WO2002081712A2

WO2002081712A2 - Artificial chromosomes comprising ehv sequences

Info

Publication number: WO2002081712A2
Application number: PCT/EP2002/003575
Authority: WO
Inventors: Nikolaus Osterrieder; Jens Rudolph
Original assignee: Boehringer Ingelheim Vetmedica Gmbh
Priority date: 2001-04-03
Filing date: 2002-03-30
Publication date: 2002-10-17
Also published as: EP1377668A2; CA2443039A1; AR035809A1; JP2004531254A; WO2002081712A3; MXPA03008971A; DE10116594A1

Abstract

The invention belongs to the field of animal health, in particular equine diseases caused by equine herpesvirus (EHV). The invention relates to artificial chromosomes comprising the genome of equine herpesviruses, methods of producing attenuated or virulent EHV with or without the insertion of foreign genes, EHV obtainable with said methods and pharmaceutical compositions comprising said viruses.

Description

Artificial Chromosomes comprising EHN Sequences

The invention belongs to the field of animal health, in particular equine diseases caused by equine herpesviras (EHN). The invention relates to artificial chromosomes comprising the genome of equine herpesviruses, methods of producing attenuated EH-viruses, EH-viruses obtainable with said methods and pharmaceutical compositions comprising said viruses.

Background of the invention

Equine herpesviras 1 (EHN-1), a member of the Λlphaheψesvirinae, is the major cause of virus- induced abortion in equids and causes respiratory and neurological disease. The entire DΝA sequence of the EHN-1 strain Ab4p has been determined (Telford, E. A. R. et al, 1992). Only few genes and gene products have been characterized for their relevance for the virulence or immunogenicity of EHN-1 because the production of viral mutants is still relying on the generation of recombinant viruses by homologous recombination between the viral genome and respective foreign DΝA to be inserted in cultured mammalian cells.

For control of EHN-1 infections, two different approaches are followed. First, modified live vaccines (MLNs) have been developed, including the strain RacH (Mayr, A. et al, 1968; Hubert, P. H. et al., 1996), which is widely used in Europe and the United States. Second, inactivated vaccines and independently expressed viral glycoproteins have been assessed for their immunogenic and protective potential. Among the glycoproteins that were expressed using recombinant baculoviruses are the glycoproteins (g) B, C, D, and H, which induced partial protection against subsequent challenge EHN-1 infection in a murine model (Awan, A. R. et al, 1990; Tewari, D. et al, 1994; Osterrieder, Ν. et al, 1995; Stokes, A. et al, 1996). However, the use of MLNs has advantages over killed and subunit vaccines. MLNs are highly efficient in inducing cell-mediated immune responses, which are most likely to be responsible for protection against disease (Allen, G. P. et al, 1995; Mumford, J. A. et al, 1995).

Herpesviras glycoproteins are crucially involved in the early stages of infection, in the release of virions from cells, and in the direct cell-to-cell spread of virions by fusion of neighboring cells. To date, 11 herpes simplex virus type 1 (HSN-l)-encoded glycoproteins have been identified and have been designated gB, gC, gD, gE, gG, gH, gl, gJ, gK, gL, and gM. HSV-1 mutants lacking gC, gE, gG, gl, gJ, and gM are viable, indicating that these genes are dispensable for replication in cultured cells. Comparison of the HSN-1 and equine herpesviras 1 nucleotide sequences revealed that all of the known HSN-1 glycoprotems are conserved in EHN-1. According to the current nomenclature, these glycoproteins are designated by the names of their HSN-1 homologs. In addition, a further envelope protein named gpl/2 and a tegument protein, the VP13/14 homolog of HSV-1, have been described to be glycosylated in case of EHN-1 (reviewed in Osterrieder et al., 1998). It is known that EHN-1 gC, gE gl, and gM are not essential for growth

5 in cell culture, whereas gB and gD appear to be essential for virus growth in cultured cells. The contributions of other EHN-1 glycoproteins to replication in cultured cells are not known (Νeubauer et al, 1997; Flowers et al, 1992).

