CA2086740A1 - Equine herpesvirus-4 tk-vaccine - Google Patents

Abstract

The present invention is concerned with an attenuated EHV-4 vaccine. The attenuation can be achieved by a deletion and/or insertion in the thymidine kinase gene of EHV-4. The invention also relates to a vector vaccine comprising an EHV-4 mutant having a foreign gene inserted into the EHV-4 genome.

Description

WO92/0104~ PCT/GB91/01100 ~S'74~
~t Equine herpesvirus-4 TK vaccine The present invention is concerned with an Equine herpesvirus-~ mutan~ (E~-4), a recomDinant DNA molecule comprising E~.l-d DNA, host cell containing said recombinant DNA molecule, process for the ~-eparation of said E~J-d mutant, cell culture infected with the EHV-4 ~ut n., a vaccine derived from the EHV-d mutant as well as a process for the prepara~ion of such a vaccine.
_quine herpesvirus-~ (E~J-4) is, like the related Equine herpesvirus-l, an alphaAerpesvirus responsible for significant economic losses within the equine industry.
EHV-~ is primarily associated with respiratory disease though _HV-4 induced abortions are occasionally reported.
The genome of EHV-4 has been characterized as a double-s~randed 'inear DNA molecule consis~ ng of two covalently linked segments (L, lO9 kbp; S, 35 kbp) the latter being 4lanked by inverted repeats.
Control by vaccinatlon of EHV-4 infection has been a long-sought goal.
Curren~ vaccines comprise chemically inactivated virus vaccines and modified live-virus vaccines.
However, inactivated vaccines generally indl~ce only a low level of immunity, requiring additional immunizations, disadvantageously require adjuvants and are expensive to produce. Further, some infectious virus particles may survive the inactivation process and causes disease after administration to the animal.
In general, attenuated live virus vaccines are preferred because they evoke a more long-last,ng immune response (often both humoral and cellular) and are easier to . .

WO 92/01045 PCI`/GB91/01100 S~O ~ .

