MXPA01001175A - Attenuated equine herpesvirus - Google Patents

Abstract

The present invention relates to novel Equine herpesvirus (EHV) mutants comprising one or more deletions, substitutions, or insertions in the endogenous promoter region of an essential viral gene, preferably the immediate early gene of EHV. The EHV mutants are stable and have reduced virulence, which makes them very suitable for use in a live vaccine. The invention furthermore relates to live vaccines comprising said EHV mutants, to DNA sequences and vectors harbouring a mutated EHV sequence, to host cells transfected with said DNA or vectors. The invention also relates to a method of attenuating EHV in general, and EHV-1 in particular.

Description

VIRUS OF HERPES AOUINO ATENUADO The present invention relates to a new equine herpes virus (EHV) mutant, to its use in vaccines, to DNA sequences, and to vectors harboring a mutated equine herpes virus sequence, to host cells transfected with these vectors, and a method to attenuate equine herpes virus in general, and EHV-1 in particular. Equine herpes viruses comprise a group of different antigenic biological agents that cause a variety of infections in horses, from a subclinical to fatal disease. The equine herpes virus, among other things, is one of the most common causes of infectious abortions, and can account for 15 percent of all maternal abortions that occur during the last six months of pregnancy. The equine herpes virus type I (EHV-1) can cause abortion, perinatal mortality, respiratory inflammation, and neurological disease. Although very similar to EHV-4 (previously classified as EHV-1 subtype 2), the main clinical sign of EHV-1 is abortion, while EHV-4 results in respiratory disease. But both cross and may be clinically indistinguishable as a disease.

The virus is contracted through inhalation. The respiratory signs can be from severe to not apparent. Abortion occurs primarily in mares with more than 7 months of pregnancy, continues infection in 14 to 120 days, and may present suddenly without signs in the mare. The virus infects the fetal lung and liver tissue, and the placental endothelial tissue of the mare. Abortion can occur due to direct effects on the fetus, or due to separation of the placenta. Near-term fetuses may be born alive, but die quickly due to a pathology of the lung. The primary infection of the upper respiratory tract of young horses results in a febrile illness lasting from 8 to 10 days. Immunologically experienced mares can be reinfected through the respiratory tract without the disease being observed, so that abortion usually occurs without warning. The neurological syndrome is associated with respiratory disease or abortion, and can affect animals of any sex of any age, leading to lack of coordination, weakness, and subsequent paralysis. Other EHV viruses are EHV-2 or Equine Cytomegalovirus which is a ubiquitous, antigenically heterogeneous group, which normally grows slowly from viruses, which cause an unknown disease, and EHV-3, the Coital Equine exanthema virus, which is the causative agent of a relatively light pregenital exanthema of both the mare and the foal. EHV-1 subtype 2 is now called EHV-4, and is primarily associated with respiratory disease, although abortions induced by EHV-4 have been reported sporadically. The genomic structure of EHV is similar to that of other alpha herpes viruses, which comprise a double-stranded linear molecule consisting of two covalently linked segments (UL and Us), where the Us segment is flanked by inverted repeats. Of the EHV, the EHV-1 is the most extensively studied. Telford, E.A.R. et al., Virology 189, 304-316 (1992) have published the complete DNA sequence of EHV-1. The genome consists of approximately 150 base pairs, and approximately 76 different genes have been recognized so far. The equine herpes virus in general, and EHV-1 in particular, are ubiquitous pathogens in horses. EHV-1 can even cause epidemics of abortion, respiratory tract disease, and central nervous system disorders. Therefore, prevention of infection with the virus is of great economic importance, because EHV can be a severe threat, especially for horses that live in closed groups, such as in stables. Current vaccines against these viruses include chemically inactivated virus or live attenuated virus. Nevertheless, these require multiple administration, and only have limited effectiveness. Inactivated vaccines generally induce only a low level of immunity. Accordingly, live attenuated vaccines are preferred, because they elicit a more lasting immune response, and are also easier to produce. The attenuation can be obtained by serial steps of the virulent strains in tissue culture of other hosts different from the natural one. However, the strains thus obtained are not very well defined, and it is considered that they are not affected. In addition, there is always a risk that viruses will revert to virulence, resulting in disease in the inoculated animals. For this reason, genetic attenuation was adopted as a novel approach to obtain safe vaccine strains. Genetic attenuation consists, for example, in the suppression of one or more non-essential genes. Examples are viruses with deletions in the thymidine kinase gene, or the gene encoding the gE glycoprotein. Both have been used successfully for the genetic attenuation of the herpes virus in general, but similar mutations in EHV-1 strongly abolished the replication in the host, and made the viruses no longer useful for vaccine purposes. In addition, the mutations often did not result in the desired level of attenuation. Accordingly, it is a first objective of the present invention to provide new strains of EHV virus that do not have the above mentioned drawbacks, when used in vaccines. It is a second object of the invention to provide a method for the more general genetic attenuation of herpes viruses. The third objective of the invention is to provide live vaccines comprising these novel EHV mutant viruses. The first objective of the present invention is achieved by an equine herpes virus (EHV) mutant, comprising one or more deletions, substitutions, or insertions in the endogenous promoter region of an essential viral gene. Preferably, this mutation is a deletion in the endogenous promoter region. Because the attenuation of EHV by mutating non-essential genes was not successful, at least for vaccine purposes, the skilled person would have considered a mutation in the endogenous promoter region of the essential genes as even less promising, because he would not have expected that the virus will replicate in the host at all. Accordingly, the discovery of the invention, that the endogenous promoter region of the essential genes can be mutated to reduce the virulence of the EHV viruses, leading to a successful attenuation, is highly surprising. The mutations according to the invention do not eliminate the replication of the virus in the host, and they do affect the virulence level of the virus. The EHV mutants according to the invention are stable and attenuated with respect to the progenitor strain, which makes them very suitable for use in a live vaccine. In a first embodiment of the invention, the promoter region is from the immediate early gene (IE) of the EHV virus.