The gpl/2 glycoprotein is encoded by gene 71 (Wellington et al, 1996; Telford et al., 1992) and was also shown to be nonessential for virus growth in vitro (Sun et al., 1996). In addition, a viral o mutant carrying a lacZ insertion in the gene 71 open reading frame was apathogenic in a murine model of infection but still able to prevent against subsequent challenge infection (Sun et al., 1996; Marahall et al. 1997). In addition, the KyA strain of EHN-1 harbors a major deletion in the coding sequences of gene 71 (Colle et al., 1996).

s The technical problem underlying this invention was to provide a new tool and procedure to generate attenuated equine herpesviruses of defined specificity.

Summary of the invention

o The above-captioned technical problem is solved by the embodiments characterized in the claims and the description.

The invention relates to artificial chromosomes comprising the genome of EHN, methods of producing attenuated EHN, EHN obtainable with said methods and pharmaceutical compositions comprising said viruses. 5

FIGURE LEGENDS Figure 1:

Cloning strategy for introduction of mini F plasmid sequences into the RacH genome (A). PCR amplification of fragments bordering gene 71 located in the US region of the genome (B) was o done and the resulting BamΗI-Kpnl and Sa^' K-Sphl fragments were consecutively cloned into vector pTZ18R (C). Mini F plasmid sequences were released from recombinant plasmid pHA2 (Adler et al., 2000) with P d and cloned to give rise to recombinant plasmid p71-pHA2 (D). This plasmid was co-transfected with RacH DNA into RK13 cells and fluorescing virus progeny was selected. Viral DNA from green fluorescing virus progeny was used to transform Escherichia coli DH10B cells from which infectious RacH-BAC was isolated. Restriction enzyme sites and scales (in kbp) are given.

Figure 2:

Restriction enzyme digests of RacH and RacH-BAC. After separation by 0.8% agarose gel electrophoresis, fragments were transferred to a nylon membrane (Pharmacia-Amersham) and hybridized with a labelled pHA2 probe (see Fig. 1). Reactive fragments which are present due to insertion of mini F plasmid sequences are indicated by asterisks. Molecular weight marker is the 1 kb ladder (Gibco-BRL). The restriction enzymes used are indicated.

Figure 3:

Plaque sizes of RacH and RacH-BAC. Plaque sizes were determined on RK13 cells by measuring diameters of 150 plaques each. Plaque sizes of RacH were set to 100%, respectively, and plaque sizes of virus progeny reconstituted from BAC were compared to those of the parental virus. Standard deviations are given.

Figure 4:

Principle of the deletion of the genes encoding for gD (a) or gM (b) in RacH-BAC by replacing the open reading frames with the kanamycin resistance gene (kan^R) using E/T cloning. The kan^R gene was amplified by PCR using the primers listed in Table 1, and the amplicon was electroporated into DH10B cells containing RacH-BAC and plasmid pGETrec which expresses the enzymes necessary for E/T cloning after arabinose induction (Schumacher et al., 2000). Kanamycin-resistant colonies were picked, DNA was isolated and subjected to Southern blot analysis using a kan^R-specific probe. In both gD-negative RacH-BAC (c) and gM-negative RacH-BAC (d), fragments of the expected sizes (gD: 20.4 kbp; gM: 9.3 kbp specifically reacted with the kan^R probe.

Detailed description of the invention

Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of such viruses, reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and s disclosing the cell lines, vectors, and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. "EHV" or "EH- virus" as used herein relates to equine herpes virus.

10

The invention relates to an artificial chromosome vector characterized in that it comprises essentially the entire genome of an EHV strain from which infectious progeny can be reconstituted after transfection into a permissive cell. With the artificial chromosome vectors according to the present invention, safe EHV-vaccines

75 comprising EHV with defined attenuations can be generated. Such viruses are useful for the preparation of a safe live vaccine for use in the prevention and/or treatment of EHV infections (see infra). The invention provides the possibility for a fast and efficient manipulation of the EHV genome which remains fully infectious for eukaryotic cells or is modified into a replication- deficient virus. There was a long lasting need in the art for such a tool to handle and manipulate

20 the huge genome of EHV. Lastly, the EHV nucleic acid can be used as a polynucleotide vaccine which is applied either topically or systemically to naive or primed horses and may also be applied in utero.

