WO1994000587A2

WO1994000587A2 - Attenuated equine herpesvirus-4 as live vaccine or recombinant vector

Info

Publication number: WO1994000587A2
Application number: PCT/GB1993/001355
Authority: WO
Inventors: Marcello Riggio; David Edward Onions
Original assignee: University Court Of The University Of Glasgow; Equine Virology Research Foundation
Priority date: 1992-06-30
Filing date: 1993-06-29
Publication date: 1994-01-06
Also published as: WO1994000587A3; GB9213882D0

Abstract

The present invention is concerned with an attenuated Equine herpesvirus-4 vaccine. The attenuation is achieved by a mutation into a gene of Equine herpesvirus-4 defined in SEQ ID NO: 1. The invention also relates to a vector vaccine comprising an Equine herpesvirus-4 mutant having a foreign gene inserted into the Equine herpesvirus-4 genome.

Description

ATTENUATED EQUINE HERPESVIRUS-4 AS LIVE VACCINE OR RECOMBINANT VECTOR

The present invention is concerned with an Equine herpesvirus-4 mutant, a recombinant DNA molecule comprising Equine herpesvirus-4 DNA, a host cell transfected with said recombinant DNA molecule, a cell culture infected with the Equine herpesvirus-4 mutant, as well as a vaccine comprising such an Equine herpesvirus-4 mutant- Equine herpesviruses comprise a group of antigenically distinct biological agents which cause a variety of infections in the horse ranging from subclinical to fatal disease.

Equine herpesvirus-4 is, like the distinct Equine herpesvirus-1, an alphaherpesvirus responsible for significant economic losses within the equine industry. Equine herpesvirus-4 is primarily associated with respiratory disease though Equine herpesvirus-4 induced abortions are occasionally reported.

The genome of Equine herpesvirus-4 has been characterized as a double-stranded linear DNA molecule consisting of two covalently linked segments (L, 109 kbp; S, 35 kbp) the latter being flanked by inverted repeats.

Control by vaccination of Equine herpesvirus-4 infection has been a long-sought goal.

Current vaccines comprise chemically inactivated virus vaccines and attenuated live virus vaccines. However, inactivated vaccines generally induce 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-lasting immune response (often both humoral and cellular) and are easier to produce.

Up to now only live attenuated, Equine herpesvirus-4 vaccines are available which are based on live Equine herpesvirus-4 viruses attenuated by serial passages of virulent strains in tissue 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 Equine herpesvirus-4 vaccines a positive serological test is obtained for Equine herpesvirus-4 infection. Thus, with the existing Equine herpesvirus-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. Furthermore, it would be advantageous if an Equine herpesvirus-4 strain could be used as a vaccine that affords protection against both Equine herpesvirus-4 infection and an other equine pathogen such as Equine herpesvirus-1. This could be achieved by inserting a gene encoding a relevant antigen of the equine pathogen into the genome of the Equine herpesvirus-4 in such a way that upon replication of the Equine herpesvirus-4 both Equine herpesvirus-4 antigens and the antigen of the other equine pathogen are expressed.

Until now, the structure and functions of only a small number of genes within the about 144.000 base pairs genome of Equine herpesvirus-4 have been elucidated.

WO 92/01045 discloses the DNA sequence of the thymidine kinase (T ) gene of Equine herpesvirus-4 and its use for the preparation of a vector vaccine. Glycoproteins gH and gC of Equine herpesvirus-4 and the genes coding for these proteins are described in WO 92/01057. These proteins are antigens which can be used as subunits in order to elicit a protective immune response against Equine herpesvirus-4 infection.

Riggio et al. (J. Virology 63, 1123-1133, 1989) disclose the DNA sequence of another glycoprotein, i.e. gB of Equine herpesvirus-4 which is involved in producing a protective immune response.

It is an object of the present invention to provide an Equine herpesvirus-4 mutant which can be used for the preparation of a vaccine against Equine herpesvirus-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 a host animal. According to the present invention such a mutant Equine herpesvirus-4 is characterized in that it comprises a mutation in the gene encoding a protein having the amino acid sequence shown in Fig. 6 resulting in the absence of the expression of said protein or in the expression of said protein in a non¬ functional form.

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 localisation on the Equine herpesvirus- 4 genome of the above region involved with the virulence of Equine herpesvirus-4, the approximate 5* and 3¹ ends of this region were not known, neither the nucleotide sequence nor the restriction sites within this region necessary to allow the introduction of controlled mutations were known, making the production of a genetic engineered attenuated Equine herpesvirus- 4 impossible.