Other essential genes from which the promoters can be used are the genes involved in DNA replication (for example, gene 57 in EHV-1 that codes for the helicase / primase complex), or transactivators of immediate early genes, such as gene 12 in EHV-1, or the genes that code for the essential structural components of virions, such as glycoproteins gB, gH, and gL in EHV-1, or other herpes viruses. The sequence upstream of the start of transcription contains several elements that regulate the expression of a gene. All these elements are defined together as the promoter. The nuclear sequence of the promoter is also known as the TATA table, and is located at approximately 40 base pairs from the start of transcription, although this structure can vary considerably from case to case. Other more or less conserved elements are located more upstream, such as the CCAAT sequence, and the repeated CG motifs. Some elements are found specifically in certain types of promoters, such as a TAATGARATTC consensus sequence in the immediate early genes of herpes viruses. Also, the sequence corresponding to the untranslated region upstream of the ATG start codon may be involved in the activity of the promoter. Many of these elements have not yet been defined, nor is the way in which they interact with the so-called transcription factors completely understood, as a result of which the transcription of the gene is regulated. For the purpose of the present invention, however, a promoter is defined as the sequence extending over several hundred base pairs upstream of the coding region of the gene, preceding the most important part located at 200 base pairs at transcription start site. Examples of sequences containing promoters from essential genes can be recovered in GenBank. The complete sequences for EHV-1 and EHV-4 have been deposited in GenBank under accession numbers M86664 and AF030027, respectively. For genes 57 and 12 from EHV-1, the promoter will be located between nucleotides 101,600-102,347 and 12,900-13,505 respectively.