produce.
Up to ~ow only live attenuated, EHV-4 vaccines are available which are based on live EHV-4 viruses attenua~ed by serial passages of virulent strains in ,issue culture. However, because of this treatment uncontrolled mutations are introduced into the viral genome, resulting in a population of virus particles heterogeneous in their virulence and immunizing properties. In addition it is well known that such traditional attenuated live virus vaccines can revert to virulence resulting in disease of the inoculated animals and the possible spread of the pathogen to other animals.
Furthermore, with the existing live attenuated EHV-4 vaccines a positive serological test is obtained for EHV-~ infec~ion. Thus, with the existing EHV-4 vaccines, it ; is not possible to determine by a (serological) test, e.g. an Elisa, whether a specific animal is a (latent) carrier of the virulent virus or is vaccinated.
., It is an object of the present invention to provide an EHV-4 mutant which can be used for the preparation of a vacc~ne against EHV-4 infection, the mutant viruses being attenuated in a controlled way in a manner which ; excludes the reversion to virulence and which still elicit a strong immune response in host animals.
According to the present invention such a mutant s EHV-4 is characterized in that it does not produce a -, functional thymidine kinase (T~ ) as a result of a deletion and/or insertion in the gene encoding thymidine kinase.
'A The development of techniques for controlled manipulation of genetic material has allowed the possibility of obtaining attenuated virus vaccines which avoid the disadvantages of the classic attenuated virus ~, vaccines.
However, up to now no information was available with respect to the exact localisation on the EHV-4 genome of .
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WO 92/0104~ PCT/GB91/01100 2~S740 a region lnvolved with the virulence of EHV-4 making the 2roouc-ion of an genetic engineere~ attenuated EHV-4 impossible.
The gene encoding thymidine kinase was mapped within _he 3amHI C fragment of the EHV-4 genome and was further localised to an about 2 kbp EcoRV/XhoI fragment thereof, with a map position of approximately 0,48 (fig.1).
The nucleic acid sequence of the TX gene was determined and is shown in SEQ ID N0: 1 from which restriction enzyme cleavage sites to be used for the genetic ~anipulation of the gene can be d~rived.
The TK gene consists of 1056 nucleotides encoding a 352 amino acid enzyme of predicted molecular weight of 38.800 D. The efficiency of the expression of TR is regulated by the presence of expression control sequences. For example promoter sequences are involved in the binding of RNA polymerase to the DNA template and control the site and onset of the mRNA. Such sequences are often found within a 100 bp region before the transcription initiation site. Downstream transcriptional control signals are inter alia, the transcription termination codon and a polyadenylation signal.
The TATA box positioned at base pair 21-25 is the putative promoter TATA box of the EHV-4 TX gene. A
potential RNA polymerase initiation site is located 22 bp downstream of the TATA box. A poly A signal is positioned 42 bp downstream of the termination codon (SEQ ID NO: 1).
It will be understood that for the DNA sequence of the EHV-4 TK gene natural variations can exist between individual EHV-4 viruses. These variations may result in a change of one or more nucleotides in the TK gene which, however still encodes a functional TK. Moreover, the potential exists to use genetic eng_neering technology to bring about above-mentioned variations resulting in a DNA
sequence related to the sequence shown in SEQ ID NO: 1.
It is clear that EHV-4 mutants comprising a deletion WO 92~01045 ;~ S~7gO P~/GB91/01100 i`,` ~ _ and/or insertion in such a related nucleic acià seouence are also included within the scope of the invention. ' , The EHV-4 deletion mutants of the present invention comprise a TK gene from which a DNA fragment has b~en ~eleted so that no functional TX enzyme is produced upon replication of the virus, e.g. as result of a change of the tertiary structure of the altered TK protein or as a result of a shift of the reading frame.
In addition the deletion in the genome of the EHV-4 ~utant may comprise the complete TK gene.
EXV-4 mutants according to the invention can also be obtained by inserting a nucleic acid sequence into the TK
coding region thereby preventing the expression of a -^unctional TK enzyme. Such a nucleic acid sequence can ~nter alia be an oligonucleotide, for example of about iO-50 bp, preferably also containing one or more - .ranslational stop codons, or a gene encoding a polypeptide. Said nucleic acid sequence can be derived from any source, e.g. synthetic, viral, prokaryotic or eukaryotic.
In another embodiment of the present invention the _HV-4 deletion mutants can contain above-mentioned nucleic acid sequence in place of the deleted EHV-4 DNA.
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It is another object of the present invention to provide a mutant E~J-4 which can be used not only for the preparation of a vaccine against EHV-4 infection but also against other equine infectious diseases. Such a vector vaccine based on a safe live attenuated EHV-4 mutant offers the possibility to immunize against other pathogens by the expression of antigens of said pathogens within infected cells of the immunized host and can be obtained by inserting a heterologous nucleic acid sequence encoding a polyp~ptide heterologous to EHV-4 in an insertion-region o~ the EHV-4 genome.

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However, the prerequisite for a useful EHv-4 vector _s that the heterologous nucleic acid seouence is - -ncorporated ln a per~issive position or region of the enomic EHV-4 sequence, i.e. a position or region which _an be used for the incorporation of a heterologous sequence ~!ithout disrupting essential functions of EHV-4 such as those necessary for infection or replication.
Such a region is called an insertion-region~ Prior to the present invention no insertion-region in the EHV-4 genome ;~as been described.
According to the present invention EHV-4 mutants are provided which can be used as a viral vector, characterized in that said mutants do not produce a -unc.ional TK as a result of an insertion of a :~eterologous nucleic acid sequence encoding a polypeptide in the gene encoding TK.
EHV-~ insertion mutants as described above having a ; heterologous nucleic acid sequence inserted in place of ~eleted TK DNA are also within the scope of the present invention.
; The term "EHV-4 insertion mutants" comprises inter alia infective viruses which have been genetically . modified by the incorporation into the virus genome of a heterologous nucleic acid sequence, i.e. a gene which codes for a protein or part thereof said gene being different of a gene naturally present in EHV-4.
on infection of a cell by said EHV-4 insertion mutant it expresses the heterologous gene in the form of a heterologous polypeptide.
The term "polypeptide" refers to a molecular chain of amino acids with a biological activity, does not refer to a specific length of the product and if required can ~ be modified in vivo or in vitro, for example by ;, glycosylation, amidation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides ;i and proteins are incll~ded within the definition of polypeptide.
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WO 92/01045 PCr/GB91/01100 2~ 0 ,. ` , The heterologous nucleic acid sequence to be incorporated into the -HV-4 genome according to the ~resent invention can be derived from any source, e.g.
virai, prokaryotic, eukaryotic or synthetic. Said nucleic acid sequence can be derived from a pathogen, preferably an equine pathogen, which after insertion into the EHV-4 genome can be applied to induce immunity against disease.
Preferably, nucleic acid sequences derived from EHV-1, equine influenza virus, -rotavirus, -infectious anemia virus, arteritis virus, -encephalitis virus, Borna disease virus of horses, Berue virus of horses, E.coli or Streptococcus equi are contemplated of for incorporation into the insertion-region of the EHV-4 genome.
Furthermore, nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic application, in particular immune modulators such as lvmphokines, interferons or cytokines, may be incorporated into said insertion-region.