The present invention is illustrated in example 1 showing the cloning of the entire genome of EHV-1 as an infectious mini F plasmid ('bacterial artificial chromosome', BAC) into

25 Escherichia coli. The generation of said BAC was not trivial and was posed many difficulties, including the preparation and extraction of sufficient amounts of circular DNA. The circularized form of recombinant viral DNA was needed to transform DH10B cells with the recombinant DNA in order to prepare the mini F plasmid-cloned EHN DΝA. To obtain sufficient amounts of circular viral DΝA, early viral transcription was blocked by the addition of 100 μg per ml of cycloheximide after infection of cells. Viral DΝA was then prepared and used for transformation of DH10B cells. Only from cells treated with cycloheximide was it possible to extract sufficient amounts of circular DΝA and to obtain DH10B clones containing the enitre RacH genome. „Essentially" means that the EHV genome is complete with the exception that it may carry a mutation as set out infra.

„Artificial chromosome" relates to any known artificial chromosomes, such as yeast, or preferably bacterial artificial chromosomes.

Preferably, a bacterial artificial chromosome (BAC) according to the invention is a vector used to clone large DNA fragments (100- to 300-kb insert size) in Escherichia coli cells which is based on naturally occurring F-factor plasmid found in the bacterium E. coli (Shizuya, H., B. Birren, U.J. Kim et al. 1992. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proceedings National Academy of Science 89: 8794-8797). The type of vector is preferably based on a F-plasmid replicon containing the origin of replication (oriS) and its own DNA polymerase (repE) as well as the genes parA and parB involved in maintaining its copy number at a level of one or two per E. coli. The antibiotic resistance marker is preferably Cm-resistance.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EETV is EHV-1.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHN is EHN-4.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHN strain is RacH.

According the invention, any type of mutation can be introduced into the EHV genome, in order to obtain a replication-deficient and/or attenuated EH-viras. Such mutations include, but are not limited to any mutation (e.g. deletion, insertion, substitution) relating to the glycoproteins gB, gC, gD, gE, gG, gl, gJ, gL and gM, gpl/2 and any combination thereof. Preferably, said mutations are deletion mutations, i.e. the respective glycoproteins such as e.g. gM are completely deleted.

Thus, the invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gB.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gC.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gD. The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gE.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gG.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gH.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gl.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gK.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gM.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the EHV strain is lacking the glycoprotein gpl/2.

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the artificial chromosome is a bacterial artificial chromosome (BAC). Said

BAC's can be propagated in any bacterium known to the skilled person, e.g and preferably

Escherichia coli T

The invention preferably relates to an artificial chromosome vector according to the invention, characterized in that the artificial chromosome is a yeast artificial chromosome (YAC).

The invention preferably relates to an artificial chromosome vector RacH-BAC according to the invention, characterized in that the artificial chromosome as deposited under the accession number ECACC 01032704 with the ECACC in Porton Down, UK (European Collection of Cell

Cultures, CAMR, Salisbury, Wiltshire SP4 0JG, UK).

Another important embodiment of the present invention is a polynucleotide vaccine encoding an an artificial chromosome vector or EHV contained therein according to the invention.

Yet another important embodiment of the present invention is the use of an artificial chromosome vector according to the invention for the generation of infectious EHV.