Alternatively, the promoter regions can be determined by nucleotide sequence analysis of the DNA fragments containing an essential gene, including the region upstream of the coding sequence. A preferred embodiment of the invention is the endogenous promoter of the IE gene of EHV-1 of which a copy is located between nucleotide 118.590 and about 119.890, in the Inverted Repetition of the short segment (IRs). In accordance with the invention, it has been found that the endogenous promoter region of the essential genes may be more suitable for attenuating the virulence of EHV. So far, this has not been considered, because it was assumed that manipulation of an essential gene was detrimental to efficient replication in the host. The term "essential gene" as used herein, is intended to encompass the genes that are required for replication in the host. "Gen" is used exclusively for the coding sequence, while the "promoter region" refers to the regulatory sequences necessary for the expression of the gene. Preferred mutations according to the invention are deletions between 1 and about 500 bases at any position within the promoter region, which reduce the level of expression of the essential gene which is located downstream, and which attenuates the virulence of the pathogen. By no means a deletion in accordance with the present invention eliminates the expression of the genetic product. For EHV-1, the preferred deletions are the deletion of the Sacl-Sacl fragment or the HindIII-Clal fragment, or the Ndel-Ndel fragment, or the Sphl-Sphl fragment of the promoter region of the immediate early gene. A deletion of the HindIII-Clal fragment from the IE promoter region is highly preferred.; this suppression eliminates the virulence of the virus without affecting the local replication in the host. An EHV-1 mutant comprising a deletion of the HindIII-Clal fragment of the promoter region of its IE gene, shows an exceptionally good level of attenuation, without any negative effect on its replication, thus making this mutant highly suitable to be used in a live vaccine. As an alternative, fragments with a size between 1 and about 500 bases in the endogenous promoter region can be inserted, provided that this insertion does not eliminate the expression of the essential gene product. These inserts, in accordance with the invention, do not interfere with the ability of the virus to replicate, and have the advantage that they can lead to a desired level of virulence reduction.

Substitutions can be made in the endogenous promoter of an essential viral gene, varying from one or more nucleotides to 500 base pairs, with the understanding that this substitution does not lead to loss of expression of the essential gene product. The substitutions according to the invention do not result in a replication of the virus in the host, but may lead to a desired level of attenuation. Substitution according to the invention should not be confused with the replacement of the entire promoter region with a cell-specific, tissue-based, or host promoter, as described in Glazenburg et al., In U.S. Pat. US-5, 580, 564. Glazenburg aims to select the cell, the tissue and / or the host, in such a way that the harmful characteristics of the microorganism are not expressed, or are only expressed to an acceptable degree. The overall virulence of the virus is not reduced, but instead, the location of the viral replication is limited to particular cells, tissues, or hosts. The substitutions according to the invention are not intended to encompass these mutations in specific promoters that lead to a modified cell, tissue, or host tropism. Suppressions can be introduced by the following methods. Based on the map of the promoter region, suitable restriction sites can be deduced to remove fragments of a defined size and at a defined position in the promoter. As an alternative, one could start at a single site, and make progressive deletions in one or two directions, using the technique described by Henikoff (Gene 28, 351-359 (1984)). Also, linker-mediated mutagenesis or polymerase chain reaction can be used (Current Protocols in Molecular Biology, eds., Ausubel et al., Chapter 8, John Whiley &Sons, Inc. (1996)). Mutations introduced into the cloned subfragments can be transferred to the virus genome, for example, as described in Maniatis T. et al. (1982) in "Molecular cloning", Cold Spring Harbor Laboratory; European Patent Application Number 74,808; Roizman, B. & Jenkins, F.J. (1987); Science 229, 1208; Higuchi, R. et al., (1988), Nucleic Acids Res. 16, 7351. Briefly, this can be done by constructing a recombinant DNA molecule to recombine with the equine herpes virus DNA. This recombinant DNA molecule comprises vector DNA that can be derived from any suitable plasmid, cosmid, virus, or phage, and contains the equine herpes virus DNA of the region identified above.

Examples of suitable cloning vectors are plasmid vectors, such as pBR322, different pUCs, and Bluescript plasmids, cosmid vectors, for example THV, pJB8, MUA-3 and Cosi, bacteriophages, for example lambda-gt. - ES-lambda B, charon 28, and the M13mp phages or viral vectors such as SV40, bovine papilloma virus, Polyoma and Adenovirus. Other vectors to be used can be retrieved from the Intelligenetics vector database accessible through the http website; // www, seqnet. < 31.ag.uk. A deletion to be introduced in the region described can first be incorporated into a recombinant DNA molecule carrying the promoter region of the essential EHV gene, by means of a restriction enzyme digestion, with one or more enzymes, of which correctly place the dissociation sites in the promoter region of the gene. The recircularization of the remaining recombinant DNA molecule would result in a derivative lacking at least part of the promoter region. Alternatively, progressive deletions may be introduced, either in one or both directions, starting from within the dissociation site with restriction enzyme present within the gene sequence. Enzymes such as BaI31 or exonuclease III can be used for this purpose.