The gene encoding the protein involved with virulence was mapped within the about 18.5 kb BamHI A fragment and was further mainly localised within a region ranging from the Hindlll site at map position 2.7 to the Sail site at map position 5.5 within the BamHI A fragment (Figure 1.).. The exact nucleic acid sequence of this gene was determined and is shown in Fig. 5 from which restriction enzyme cleavage sites to be used for the genetic manipulation of the gene can be derived. The gene defined by the sequence of Fig. 5 encodes the enzyme ribonucleotide reductase (RR) and consists of about 3678 nucleotides. The enzyme reduces ribonucleot des to the corresponding deoxyribo- nucleotides and is essential for the formation of substrates for DNA synthesis in prokaryotic and eukaryotic systems. RR is composed of two non- identical subunits designated the large subunit (RR1) and the small subunit (RR2) .

The amino acid sequence of RR1 and RR2 are shown in Fig. 6 and correspond with the amino acid positions 1-789 and 1-320, respectively.

The DNA sequences encoding the two subunits are shown in Fig. 5 and correspond with the nucleotide positions 77-2443 and 2435-3444, for RR1 and RR2 respectively.

It will be understood that for the DNA sequence of the gene shown in Fig. 5 natural variations can exist between individual Equine herpesvirus-4 viruses. These variations may result in a change of one or more nucleotides in the RR gene' which, however still encodes for a functional enzyme. Moreover, the potential exists to use genetic engineering 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 Equine herpesvirus-4 mutants comprising a mutation in such a related nucleic acid sequence are also included within the scope of the invention.

The term "mutation" means any change introduced into the gene encoding the enzyme RR resulting in a mutated gene not capable of expressing a functional enzyme upon replication of the virus, e.g. as a result of a change of the tertiary structure of the altered enzyme or as a result of a shift of the reading frame. The presence or absence of RR enzyme activity can be assayed according to the method described by Darling et al. (1987) .

The mutation may be an insertion, deletion and/or substitution of nucleotides in the gene encoding the enzyme.

The Equine herpesvirus-4 mutants of the present invention preferably comprise a gene from which a fragment has been deleted so that no functional RR enzyme is produced upon replication of the virus.

In another embodiment the deletion in the genome of the Equine herpesvirus-4 mutant may comprise the complete gene encoding the enzyme disclosed in Fig. 6.

Equine herpesvirus-4 mutants according to the invention can also be obtained by inserting a nucleic acid sequence into the gene encoding the enzyme shown in Fig. 6 thereby preventing the expression of a functional enzyme. Such a nucleic acid sequence can inter alia be an oligonucleotide, for example of about 10-60 bp, preferably also containing one or more translational 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 Equine herpesvirus-4 deletion mutants can contain above-mentioned nucleic acid sequence in place of the deleted Equine herpesvirus-4 DNA.

It is another object of the present invention to provide a mutant Equine herpesvirus-4 which can be used not only for the preparation of a vaccine against Equine herpesvirus-4 infection but also against other equine infectious diseases. Such a vector vaccine based on a safe live attenuated Equine herpesvirus-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 polypeptide heterologous to Equine herpesvirus-4 in an insertion- region of the Equine herpesvirus-4 genome.

However, _the prerequisite for a useful Equine herpesvirus-4 vector is that the heterologous nucleic acid sequence is incorporated in a permissive position or region of the genomic Equine herpesvirus-4 sequence, i.e. a position or region which can be used for the incorporation of a heterologous sequence without disrupting essential functions of Equine herpesvirus-4 such as those necessary for infection or replication. Such a region is called an insertion- region.

According to the present invention Equine herpesvirus-4 mutants are provided which can be used as a viral vector, characterized in that said mutants comprise a heterologous nucleic acid sequence encoding a polypeptide inserted into the genome of Equine herpesvirus-4, the insertion-region being identified as the gene encoding the enzyme defined by the amino acid sequence shown in Fig. 6.

Equine herpesvirus-4 insertion mutants as described above having a heterologous nucleic acid sequence inserted in place of deleted DNA representing the whole or a fragment of said gene are also within the scope of the present invention.

The term "Equine herpesvirus-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 Equine herpesvirus-4. Upon infection of a cell by said Equine herpesvirus-4 insertion mutant it expresses the heterologous gene in the form of a heterologous polypeptide.

The term "polypeptide¹' 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 and proteins are included within the definition of polypeptide.