An essential requirement for the expression of the heterologous nucleic acid sequence in a EHV-4 mutant is ; an adequate promoter operably linked to the heterologous ` ~ nucleic acid sequence. It is obvious to those skilled in - the art that the choice of a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells infected by the -EHV-4 mutant, such as the SV-40 promoter (Science 222, 524-527, 1983) or, e.g., the metallothioneîn promoter (Nature 296, 39-42, 1982) or a heat shock promoter - (Voellmy et al., Proc. Natl. Acad. Sci. USA 82, 4949-53, 1985) or the human cytomegalovirus IE promoter or promoters present in EHV-4, e.g. the TK promoter.
Well-known procedures for inserting DNA sequences into a cloning vector and in vivo homologous recombination can be used to introduce a deletion and/or an insertion into the EHV-4 genome (Maniatis, T. et al.
(1982) in "Molecular cloning", Cold Spring Harbor ' ~
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Laboratory; European Patent Application 74.808; Roizman, 3. and Jenkins, F.J. (1985), Science 229, 1208; Higuchi, . el al. (1988), Nucleic Acids Res. 16, 7351).
3riefly, this can be accomplished by constructing a recombinant DNA molecule for recombination with EHV-4 DNA. Such a recombinant DNA molecule may be derived from any suitable plasmid, cosmid, virus or phage, plasmids being most preferred, and contains EHV-4 DNA possibly having a nucleic acld sequence inserted therein if desired operably linked to a promoter. Examples of suitable cloning vectors are plasmid vectors such as pBR322, the various pUC and Bluescript plasmids, bacteriophages, e.g. ~ gt-WES-~ B, charon 28 and the ~13mp phages or viral vectors such as SV40, Bovine papillomavirus, Polyoma and Adeno viruses. Vectors to be ~sed in the present invention are further outlined in the art, e.g. Rodriguez, R.L. and D.T. Denhardt, edit., Vectors: A survey of molecular cloning vectors and their uses, Butterworths, 1988. - -First, an EHV-~ DNA fragment comprising the insertion region, i.e. the TX gene, is inserted into the _loning vector according to recDNA techniques. Said DNA
_raoment may comprise part of the TK gene or suDslanlially the complete TX gene, and if desired flanking sequences thereof.
Second, if an EHV-4 TX deletion mutant is to be obtained at least part of TX gene is deleted from the recombinant DNA molecule obtained from the first step.
This can be achieved for example by appropriate ~ -exonuclease III digestion or restriction enzyme treatment of the recombinant DNA molecule from the first step.
In the case an EHV-4 insertion mutant is to be obtained the nucleic acid sequence is inserted into the TK gene present in the recombinant DNA molecule of the first~step or in place of the TK DNA deleted from said recombinant DNA molecule. The EHV-4 DNA sequences which flank the deleted TX D`A or the inserted nucleic acid sequence . . .