The recircularized molecules are transformed into E. coli cells, and the individual colonies can be analyzed by restriction mapping, in order to determine the size of the suppression introduced in the promoter region. Accurate placement of the deletion can be obtained by sequence analysis. In the case that the insertion of a heterologous nucleic acid sequence is desired, the recombinant DNA molecule comprising the essential EHV gene can be digested with the appropriate restriction enzymes to produce linear molecules, after which can bind the heterologous nucleic acid sequence with the linear molecules, followed by recircularization of the recombinant DNA molecule. Optionally, a deletion is introduced into the promoter region of the EHV gene in a manner concomitant with the insertion of the heterologous nucleic acid sequence. In the case where the homologous recombination method is applied in vivo for the preparation of an EHV mutant according to the invention, the EHV sequences flanking the deleted genetic sequences or the inserted heterologous nucleic acid sequences should be an appropriate length, for example 50 to 3,000 base pairs, to allow homologous recombination to occur in vivo with the viral EHV genome. Subsequently, cells, for example equine cells, such as equine dermal cells (NBL-6), or cells of other species, such as RK13, Vero, and BHK cells, can be transfected with EHV DNA in the presence of the recombinant DNA molecule containing the mutation flanked by the appropriate EHV sequences, whereby recombination occurs between the EHV sequences in the recombinant DNA molecule, and the corresponding sequences in the EHV genome. The recombinant viral progeny is then produced in the cell culture, and may be selected, for example, genotypically or phenotypically, for example, by hybridization. The selected EHV mutant can be cultured on a large scale in a cell culture, after which the material containing the EHV mutant can be harvested therefrom. The genome of EHV-1 contains two copies of the immediate early gene, in opposite orientation. In this case, a convenient method to mutate the promoter is the construction of a set of cosmids that house fragments of the EHV genome, including only one of the two copies of the IE gene and its promoter region. This single copy of the promoter region can then be mutated in a routine manner. The set of cosmids is transfected to a confluent monolayer of host cells. Viruses will be formed by recombination, and can be recovered from the plates formed in the monolayer. It is necessary to use a copy of the promoter region IE and the corresponding gene, for the preparation of a mutant according to the invention, to prevent replacement of the mutation by the non-mutated promoter region. The mutant virus will reestablish a second identical copy of the mutated promoter region of the IE gene, while assembling a full-length functional genome containing both inverted repeats flanking the Us. It is a conventional procedure to test the efficacy and safety of a live vaccine. The initial test of the mutant EHV viruses can be performed in a well-known mouse model, as described by van Oensel et al., J. Virological Methods 54, 39-49 (1995), and Osterrieder et al., Virology 226, 243 -251, (1996). The final test of the safety and efficacy of the virus strain takes place live in horses. Mutations in the endogenous promoter of one or more essential genes can be combined with one or more mutations in one or more different genes and / or their promoters. Examples of these are mutations that create a marker vaccine that allows the differentiation of vaccinated animals from infected animals. In this way, additional optimization of the properties of the vaccine can be obtained.