The invention furthermore relates to a method for the generation of an infectious EHV, characterized in that an artificial chromosome vector according to the invention is used to infect a suitable cell line and the shedded virus is collected and purified. The invention furthermore relates to a method for the generation of an attenuated EHV, characterized in that the EHV sequence contained in an artificial chromosome vector according to the invention is specifically modified by molecular biology techniques. Said modifications may be carried out by methods known in the art, e.g. site directed mutagenesis see e.g. Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Furthermore, the invention relates to a EHV obtainable by a method according to the invention. Another very important embodiment is a pharmaceutical composition comprising a polynucleotide according to the invention and optionally pharmaceutically acceptable carriers and/or excipients. Such a polynucleotide according to the invention may also be used in a pharmaceutical composition within the scope of this invention, e.g. for DNA vaccination. One example of a targeted system of administration, e.g. for polynucleotides according to the invention is a colloidal dispersion system. Colloidal dispersion systems comprise macromolecule complexes, nanocapsules, microspheres and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes or liposome formulations. Liposomes are the preferred colloidal system according to the invention. Liposomes are artificial membrane vesicles which are useful as carriers in vitro and in vivo. These formulations may carry a cationic, anionic or neutral charge. It has been shown that large unilamellar vesicles (LUV) ranging from 0.2-4.0 μm in size may enclose a major part of an aqueous buffer solution with large macromolecules. RNA, DNA and intact virions can be encapsulated in the aqueous phase inside and transported to the target in a biologically active form (Fraley R et al., 1981, Trends Biochem Sci 6, 77-80). In addition to mammalian cells, liposomes have also proved suitable for the targeted transporting of nucleotides into plant, yeast and bacterial cells. In order to be an efficient gene transfer carrier the following properties should be present: (1) the genes should be enclosed with high efficiency without reducing their biological activity; (2) there should be preferential and substantial binding to the target cell compared with non-target cells; (3) the aqueous phase of the vehicle should be transferred highly efficiently into the target cell cytoplasm; and (4) the genetic information should be expressed accurately and efficiently (Mannino RJ et al., 1988, BioTechniques 6, 682-690).

The composition of the liposomes usually consists of a combination of phospholipids, particularly high phase transition temperature phospholipids, e.g. combined with steroids such as cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of the liposomes depend on the pH, the ion concentration and the presence of divalent cations. The pharmaceutical composition according to the invention may also contain a vector according to the invention, e.g. a BAC vector comprising an EHN genome as described supra, as a naked

"gene expression vector". This means that the vector according to the invention is not associated with an adjuvant for targeted administration (e.g. liposomes, colloidal particles, etc.). A major advantage of naked DΝA vectors is the absence of any immune response caused by the vector itself.

The EHN nucleic acid can be used as a polynucleotide vaccine (see pharmaceutical composition, supra) which is applied either topically (e.g. intranasally) or systemically to naive or primed horses and may also be applied in utero.

Another very important embodiment is a pharmaceutical composition comprising an EHV according to the invention and pharmaceutically acceptable carriers and/or excipients.

A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption or form part of a slow release formulation of the EHV or the polynucleotide according to the invention. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also e.g. Remington's Pharmaceutical Sciences

(1990). 18th ed. Mack Publ., Easton). One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.

Furthermore, the invention relates to the use of a polynucleotide according to the invention in the manufacture of a vaccine for the prevention and/or treatment of EHV infections.

Furthermore, the invention relates to the use of an EHV according to the invention in the manufacture of a vaccine for the prevention and/or treatment of EHV infections.

Furthermore, the invention relates to the use of the BAC technology to establish a highly virulent and genetically well characterized EHV which can be used for immunization and challenge studies for use e.g. in vaccine potency studies.

Furthermore, the invention relates to the use of EHV BACs according to the invention to generate mutant BACs that are generated taking into account appearing genetic or antigenetic variants of EHV. This relates to one or more mutations present withing ,new variants' of EHV which can be easily introduced in the existing EHV BAC. The following example is intended to aid the understanding of the invention and should in no way be regarded as limiting the scope of the invention.