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W092/0l045 PCT/GB91/01100 2 ~ 4 0 8 should be of appropriate length as to allow homologous recombination with the viral EHv-4 genome to occur.
If desired, a construct can be made which contains two or more different inserted (heterologous) nucleic acid sequences derived from e.g. the same or different pathogens said sequences being flanked by insertion-region sequences of EHV-4 defined herein. Such a recombinant DNA molecule can be employed to produce an EHV-4 mutant which expresses two or more different antigenic polypeptides to provide a multivalent vaccine.
Thereafter, cells, for example rabbit cells, T~+ or TK phenotype, or equine cells, e.g. equine dermal cells, can be transfected with EHV-4 DNA in the presence of the recombinant DNA molecule containing the deletion and/or insertion of (heterologous) nucleic acid sequence flanked by appropriate EHV-4 sequences whereby recombination occurs between the corresponding regions in the recombinant DNA molecule and the EHV-4 genome.
Recombination can also be brought about by transfecting EXV-4 genomic DNA containing host cells with a DNA
containing the (heterologous) nucleic acid sequence flanked by appropriate flanking insertion-region sequences without vector DNA sequences. Recombinant viral progeny is thereafter produced in cell culture and can be selected for example genotypically or phenotypically, e.g. by hybridization, detecting enzyme activity encoded by a gene co-integrated along with the (heterologous) nucleic acid sequence, screening for EHV-4 mutants which do not produce functional TK (Roizman, B. and Jenkins, F.J. (1985), ibid~) or detecting t~e antigenic heterologous polypeptide expressed by the EHV-4 mutant immunologically. The selected EHV-4 mutant can be cultured on a large scale in cell culture whereafter EHV-mutant con~aining material or heterologous polype~;~idesexpressed by said EHV-4 can be collected therefrom~
Alternatively, mutant EHV-4 could be generated by cotransfection of several cosmids, containing between , .

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According to the present invention a live attenuated EHV-4 mutant which does not produce a functional TK, and if desired expresses one or more different heterologous polypeptides of specific pathogens can be used to vaccinate horses, susceptible to EHV-4 and these pathogens.
Vaccination with such a live vaccine is preferably followed by replIcation of the EHV-4 mutant within the inoculated host, expressing in vivo EHV-4 polypeptides, and if desired heterologous polypeptides. An immune response will subsequently be elicited against EHV-4 and the heterologous polypeptides. An animal vaccinated with such an EHV-4 mutant will be immune for a certain period to subsequent infection of EHV-4 and above-mentioned pathogen(s).
An EHV-4 mutant according to the invention optionally containing and expressing one or more different heterologous polypeptides can serve as a monovalent or multivalent vaccine.
An EHV-4 mutant according to the invention can also be used to prepare an inactivated vaccine.
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For administration to animals, the EHV-4 mutant according to the presentation can be given inter alia by aerosol, spray, drinking water, orally, intradermally, subcutaneously or intramuscularly. Ingredients such as skimmed milk or glycerol can be used to stabilise the virus. It is preferred to vaccinate horses by intranasal administration. A dose of 103 to 108 TCTD50 of the EHV-4 mutant per horse is recommended in general It is a further object of the present invention to produce subunit vaccines, pharmaceutical and diagnostic preparations comprising a heterologous polypeptide expressed by an EHV-4 mutant according to the invention.
This can be achleved by culturing cells infected with ~' . .

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WO 9~/01045 PCl/GB91/01100 2C~6~ ~) lo ~r-;
said EHV-4 mutant under conditions that promote expression of the heterologous polypeptide. The heterologous polypeptide may then be purified with conventional techniques to a certain exten~ depending on its intended use and processed further into a preparation with immunizing therapeutic or diagnostic activity.
The above described active immunization against specific pathogen_ will be applied as a protective treatment in healthy animals. It goes without saying that anlmals already infected with a specific pathogen can be treated with antiserum comprising antibodies evoked by an EHV-4 mutant according to the invention. Antiserum directed against an EHV-4 mutant according to the invention can be prepared by immunizing animals with an effective amount of said EHV-4 mutant in order to elicit an appropriate immune response. Thereafter the animals are bled and antiserum can be prepared.