The invention further relates to a nucleic acid sequence, comprising the endogenous promoter region of an essential EHV gene, and optionally one or more flanking sequences, whose promoter region comprises one or more deletions, substitutions, or insertions. For example, the gene is an immediate early gene, or any other essential gene, such as those described hereinabove. Specific deletions are deletions of the Sacl-Sacl fragment or the HindIII-Clal fragment, or the Ndel-Ndel fragment, or the Sphl-sphl fragment of the promoter region of the immediate early gene. Particularly preferred are deletions of the HindIII-Clal fragment from the promoter region of the immediate early EHV gene. The nucleic acid sequence in a specific embodiment comprises the promoter region of EHV-1 or EHV-4 more specifically EHV-1. The nucleic acid sequence can be incorporated into a recombinant DNA molecule, which is also part of the invention. The selection of suitable vectors for preparing the recombinant DNA molecule is well within the scope of the person skilled in the art. In addition, the invention relates to a host cell that hosts this recombinant DNA molecule. In addition, the present invention provides a vaccine comprising an EHV mutant of the invention, and a pharmaceutically acceptable carrier or diluent. A live attenuated mutant according to the present invention can be used to vaccinate equines, particularly domestic and non-domestic, and more specifically horses. Vaccination with this live vaccine is preferably followed by replication of the mutant inside the inoculated host, whose host will then elicit an immune response against the EHV, and the animal inoculated with the EHV mutant according to the invention, will be immune to infection. by EHV. Accordingly, a mutant according to claim 1, can serve as a live vaccine. For the preparation of a live vaccine, the recombinant EHV mutant according to the present invention can be cultured in a cell culture, for example of equine, rabbit, hamster, or calf origin. Viruses that grow in this way can be harvested by harvesting the fluids and / or cells from the tissue cell culture. The live vaccine can be prepared in the form of a suspension, or it can be lyophilized. The vaccine according to the invention can be prepared using conventional techniques available in the art. In general, the vaccine is prepared by mixing the virus with a pharmaceutically acceptable carrier or diluent. For administration to animals, the EHV mutant according to the present invention can be given, inter alia, intranasally, intradermally, subcutaneously, or intraramously. The pharmaceutically acceptable carriers or diluents that can be used to formulate a vaccine according to the invention, are sterile and physiologically compatible, such as, for example, sterile water, serum, aqueous regulators, such as alkali metal phosphates (e.g. serum regulated with phosphate), alcohols, polyols, and the like. In addition, the vaccine according to the invention may comprise other additives, such as adjuvants, stabilizers, antioxidants, preservatives, and the like. Suitable adjuvants include, but are not limited to, aluminum salts or gels, carbomers, non-ionic block copolymers, tocopherols, monophosphoryl lipid A, muramyl dipeptide, oil emulsions (water / oil or oil / water), cytokines . The amount of adjuvant added depends on the nature of the adjuvant itself. Suitable stabilizers for use in a vaccine according to the invention are, for example, carbohydrates, including sorbitol, nanitol, starch, sucrose, dextrin and glucose, proteins, such as albumin or casein, and regulators, such as alkaline phosphates. Suitable conservatives include, among others, thimerosal, mertiolate, and gentamicin. The live vaccines according to the invention comprise an effective amount of the aforementioned EHV mutant virus, and a pharmaceutically acceptable carrier. The term "effective", as used herein, is defined as the amount sufficient to induce an immune response in the target animal. The amount of virus will depend on the route of administration, the time of administration, as well as the age, general health, and diet of the subject to be vaccinated. Dosages in which live vaccines according to the invention can prevent infectious disease, can be easily determined by routine tests with appropriate controls, and are well within the practitioner's routine skills. The useful dosage to be administered will vary depending on age, weight, mode of administration, and the type of pathogen against which vaccination is sought. A suitable dosage can be, for example, from about 103'0 to 107'0 plaque / animal forming units. In accordance with a further aspect, the invention provides a process for the preparation of EHV mutants, which comprises transfecting a cell culture with a recombinant DNA molecule, and EHV genomic DNA.

Therefore, the invention provides a method for genetically attenuating equine herpes viruses which comprises the mutation of the endogenous promoter region of an essential gene, which mutation consists of one or more deletions, substitutions, or insertions in the promoter region of an essential gene. The present invention will be further illustrated in the following non-limiting examples. All the molecular biological techniques used in the examples are conventional methods, as for example described in Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press (1989). These techniques will not be described in detail.

LEYSNPAS Figure 1 shows a map of the EHV-1 genome, with the position of five cosmid DNA inserts that restore a viable virus after cotransfection and recombination between its overlapped sequences. Suppressions were introduced into the IE gene promoter in an 8 kb Spel fragment of the IRs, which was reassembled with a 21 kb Spel, and used as such to replace the cosmid 2D9 in the cotrasfection experiment. Figure 2 shows a schematic display of the promoter region of the IE gene in the IRs from EHV-1. Different elements of sequences that can regulate the start of transcription are indicated. Below we show the deletions that were introduced and the mutants that were generated by incorporating these deletions in the viral genome after co-tranfection with the set of overlapping cosmid DNA inserts. Strain EHV-4 was restored in a similar manner, but did not contain suppressions in the promoter region.