The heterologous nucleic acid sequence to be incorporated into the Equine herpesvirus-4 genome according to the present invention can be derived from any source, e.g. viral, prokaryotic, eukaryotic or synthetic. Said nucleic acid sequence can be derived from a pathogen, preferably an equine pathogen, which after insertion into the Equine herpesvirus-4 genome can be applied to induce immunity against disease. Preferably, nucleic acid sequences derived from Equine herpesvirus-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 Equine herpesvirus-4 genome.

Furthermore, nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic application, in particular immune modulators such as lymphokines," interferons or cytokines, may be incorporated into said insertion-region. An essential requirement for the expression of the heterologous nucleic acid sequence in a Equine herpesvirus-4 mutant infected cell 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 Equine herpesvirus-4 mutant, such as the SV-40 promoter (Science 222. 524-527, 1983) or, e.g., the metallothionein promoter (Nature 296, 39-42, 1982) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA 8_2, 4949-53, 1985) or the human cytomegalovirus IE promoter or promoters present in Equine herpesvirus-4.

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 Equine herpesvirus-4 genome (Maniatis, T. et al. (1982) in "Molecular cloning", Cold Spring Harbor Laboratory; European Patent Application 74.808; Roizman, B. and Jenkins, F.J. (1985), Science 229, 1208; Higuchi, R. et al. (1988), Nucleic Acids Res. 16, 7351).

Briefly, this can be accomplished by constructing a recombinant DNA molecule for recombination with Equine herpesvirus-4 DNA. Such a recombinant DNA molecule comprises vector DNA which may be derived from any suitable plasmid, cos id, virus or phage, plasmids being most preferred, and contains Equine herpes¬ virus-4 DNA of the insertion-region identified above, possibly having a nucleic acid sequence inserted therein if desired operably linked to expression control sequences. Examples of suitable cloning vectors are plasmid vectors such as pBR322, the various pUC and Bluescript plasmids, bacteriophages, e.g. λgt-WES-λ B, charon 23 and the M13mp phages or viral vectors such as SV40, Bovine papillomavirus, Polyoma and Adeno viruses. Vectors to be used 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 Equine herpesvirus-4 DNA fragment comprising the insertion region identified above, is inserted into the cloning vector using well known recDNA techniques. Said DNA fragment may comprise essentially the complete DNA sequence of said gene shown in Fig. 5, and if desired flanking sequences thereof.

Second, if an Equine herpesvirus-4 deletion mutant is to be obtained at least part of said 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 Equine herpesvirus-4 insertion mutant is to be obtained the heterologous nucleic acid sequence is inserted into the insertion-region present in the recombinant DNA molecule of the first step or in place of the DNA deleted from said recombinant DNA molecule prepared in the second step. The Equine herpesvirus-4 DNA sequences which flank the deleted DNA or the inserted nucleic acid sequence should be of appropriate length as to allow homologous recombination with the viral Equine herpesvirus-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. different pathogens said sequences being flanked by insertion- region sequences of Equine herpesvirus-4 defined herein. Such a recombinant DNA molecule can be employed to produce an Equine herpesvirus-4 mutant which expresses two or more different antigenic polypeptides to provide a multivalent vaccine.

In order to obtain an Equine herpesvirus-4 mutant according to the invention, cells, for example rabbit cells, or equine cells, e.g. equine dermal cells, can be transfected with Equine herpesvirus-4 genomic DNA in the presence of the recombinant DNA molecule containing the deletion and/or insertion of (heterologous) nucleic acid sequence flanked by appropriate Equine herpesvirus-4 sequences whereby recombination occurs between the corresponding regions in the recombinant DNA molecule and the Equine herpesvirus-4 genome. Recombination can also be brought about by transfecting Equine herpesvirus-4 genomic DNA containing host cells with a DNA fragment 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 Equine herpesvirus-4 mutants which do not produce functional RR (Darling et al., 1987) or detecting the antigenic heterologous polypeptide expressed by the Equine herpesvirus-4 mutant immunologically. The selected Equine herpesvirus-4 mutant can be cultured on a large scale in cell culture whereafter Equine herpesvirus-4 mutant containing material or heterologous polypeptides expressed by said Equine herpesvirus-4 can be collected therefrom.

Alternatively, mutant Equine herpesvirus-4 could be generated by cotransfection of several cosmids, containing between them the entire Equine herpesvirus- 4 genome, where an insertion and/or deletion has been engineered into the cos id possessing Equine herpesvirus-4 insertion region DNA.