, W092/0104~ PCT/CB91/~1100 1'1 ' 2~ n Exam~le l Isolation and characterization of E~J-a insertion reqion.
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1. Culturinq of EHV-4 virus Roller ~ottles of slightly sub-confluent monolayers of equine dermal cells (NBL-6) grown in Earle's Minimum Essential Medium (Flow) supplemented with 0,2% sodium bicarbonate, 1% non-essential amino acids, 1% glutamine, lO0 units/ml penicillin, lO0 mg/ml streptomycin and 10%
foetal calf serum were infected with virus of the EHV-4 strain 1942 at a m.o.i. of 0,003 and allowed to adsorb for 60 min at 37 C. They were incubated at 31 C until extensive c.p.e. was evident and the majority of cells had dstached from the bottle surface (2-6 days). The infected cell medium was centrifu~ed at 5.000 r.p.m. for 5 min to pellet the cells, and the supernatant was centrifuged at 12.000 r.p.m. for 2 hours in a Sorvall GSA
6 X 200 ml rotor. The pellet was resuspended in 5 ml PBS, sonicated and centrifuged at ll.000 r.p.m. in a Sorvall SS34 rotor for 5 min to spin down cellular debris. Virus was then pelleted by centrifugation at 18.000 r.p.m. in a Sorvall SS34 rotor for l hour. Ratios of virus particles to plaoue-forming units were approximately l.000 to 5.000.
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2. Pre~aration of EHV-4 DNA
The pelleted virus was resuspended in lO ml NTE
(NaCl/Tris/EDTA) and briefly sonicated. Contaminating cellular DNA was degraded by adding DNase at lO ~g/ml and incubating at 37 C for l hour. SDS was added to a final concentration of 2%, and the preparation was extracted approximately 3 times with NTE equilibrated phenol until a clear interphase was obtained.
A chloroform extraction was followed by ethanol precipitation of the DNA as described above. The DNA was '..
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W O 92/01045 P ~ /G B91/01100 2 ~ n 12 pelleted, washed with 70% ethanol, resuspended in 10 ml of 100 mM NaCl and 10 ~g/ml RNase and left overnight at room temperature. Further purification was achieved by treatment with 1 mg/ml proteinase K for 2 hours at 31 C.
The DNA was extracted once with phenol:chloroform (1:1 vol/vol), once with chloroform, ethanol precipitated, drained well and resuspended in 0.1 X SSC.

3. Cloning of EHV-4 DNA
EHV-4 BamHI DNA fragments were ligated into the vector pUC9, a plasmid which includes the ampicillin-resistance gene from pBR322 and the polylinker region from M13mp9 (Vieira, J. and Messing, J. (1982), Gene 19, 259). 5 ~g of EHV-4 DNA and S ~g pUC9 DNA were separately digested with BamHI.
Complete digestion was verified by gel electrophoresis of aliquots of the reactions and then the DNA was extracted twice with an equal volume of phenol:chloroform (1:1) and ~- ethanol-precipitated. Ligation was performed essentially by the method of Tanaka and Weisblum (J. Bact. 121, 354, 1975). Approximately 0.1 ~g of BamHI digested pUC9 and 1 ~g of BamHI-digested EHV-4 DNA were mixed in 50 mM Tris-HCl pH 7,~, 8 mM MgC12, 10 mM dithiothreitol, 1 mM ATP in a final volume of 40 ~1. 2 units of T4 DNA ligase (0,5 ~1) were then added. The reaction was incubated at 4 C
for 16 hours.

Calcium-shocked E.coli DHI cells (Hanahan, D. (1983), J.
Mol. Biol. 166, 557) were transformed with the recombinant plasmids essentially described by Cohen et al. (Proc. Natl. Acad. Sci., USA 69, 2110, 1972).
Additional clones were derived by restriction digestion of recombinant plasmid pUC9 containing BamHI C fragment 'fig. lb), followed by recovering of the specific E~'-4 restriction fragments and sub-cloning thereof within the multi-cloning site of the Bluescript M13+ plasmid vector (Stratagene; Maniatis, T. et al. ibid).