EXAMPLE 1: Construction of the cosmid set for general EHV-1 virus. The superCos 1 cosmid vector kit was purchased from Stratagene (Catalog # 251301), and the enzymes were purchased from New England Biolabs. The vector was further modified by the addition of extra restriction enzyme sites thereto. For this, a DNA linker was purchased in Pharmacia, which contained the following restriction sites: BamHI, I-Scel, PacI, AscI, EcoRV, PacI, AscI, I-Scel and BamHI. The SuperCos 1 vector and the linkers were both cut with BamHI. The vector digested with BamHI was dephosphorylated with alkaline phosphatase, after which, the BamHI-digested linker was ligated into the SuperCos 1 vector by DNA-T4 ligase, all according to the manufacturer's instructions. The resulting vector was designated as THM, and was used for the cloning of the EHV-1 inserts. Viral DNA was obtained from the M8 strain of EHV-1, a pathogenic strain of EHV-1 isolated from a horse with severe signs of an EHV-1 infection, but could be any pathogenic strain of EHV-1. . This particular virus was grown in Vero cell cultures, and recovered by centrifugation. The viral DNA was prepared by lysis with EDTA and SDAS, extraction with phenol, and precipitation with alcohol. The first set of cosmids was constructed by digestion of the EHV-1 DNA with Pací. After the phenol extraction of the Paci digests of M8, the ends were filled in with T4 DNA polymerase, and then dephosphorylated with alkaline phosphatase. The vector of cosmid THM was digested with EcoRV, and the inserts were ligated into the vector with T4 DNA ligase. The ligation mixture was added to a packing mixture (Gigapack, Stratagene) according to the manufacturer's instructions. The packaged DNA was added to a fresh overnight culture of E. coli DH1, and placed for one hour at 37 ° C. Then the bacterial suspension was spread on the agar plates containing ampicillin. The plasmid DNA from all the colonies was subjected to restriction enzyme analysis on the insert. For the construction of other cosmids, the same procedure was followed, only now the viral DNA was digested with AscI, Asel, Rsrl, or Notl, then all the ends were filled with T4 DNA polymerase, and the inserts were ligated into the EcoRV site of the vector. To obtain a third generation of cosmids, the viral DNA was torn twice through a 19G needle, then the ends were filled in with T4 DNA polymerase, and after phenol extraction and precipitation, the inserts were cloned back into the EcoRV site of the cosmid vector THM. Then the vector with the inserts was packed, put into bacteria, and the colonies were analyzed. Of all the colonies obtained, restriction maps were determined by multiple digestions. Then the location of the different clones was determined, comparing the restriction map of the clones with the restriction map of the Ab4 strain of EHV-1 (Telford et al., 1992). Cosmids 2D3, 1A12, 1F4, 2C12, and 2D9 were selected in such a way that the complete genome could be reconstituted by recombination between the overlapped sequences (Figure 1). The viable virus was generated after transfecting the EHV-1 inserts separated from the cosmids by digestion with I-Scel (Boehringer) in equine thermal cell cultures (ATCC number CCL-57), using the calcium phosphate method. The viral progeny was passed, and the plate was purified on rabbit kidney monolayers (RK13, ATCC number CCL-37), or other kidney cells that supported viral replication.