According to the present invention a live attenuated Equine herpesvirus-4 mutant which does not produce a functional RR, and if desired expresses one or more different heterologous polypeptides of specific equine pathogens can be used to vaccinate horses, susceptible to Equine herpesvirus-4 and these pathogens.

Vaccination with such a live vaccine is preferably followed by replication of the Equine herpesvirus-4 mutant within the inoculated host, expressing in vivo Equine herpesvirus-4 polypeptides, and if desired heterologous polypeptides. An immune response will subsequently be elicited against Equine herpesvirus-4 and the heterologous polypeptides. An animal vaccinated with such an Equine herpesvirus-4 mutant will be immune for a certain period to subsequent infection of Equine herpesvirus-4 and above-mentioned pathogen(s) .

An Equine herpesvirus-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 Equine herpesvirus-4 mutant according to the invention can also be used to prepare an inactivated vaccine. For administration to animals, the Equine herpesvirus-4 mutant according to the presentation can be given inter alia by aerosol, spray, drinking water, orally, intradermally, subcutaneously or intra¬ muscularly. 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 TCID₅₀ of the Equine herpesvirus-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 Equine herpesvirus-4 mutant according to the invention. This can be achieved by culturing cells infected with said Equine herpesvirus-4 mutant under conditions that promote expression of the heterologous polypeptide. The heterologous polypeptide may then be purified with conventional techniques to a certain extent depending on its intended use and processed further into a preparation with immunizing therapeutic or diagnostic activity.

The above described active immunization against specific pathogens will be applied as a protective treatment in healthy animals. It goes without saying that animals already infected with a specific pathogen can be treated with antiserum comprising antibodies evoked by an Equine herpesvirus-4 mutant according to the invention. Antiserum directed against an Equine herpesvirus-4 mutant according to the invention can be prepared by immunizing animals with an effective amount of said Equine herpesvirus-4 mutant in order to elicit an appropriate immune response. Thereafter the animals are bled and antiserum can be prepared. Example 1

Isolation and characterization of Equine herpesvirus-4 insertion region.

1. Culturinσ of Eσuine herpesvirus-4 virus

Roller bottles 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, 100 units/ml penicillin, 100 mg/ml streptomycin and 10% foetal calf serum were infected with virus of the Equine herpesvirus-4 strain 1942 at a .o.i. of 0,003 and allowed to adsor ^~for 60 in at 37 °C. They were incubated at 31 °C until extensive c.p.e. was evident and the majority of cells had detached from the bottle surface (2-6 days) . The infected cell medium was centrifuged 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 11.000 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 1 hour. Ratios of virus particles to plaque-forming units were approximately 1.000 to 5.000.

2. Preparation of Eσuine herpesvirus-4 DNA

The pelleted virus was resuspended in 10 ml NTE (NaCl/Tris/EDTA) and briefly sonicated. Contaminating cellular DNA was degraded by adding DNase at 10 μg/ml and incubating at 37 °C for 1 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 pelleted, washed with 70% ethanol, resuspended in 10 ml of 100 mM NaCl and 10 μg/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. Cloninσ of Eσuine herpesvirus-4 DNA

Equine herpesvirus-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 μg of Equine herpesvirus-4

DNA and 5 μg 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 Weisblu (J. Bact. 121. 354, 1975).

Approximately 0.1 μg of BamHI digested pUC9 and 1 μg of

BamHI-digested Equine herpesvirus-4 DNA were mixed in 50 mM Tris-HCl pH 7,5, 8 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP in a final volume of 40 μl. 2 units of T4 DNA ligase (0,5 μl) 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). Three subclones of an Equine herpesvirus-4 clone containing the 13.5 kb BamHI A fragment (Figure 1) were constructed which contained the RR genes:- p4.2EcoA, pl.7HindA and pBS1.3ESA (Figure 2) . The vast majority of the RR sequence was obtained by sequencing the Equine herpesvirus-4 inserts in clones pl.7HindA and pBS1.3ESA, covering the region from the Hindlll site at 2.7 to the Sail site at 5.5. The sequence of the start of the RR1 gene was obtained by using intact Equine herpesvirus-4 BamHI A as a template; the end of the RR2 gene was sequenced using p4.2EcoA as a template. The clones were constructed as follows: p4.2EcoA: digestion of Eσuine herpesvirus-4 BamHI A (contained in pUC9) with EcoRI and religation of the 6.9 kb fragment which contains the leftmost 4.2 kb of Equine herpesvirus-4 BamHI A attached to pUC9 vector. pl.7HindA: derived from p4.2EcoA by digestion with Hindlll and religation of the 4.2 kb fragment, thereby deleting the 2.7 kb BamHI/Hindlll subfragment at the left end of p4.2EcoA and Equine herpesvirus-4 BamHI A. pBS1.3ESA: 1.3 kb EcoRI/Sall subfragment of p4.2EcoA cloned between the EcoRI and Sail sites of Bluescript M13+ vector.