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WO 92~01045 PCI`/GB91/01100 20 ~g of eacn construct was transfected into monolayer BHK TK cells by a modification of the technique of Graham and van der Eb (Virology 52, 456, 1973; CellPhect Transrection Kit, according to manufactures instructions) TXT colonies were selected in HAT supplemented medium (Hypoxanthine 10 4 M, Aminopterin 4 x 10 S M, th,vmidine 1,6 x 10 5 M).
TK transforming activity was thus localised to a 2 kbp EcoRV/XhoI fragment (RX2), cloned in construct pBSRX2, -~ith a map position of approximately 0,48 (fig. lb).

The nucleotide seouence of both strands of fragment RX2 was determined by using single stranded plasmid DNA as template and Bluescript-derived custom-made ; oligonucleotides as primers in a Sanger dideoxy sequencing strategy (Sanger et al., Proc. Natl. Acad.
Sci: 74,5463,1977) (fig.lc). The exact localisation, ; nucleic acid sequence and corresponding amino acid seouence of the TK gene is shown in the SEQ ID NO~

~ Exam~le 2 : Preparation of ~K-deleted ~lasmids.

Restriction mapping and sequence analysis of DNA spanning the EHV-4 TX gene indicated that unique SmaI and BstXI
sites exist within fragment RS3 (fig 1,2) and unique SmaI
and BstEII sites exist within RX2, all of which map within the TK coding region. A 0,73kbp deletion within the TK gene was achieved by cloning RS3 into pUC 8 (at the SmaI and SalI sites within the multicloning site) and digesting the contruct with SmaI and BstXI. The vector ; fragment plus EHV-4 DNA flanking the deletion was isolated and the overhang generated by BstXI filled in using T4 pol. The linear plasmid was then self ligated to produce a plasmid containing RS3 fragment deleted from the SmaI-8stXI site (fig. 2b).

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~`3~ 14 A 0,52kbp deletion within the TK gene was achieved by .
cloning EHV-4 RX2 into a Bluescript vector (at the SmaI
and XhoI sites within the multicloning site) and deleting from the SmaI-BstEII sites within the TX gene by restriction digestion with these enzymes. The larger vector fragment was separated from the 0,52 ~bp EHV-4 fragment, the overhang filled in and the plasmid religated. The resultant plasmid possesses the 5' and 3' coding regions of the EHV-4 TK gene but is deleted from the SmaI-BstEII sites (figure 2c).
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W092/0104~ PCT/GB9l/Ot100 2~

ExamPle 3 .
Use of Recombinant PCR to Produce TK- Constructs ~ .
The two plasmid constructs preferred in Example 2 contain EHV-4 DNA with distinct deletions within the TK gene. The positions of these deletions are dictated by the availability of restriction endonuclease sites within the -TK gene which could be utilised in the deletion strategy.
Both deletions span the N-terminal coding region of the TX
gene. Given that this region is likely to contain the promoter 'or the adjacent UL24-type gene, incorporation of these DNAs into the EHV genome could possibly result in the altered expression of both the UL24-type gene and of the TK gene. DNAs were therefore constructed with deletions within the C-terminal coding region of the TK
gene in order to ultimately produce EHV recombinants affected solely in TK expression. Recombinant polymerase chain reaction (PCR) was utilised to prepare these DNAs since, this technique permits the deletion to be localised anywhere within the gene. As shown in Figures 2 and 3 we synthesised two primer sets (primers 1,2 and 3,4) which were utilised independently to prepare DNA fragments mapping to the left and right of the deletion site (step 1). The external primers (primers 1,4) were then utilised to amplify across the products of the first round PCR reactions (primers 2,3 having complementary annealable end regions) to produce the TK-construct (steps 2,3).
Such a strategy has the added advantage that restriction endonuclease sites can be inserted at the termini and at the deletion site by incorporation within the primers.
These sites can be utilised to facilitate cloning of the TX- DNA PCR product into a suit2ble plasmid vector (step 4) and to effect cloning within the deletion site.
, 5 PCl/GB91/01100 Z~ " A ~ -- 16 Primers The 3' terminal sequences of the following primers were derived from the published sequence information on the TX
genes of EHV-l and EHV-4 (sequences underlined below). 5' sequences incorporating a 'GC' clamp (to enhance the efficiency of cleavage with intra-primer restriction enzymes), and 3 restriction enzyme sites. In the case of primers 2 and 3 the 5' regions of the primers are complementary in order to facilitate annealing of denatured first round amplification products at step 2.