EXAMPLE 2: Introduction of suppressions in the promoter of the IE gene. The genome of EHV-1 contains two copies of the immediate early gene (IE) located in the repeated sequence elements IRs and TRs flanking the single short region. It is necessary to introduce stable mutations that affect the expression of the IE gene in both positions simultaneously, before it is expressed in a new phenotype. Accordingly, one of the cosmids termed "2D9" was modified, such that a fragment of approximately 7 kb was deleted at the far right of the TRs, and that it contained a single copy of the IE gene. This was obtained by subcloning the two Spel fragments of 8 and 21 kb from 2D9 into pGEM-9Zf (-) (Promega), using the Spel or Spel / Xbal sites, respectively (Figure 1). Insertion of the 21 kb fragment resulted in pEHV17, and restored a single Spel site, where the 8 kb Spel fragment can subsequently be inserted to regenerate a 29 kb EHV-1 fragment used to replace the 2D9 in the transfection of the cosmid described in Example 1. The 8 kb Spel contains the entire coding sequence of the IE gene, and approximately 2 kb of the upstream sequences, including the IE gene promoter. Mutations were introduced into the promoter region in a plasmid designated pEHV06, which contained a 2 kb subfragment from the 8 kb Spel. This insert included a 1 kb EcoRV fragment, which essentially represented the promoter and other regulatory elements located in the upstream sequence of the coding region of the IE gene (Figure 2). By selecting the appropriate restriction enzymes having two sites in the EcoRV fragment of pEHV06, and relatively close to each other, small deletions with an exact size could be introduced at different positions of the promoter sequence of the IE gene. The enzymes Sacl, Ndel, Sphl and a combination of Clal and HindIII were suitable for this purpose. The deletions introduced in pEHV06 by digestion with each of these enzymes, and the recircularization of the plasmid DNA with T4 DNA ligase, are represented in Figure 2. The mutated EcoRV inserts were exchanged with the original EcoRV insert in the Spel fragment of 8 kb, and these were subsequently assembled with the 21 kb Spel in pEHV17, resulting in plasmids that were designated pEHV22, pEHV23, pEHV25, and pHV26 for the deletions generated with the Sacl, HindIII / Clal, Ndel, and Sphl enzymes , respectively (Figure 2). pEHV24 contained the reassembled Spel fragments of 8 and 21 kb, but without any deletion in the promoter region. These plasmids were used to replace 2D9 in the co-tranfection of equine skin cells, with the cosmid inserts starting from 2D3, 1A12, 1F4 and 2C12, generating the viable virus with small deletions defined in the promoter region of both copies of the IE gene. The viral progeny were plaque purified and amplified in RK13 cells. The presence and size of the deletions were confirmed by DNA stain analysis and polymerase chain reaction. The mutant strains were designated as EHV22, EHV23, EHV25 or EHV26, the number corresponding to the plasmid used in the cotransfection experiment. Strain EHV24 represents the control virus, and contains the non-mutated promoter region. The mutant EHV strains derived from cosmid 22, 25, and 26, and the control strain EHV24, grow to titers between 2 and 4 x 10e plaque forming units / milliliter in RK13 cells, which is slightly lower than the M8 strain. progenitor that grows up to titrations of 5 x 106 plaque forming units / milliliter. Strain EHV23 was replicated in a less efficient manner, and resulted in titers of approximately 2 x 105 plaque forming units / milliliter.

EXAMPLE 3: Analysis of virulence in mice Infection with EHV-1 can cause pyrexia and clinical signs of respiratory disease, but more importantly, the induction of abortion. However, given the complexity of vaccination trials in pregnant horses, models have been developed in mice, measuring the pathogenicity of EHV-1 strains based on weight reduction (van Woensel et al., J. Virological Methods 54, 39 -49, (1995); Osterrieder et al., Virology 226, 243-251 (1996)). Mice 4 to 5 weeks of age are inoculated intranasally with a dose of 10S'5 plaque forming units per animal. Body weights are determined at 9 days after the infection, and in a graph the weight gain is plotted as a percentage of the weight on day zero. The degree of pathogenicity of different strains of EHV-1 is deduced from the position of the resulting curves, some in relation to the others. Significant losses in body weight, particularly on days 3 and 4 after infection, correlate with high virulence of the strain of interest. One can analyze series of EHV-1 isolates that carry deletions in the promoter region IE (see Example 2) to determine their virulence, making a comparison with the progenitor strain M8 or the EHV-1 yirus generated by the cosmid containing the intact promoter sequence.