The actual experimental procedures for cloning were as follows: i) Agarose Gel Electrophoresis

Agarose gel electrophoresis of DNA restriction fragments was carried out on horizontal 0.8% agarose gel slabs submerged in lxTBE buffer (0.09 M Tris, 0.09 M borate, 2 mM EDTA) . Gels were srained with ethidium bromide (1 μg/ml in water) for 30 minutes and destained for 30 minutes in water. DNA was visualised on a 302 nm UV transilluminator. ii) Purification of DNA Fragments From Agarose Gels The desired DNA fragments were excised from the gel using a sterile scalpel. Excised gel slices were transferred to Spin-X filter centrifuge tubes (Costar) , stored at -20 °C for 20 minutes and then centrifuged at 13000 rpm in a benchtop microcentrifuge for 30 minutes. The eluate was successively extracted with an equal volume of phenol (equilibrated with 1 M TrisHCl, pH 8.0), phenol/chloroform (1:1), chloroform and then ether and the DNA precipitated with two volumes of ethanol in the presence of 0.3 M sodium acetate (pH 6,0) at -70 °C for 30 minutes). DNA was pelleted by centrifugation, washed with 70% ethanol and dried in a vacuum dessicator. This precipitation procedure was repeated once more, the DNA finally resuspended in 20 to 50 μl of water and stored at -20 °C until required.

iii) Ligation Reaction

Recombinant plasmid pBS1.3ESA was constructed by ligation of vector and insert DNA fragments as follows. 100 ng of vector DNA (Bluescript double- digested with EcoRI and Sail) was ligated to a three molar excess of insert DNA (1.3 kb EcoRI/Sall subfragment of Equine herpesvirus-4 BamHI A) using 3 units of T4 DNA ligase in the presence of 50 mM TrisHCl pH 7.6, 10 mM MgCl₂, 1 mM ATP, 1 mM DTT, 5% w/v PEG-8000 (T4 DNA ligase buffer, BRL) , in a total volume of 30 μl at 14 "C for 16 to 20 hours. The construction of recombinant plasmids p4.2EcoA and pl.5HindA was accomplished by simple religation of 100 ng of the appropriate purified fragments mentioned previously. iv) Transformation of Competent E.Coli Cells With Plasmid DNA

E.coli JM101 cells competent for DNA uptake were prepared by the calcium chloride method (Cohen et al., 1972) .

The contents of each ligation reaction were added to 0.2 ml of competent E.coli cells prepared as described above, and incubated on ice for 30 minutes. This cell-DNA mixture was heat shocked by incubating at 37 "C for 5 minutes to facilitate entry of DNA into the bacterial cells. To the heat shocked mixture was added 1 ml of L broth and the bacteria were incubated at 37 ° C for 1.5 hours to allow expression of antibiotic resistance genes. Cells were spread onto five L agar plates (200 ml L broth, 3 g agar, sterilise by autoclaving) containing ampicillin at 100 μg/ml and plates were incubated for 15 minutes at room temperature to allow the liquid to be absorbed. Plates were inverted and incubated at 37 "C for 16 to 20 hours. Transformants appeared as separate and well- defined colonies on the surface of the agar plates.

Transformants were screened for the presence of the desired recombinant plasmid by small scale isolation of plasmid DNA by the boiling method (Holmes and Quigley, 1981) .

The start and end points of the RR1 and RR2 genes was predicted by sequencing the ends of the Equine herpesvirus-4 DNA inserts in plasmids p4.2EcoA, pl.7HindA and pBS1.3ESA (Figure 1). Restriction mapping of Equine herpesvirus-4 BamHI A showed that the Equine herpesvirus-4 RR genes are located near the left terminus of this fragment (Figure 1) . DNA was sequenced by the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. 7_4, 5463, 1977), using the double-stranded DNA sequencing technique in which single-stranded DNA template was produced by alkaline denaturation of plasmid DNA. DNA fragments to be sequenced were cloned into the Bluescript M13+ (1.3 kb EcoRI/Sall subfragment of BamHI A) or were already contained within pUC9 (1.5 kb Hindlll/EcoRI subfragment of BamHI A) . The enzyme kits used for sequencing was the T7 DNA polymerase kit obtained from Pharmacia. The DNA sequence of the RR genes and corresponding amino acid sequence are shown in Fig. 5 and Fig. 6.