LMU (Primer 1) 5'-GCGGATCGATAGATCTGCGGCCGCTGCGTTAGTGGTGTT-3' ClaI BglII NotI

-~ LIL (Primer 2) 5'-GAGCTCGATATCTCTAGAGTAGGGCGTGGTAAAGC-3' SstI EcoRV XbaI
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;: RIU (Primer 3) 5'-TCTAGAGATATCGAGCTCATATTGGAAGTTCACGC-3' XbaI EcoRV SstI
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RML (Primer 4) 5'-CCGGGATCCAGATCTGCGGCCGCTCAGAAGATGTGTACGA-3' BamHI BglII NotI

The PCR technique is carried out as follows.
First primers 1 and 2 are hybridized onto the single stranded EHV genome. Then the second strand is extended along the first strand starting from primer 1 using a DNA
polymerase until the primer 2 is encountered, when DNA
synthesis stops. Similarly a second DNA oligonucleotide strand is synthesised from primer 3 up to primer 4. The strands are then dehybridised into single DNA strands by heating. If necessary the process can be repeated using further quantities of primer in order to amplify the amount of PCR product.

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Leqends iaure l. Strategy for the localisation and sequencing of the EHV-~ thymidine kinase qene.
(a) the TX gene was localised on the BamHI C fragment mapping between 0,43 and 0,53.
(b) subfragments of EHV-4 BamHI C were testPd for their capacity to biochemically transfrom BHX TX cells to TX+
phenotype. TK transforming actlvity was localised to a 2 kbp EcoRV/XhoI fragment, RX2.
(c) subcloning of RX2 and sequencing of overlapping fragments resulted in the exact localisation and nucleotide sequence of the TX gene.

Fiqure 2.
(a) Restriction enzyme pattern of fragment RS3 containing TK gene.
(b) 0,73 kbp deletion in TK gene (SmaI-BstXI) deleted from RS3.
(c) 0,52 kbp deletion in TK gene (SmaI-Bst~II) deleted from RX2.
Fiqure 3.
shows the strategy for deletion of a region of the TK gene using a polymerase chain reaction (PCR) technique according to steps l to 3 of Example 4; and Figure 4.
shows the strategy for cloning of the TX- DNA PCR product obtained from Example 4 into a suitable plasmid vector ~step 4).

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Seauence Listing SEQ ID NO: 1 Sequence type : nucleotide wi~h corresponding protein Sequence lengt~: 1260 base pairs; 352 amino acids.

Strandness : single Topology : linear Molecule type : genomic DNA

Original source organism : Equine herpesvirus -4 Immediate experimental source : genomic BamHI library.

Features:
from 21 to 25 bp putative TATA signal bp 47 putative polymerase initiation site from 109 to 114 bp poly A signal Properties: thymidine kinase gene.
:

Met Ala Ala 3 ~, Cys Val Pro Pro Gly Glu Ala Pro Arg Ser Ala Ser Gly Thr Pro Thr 19 Arg Arg Gln Val Thr lle Val Arg Ile Tyr Leu Asp Gly Val Tyr Gly 35 Ile Gly Lys Ser ~hr Thr Gly Arg Val Met Ala Ser Ala Ala Ser Gly ~1 Gly Ser Pro Thr Leu Tyr Phe Pro Glu Pro Met Ala Tyr Trp Arg Thr 67 Leu Phe Glu Thr Asp Val Ile Ser Gly Ile Tyr Asp Thr Gln Asn Arg 83 Lys Gln Gln Gly Asn Leu Ala Val Asp Asp Aid Ala Leu Ile Thr Ala 99 His Tyr Gln Ser Arg Phe Thr Thr Pro Tyr Leu Ile Leu His Asp His 115 ,. - , , .. ~ . .. - .