EXAMPLE 4: Analysis of virulence in horses Although the pathogenicity of the individual EHV-1 strains using the mouse model can be correlated with the behavior in the natural host, more conclusive evidence can be obtained from the vaccination trials on horses. In addition, the level of protection against aggressive infection, and finally in the prevention of abortion in pregnant mares, can only be established in the white animal. The experiment was performed in four conventional stables at the age of approximately 1 year, and previously tested by the absence of antibodies in EHV-1 serum. The animals were divided into groups of two, one group receiving the mutant strain EHV23, the other receiving the mutant strain EHV24. The foals were inoculated intranasally with a single dose of approximately 5 x 105 plaque forming units, reconstituted in 2 milliliters of Unisolve diluent, and applying 1 milliliter in each nostril. After 10 days, the animals were monitored to determine the clinical reactions. Antibodies were measured at various points in time, either by neutralization of the virus, or by using a complement fixer antibody assay. The infected blood leukocytes (viremia), and the virus excreted in the nasal swabs, were determined by titration. After three weeks, a fifth horse was added seronegative for EHV-1, and two weeks later, the five horses were assaulted intranasally with 7 x 105 plaque-forming units of the pathogenic AB4p strain of EHV-1. The animals were monitored again to determine clinical reactions, virus excretion, and infected blood leukocytes. Horses vaccinated intranasally with a high dose of the mutant strain EHV23 virtually showed no signs of disease, while horses vaccinated with the mutant control strain EHV24 showed severe signs of infection with EHV-1 including fever and ocular or nasal discharges during several days . Both mutant strains were obtained in an identical manner, in the case of EHV23, introducing a deletion of 160 base pairs in the IE promoter, and in the case of EHV24 without any deletion (see Example 2). Therefore strain EHV24 represents the control virus, which contains the non-mutated IE promoter. Surprisingly, both strains replicated in the nasal cavity, although it appeared that the replica of strain EHV23 was slightly delayed.

The aggression of all the horses with the AB4p strain of EHV-1 did not induce significant signs of the disease in the animals that had previously been inoculated with the strain EHV23 or EHV24. No viremia was detected in any of the horses inoculated with any of the EHV23 or EHV24 strains, which indicates a solid systemic protection of these animals against an aggressive infection with the AB4p strain of EHV-1.

Claims (15)

REIVINPICATIONS

1. An equine herpes virus (EHV) mutant, which comprises one or more deletions, substitutions, or insertions in the endogenous promoter region of an essential viral gene relative to the progenitor strain.

2. The EHV mutant as claimed in claim 1, wherein deletions are introduced into the promoter.

3. The EHV mutant as claimed in claims 1 and 2, wherein the gene is the immediate early gene.

4. The EHV mutant as claimed in claims 1 to 3, wherein the mutant virus is the EHV-1 virus or the EHV-4 virus.

5. The EHV mutant as claimed in claims 1 to 4, which further comprises one or more mutations in one or more different genes and / or their promoters.

6. The EHV-1 mutant as claimed in claims 1 to 5, which comprises a deletion of the Sacl-Sacl fragment or the HindIII-Clal fragment, or the Ndel-Ndel fragment, or the Sphl-Sphl fragment of the promoter region of the immediate early gene.

7. A nucleic acid sequence comprising the endogenous promoter region of the immediate early gene from EHV, and optionally one or more flanking sequences, whose promoter region comprises a deletion of the Sacl-Sacl fragment or the HindIII-Clal fragment, or of the Ndel-Ndel fragment, or of the Sphl-Sphl fragment of the promoter region of the immediate early gene.

8. The nucleic acid sequence as claimed in claim 7, wherein the EHV is EHV-1 or EHV-4.

9. A recombinant DNA molecule comprising a nucleic acid sequence as claimed in claims 7 or 8.

10. A host cell harboring a recombinant DNA molecule as claimed in claim 9.

11. A vaccine comprising an EHV mutant as claimed in claims 1 to 6, and a pharmaceutically acceptable carrier or diluent.

12. A process for the preparation of an EHV mutant as claimed in claims 1 to 6, which comprises the transfection of a cell culture with a recombinant DNA molecule as claimed in claim 11, and genomic DNA of EHV .

13. A method to genetically attenuate EHV, which comprises the mutation of the endogenous promoter region of an essential gene, whose mutation consists of one or more deletions, substitutions, or insertions in the promoter region of an essential gene.

14. The method as claimed in claim 13, wherein the EHV is EHV-1 or EHV-4.

15. The method as claimed in claims 14, wherein the gene is an immediate early gene.