV gB open reading frame could be excised from plasmid MSB27 by digestion with Hincll and Nael and the products ligated to HVT TK plasmid pTK1B. cleaved partially with Hpal. Recombinant plasmids containing both TX and gB sequences could be identified by hybridisation and further characterised by Southern blotting. The recombinant ,plasmids are then introduced into cells containing HVT virus (viral DNA) and homologous recombination will introduce the gB gene into the TK gene. HVT viral recombinants can be selected with acyclovir or FMAU or alternatively detected with labelled gB probes.

EXAMPLE 9: RBIB gC (A antigen) gene into HVT Blunt ended PTK1B is prepared as in Example 8. The RBIB gC is cleaved from the plasmid pMB419 (Example 4) with the (site within the EcoR1 and HindIII restriction endonucleases pUC13 polylinker). The sticky ends generated are again end- repaired by standard protocols. The end—repaired gC fragment is into the linearized cloned pTKlB as in then end-repaired Example 8. (The cloning can be verified by analysis of the resulting clones with restriction enzymes, probing with radio- actively labelled fragments. or DNA sequencing, or any combination of these).

The resulting plasmid with the RBIB gC gene cloned into the HVT TK gene can then be introduced into the HVT genome by transfecting the plasmid into HVT—infected cells using calcium phosphate precipitation or electro—poration. Homologous recombination. involving cross-overs either side of the g0 gene, between the HVT virus and the flanking sequences of the HVT TK plasmid will carry the RBIB gC gene into the HVT viral genome. Viral recombinants can be selected for (as they are TK‘) or identified (eg by probing) as described above.

In analogous ways, the sequence information given above and in the Figures can be used to design cloning strategies for the others into the non—essentia1 insertion of these genes and genes of the HVT described here or to generating combinations of antigen genes in HVT.

EXAMPLE 10: MDV gD gene Figure 15 shows part of the sequence of the MDV gD gene. The sequence was obtained by sequencing random fragments of the Us region MDV DNA and comparing the sequence to the sequence of known herpesvirus genes (see Buckmaster g; 1;, L_c. gLg.I. The sequence gave homology scores of 189 and 216 respectively with HSV gD and PRV gp50. The sequence information assists in the preparation of suitable probes to isolate and characterise the gene.

Claims

1. A vaccine against Marel<’s disease comprising a recombinant viral vector which is capable of expressing an antigenic portion of an MDV homologue of the HSV gD gene wherein said MDV homologue of the HSV gD gene comprises the nucleotide sequence disclosed in