2~S~
19 -- , Thr Cys Thr Leu Phe Gly Gly Asn Ser Leu Gln Arg Gly Thr Gln Pro 131 Asp Leu Thr Leu Val Phe Asp Arg His Pro Val Ala Ser Thr Val Cys 147 Phe Pro Ala Ala Arg Tyr Leu Leu Gly Asp Met Ser Met Cys Ala Leu 163 Met Ala Met Val Ala Thr Leu Pro Arg Glu Pro Gln Gly Gly Asn Ile 179 Val Yal Thr Thr Leu Asn Val Glu Glu His Ile Arg Arg Leu Arg Thr 195 Arg Ala Arg Ile Gly Glu Gln Ile Asp Ile Thr Leu Ile Ala Thr Leu 211 Arg Asn Val Tyr Phe Met Leu Val Asn Thr Cys His Phe Leu Arg Ser 227 Gly Arg Val Trp Arg Asp Gly Trp Gly Glu Leu Pro Thr Ser Cys Gly 243 Ala Tyr Lys His Arg Ala Thr Gln Met Asp Ala Phe Gln Glu Arg Val 259 Ser Pro Glu Leu Gly Asp Thr Leu Phe Ala Leu Phe Lys Thr Gln Glu 275 CTG CTA GAC GAT CGC GGT G~A ATA TTG GAA GTT CAC GCT TGG GCG TTG 981 Leu Leu Asp Asp Arg Gly Val Ile Leu Glu Val His Ala Trp Ala Leu 291 Asp Ala Leu Met Leu Lys Leu Arg Asn Leu Asn Val Phe Ser Ala Asp 307 Leu Ser Gly Thr Pro Arg Gln Cys Ala Ala Val Val Glu Ser Leu Leu 323 Pro Leu Met Ser Ser Thr Leu Ser Asp Phe Asp Ser Ala Ser Ala Leu 339 GAG CGG GCG GCA CGC ACC TTT AAC GCG GAG AT& GGC GTC TGA AGCTATATGT 1177 Glu Arg Ala Ala Arg Thr Phe Asn Ala &lu Met Gly Val 352 ..:. : . : :
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Claims (15)

1. An EHV-4 mutant, characterized in that it does not produce a functional thymidine kinase as a result of a deletion and/or insertion in the gene encoding thymidine kinase.

2. An EHV-4 mutant according to claim l wherein the ends of the region of deletion or insertion do not correspond to endonucleace restriction sites of the thymidine kinase gene.

3. An EHV-4 mutant according to claim 2 wherein the deletion or insertion is in the C-terminal coding region of the thymidine kinase gene.

4. An EXV-4 mutant according to any preceding claim, characterized in that at least one heterologous nucleic acid sequence encoding a polypeptide is inserted.

5. An EHV-4 mutant according to claim 4 characterized in that the heterologous nucleic acid sequence is under control of a promoter regulating the expression of said nucleic acid sequence in a cell infected with said EHV-4 mutant.

6. An EHV-4 mutant according to claims 4 or 5 characterized in that the heterologous nucleic acid sequence encodes an antigen of an equine pathogen.

7. An EHV-4 mutant according to claim 6 characterized in that the antigen is an EHV-1, or equine influenza antigen.

8. Recombinant DNA molecule comprising a vector molecule and part of the EHV-4 thymidine kinase gene region.

9. Recombinant DNA molecule according to claim 8 characterized in that it has a deletion and/or insertion in the thymidine kinase gene.

10. Host cell containing a recombinant DNA molecule according to claim 9.

11. Process for the preparation of an EHV-4 mutant according to any of claims 1-7 characterized in that cell culture is co-transfected with EHV-4 DNA and a recombinant DNA molecule according to claim 7.

12. Cell culture infected with an EHV-4 mutant according to any of claims 1-7.

13. Vaccine derived from an EHV-4 mutant according to any of claims 1-7.

14. Vaccine which comprises an EHV-4 mutant according to any of claims 1-7 together with a pharmaceutically acceptable carrier therefor.

15. Process for the preparation of an EHV-4 vaccine, characterized in that EHV-4 containing material is collected from a cell culture according to claim 12 and processed into a preparation with immunizing activity.