Example 2

Construction of EHV-4 RR-neσative plasmids

Two ribonucleotide reductase (RR) negative plasmids were constructed for EHV-4.

1. RRl-neσative (Fiσ. 3)

A 0.9 kb Pstl - Hindlll fragment containing most of the RR2 gene (bp 2434-3357) was cloned into vector pIC20R to give plasmid pIC0.9PH.

An oligonucleotide adaptor AB(EcoRI-PstI ends) was cloned into the plasmid pBS2.3ESA, which contained most of the RR1 gene (bp 266-1632), cloned as a 1.3 kb Sall-EcoRI fragment into pBluescript M13+, to give plasmid pBSI.3AB.

An 1.4 kb Sall-Pstl fragment from pBSI.3AB containing most of the RRl-gene and oligonucleotide adaptor AB was cloned into pIC0.9PH to give plasmid pICRRl^". From this plasmid pICRRl^" a 2.3 kb Sall-Hindlll fragment was cloned into pAT153 to give plasmid pATRRl^". Finally the lacZ gene was cloned between the EcoRV and BamHI sites of pATRRl^".

_" RRl-neσative/RR2-neσative (Fig. 4)

An 1.3 kb Sall-EcoRI fragment containing most of the

RRl gene (bp 266-1632) was cloned into vector pRIT2T to give plasmid pRIT1.3.

An oligonucleotide adaptor CD (EcoRI-Hindlll ends) was cloned into pRIT1.3 to give plasmid pRIT1.3CD.

From plasmid pRIT1.3CD an 1.4 kb Hindlll fragment containing most of the RRl gene and the oligonucleotide adaptor CD was cloned into pAT153 to give plasmid pAT1.3CD.

An 1.9 kb Hindlll fragment containing the C-terminal

94 bp of the RR2 gene (bp 3351-3444) was cloned in ■ correct orientation into pAT1.3CD to give plasmid pATRRl^"/RR2~.

Finally the lacZ gene was cloned between the EcoRV and BamHI sites of pATRRl ~/lRR2~ .

Legends

Figure l. Shows a restriction enzyme map of the Equine herpesvirus-4 genome and the location of the RR genes within the BamHI A restriction enzyme fragment of the Equine herpesvirus-4 genome.

Figure 2. Represents the restriction enzyme maps of the recombinant plasmids p4.2EcoA, pl.7HindA and pBS1.3ESA.

Figure 3. Represents the construction of the plasmid pATRRl^".

Figure 4. Represents the construction of the plasmid pATRRl^"/RR2^".

Figure 5. Represents the nucleotide sequence of the RR gene of EHV-4.

Figure 6. Represents the amino acid sequence of the enzyme ribonucleotide reductase of EHV-4. The enzyme consists of 2 subunits RRl and RR2.

ADDITIONAL REFERENCES

Cohen, S.N., Chang, A.C.Y. and Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA £9:2110.

Darling, A.J., Dutia, B.M. and Marsden, H.S. (1987). Improved method for the measurement of ribonucleotide reductase activity. J. Virological Methods 180:231-290.

Holmes, D.S. and Quigley, M. (1981) . A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

Claims

1. An Equine herpesvirus-4 mutant comprising a mutation in the gene encoding a protein having the amino acid sequence shown in SEQ ID NO: 2.

2. An Equine herpesvirus-4 mutant according to claim 1, characterized in that the mutation is located within the DNA sequence shown in SEQ ID NO: 1.

3. An Equine herpesvirus-4 mutant according to claim 1 or 2, characterized in that the mutation is a deletion and/or insertion.

4. An Equine herpesvirus-4 mutant according to claim 3, characterized in that the insertion comprises a heterologous nucleic acid sequence encoding a polypeptide.

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

6. An Equine herpesvirus-4 mutant according to claim 5, characterized in that the heterologous nucleic acid sequence encodes an antigen of an equine pathogen.

7. Recombinant DNA molecule comprising vector DNA and at least part of the gene encoding the protein having the amino acid sequence shown in SEQ ID NO: 2.

8. Host cell transfected with the recombinant DNA molecule according to claim 7.

9. Cell culture infected with an Equine herpesvirus-4 mutant according to claims 1-6.

10.Vaccine comprising an Equine herpesvirus-4 mutant according to claims 1-6.