Example 1

Construction of an EHV-1 bacterial artificial chromosome

A genetically uniform population of RacH (256^th passage) was isolated. With RacH, passage 257, Rkl3 cells were infected and a mother pool was established. Viras of one additional passage on RK13 cells was used to infect RK13 cells, from which viral DNA was prepared. Ten micrograms (μg) of viral DNA were co-transfected with 10 μg of plasmid p71-pHA2 (Fig. 1) into RK13 cells. For construction of plasmid p71-pHA2, 2.0 and 2.4 kbp fragments on either side of the EHV-1 gene 71 (Fig. 1; Table 1) were amplified by polymerase chain reaction (PCR) using primers containing appropriate restriction enzyme sites (Table 1). Both fragments were subsequently cloned into pTZ18R (Pharmacia- Amersham) to obtain plasmid p71 (Fig. 1). A BAC vector (pHA2; Messerle et al., 1997) containing the Eco-gpt and GFP (green flourescent protein) genes under the control of the HCMV (human cytomegaloviras) immediate early promoter was released as a Pad fragment from plasmid pHA2 and inserted into the Pad sites of the 2.0 and 2.4 kbp fragment cloned in p71 (Fig. 1; Table 1). Viras progeny was harvested and individual plaques expressing the green fluorescent protein (GFP) were isolated and subjected to three rounds of plaque purification until viras progeny stained homogenously green under the fluorescent microscope (Seyboldt et al., 2000). Similarly, co-transfections of p71-pHA2 and DNA of EHV-1 strain Kentucky A (KyA) were performed and the recombinant viras was purified to homogeneity. Recombinant viras DNA was prepared (Schumacher et al., 2000) and electroporated into Escherichia coli strain DH10B (Messerle et al., 1997; Schumacher et al., 2000). Electrocompetent bacteria were prepared as described (Muyrers et al., 1999; Narayanan et al., 1999; Zhang et al., 1998) and electroporation was performed in 0.1 cm cuvettes at 1250 V, a resistance of 200 Ω, and a capacitance of 25 μF (Easyject electroporation system, Eurogenentec). Transformed bacteria were incubated in 1 ml of Luria-Bertani (LB) medium (28) supplemented with 0.4%) glucose for 1 hr at 37°C, and then plated on LB agar containing 30 μg/ml chloramphenicol. Single colonies were picked into liquid LB medium, and small scale preparations of BAC DNA were performed by alkaline lysis of Escherichia coli (Schumacher et al, 2000). Large scale preparation of BAC DNA was achieved by silica-based affinity chromatography using commercially available kits (Qiagen, Macherey & Nagel). From the chloramphenicol-resistant bacterial colonies, one colony each was chosen and named RacH-BAC which contained the EHV-1 RacH genome. RACH-BAC DNA was cleaved with restriction enzymes BamHI, EcόRI and HindZZZ and the restriction enzyme patterns were compared to those of parental viral DNA. (Schumacher et al., 2000). The calculated and expected changes in the banding pattern after insertion of the mini F plasmid into the gene 71 locus were observed in RacH-BAC. In contrast, no other differences in restriction enzyme patterns as compared to the parental viras were obvious (Fig. 2). After purification of RacH-BAC DNA using affinity chromatography, RK13 cells were transfected with 1 μg of recombinant DNA. At one day after transfection, foci of green fluorescent cells were visible which developed into plaques on the following days after infection (Fig. 3). From these results we concluded that the RacH strain of EHV-1 was cloned as an infectious full-length viral DNA in Escherichia coli. Deletion of gene 71 in RacH-BAC resulted in a less than 10% reduction in plaque size (Fig. 3).

Table 1:

Sequence

Primer Fragment or plasmid generated

Gen71 l.Fr. 5 '-GG4ggtaccTTTGCACAACTTTAGGATGAC-3 ' 2.0-kb flank for p71-pHA2 for

Gen71 l.Fr. 5 '-GA rggatccCrttaattaaGTAGACGCGGCTGTAGTAAC-3 ' 2.0-kb flank for p71-pHA2 rev

Gen71 2.Fr. 5'- C gtcgacCrttaattaaTCGGGGAACTACTCACACTC-3' 2.4-kb flank for p71-pHA2 for

Gen71 2.Fr. 5 '-CGΛgcatgcAGTTTTACGCGAAGGATATAC-3 ' 2.4-kb flank for p71-pHA2 rev

Kan950 for 5 '-GCCAGTGTTACAACCAATTAACC-3 ' Kan^r950 gene

Kan950 rev 5 '-CGATTTATTCAACAAAGCCACG-3 ' Kan^r950 gene gM950EHV S'-GGTTTCAAATTCCTCGCTCACCACGTCGTAAATTGGCTCT Kan^r950 gene for gM for TCTGCGTCCGGCCAGTGτTACAACCAAτTAAC-3 ' deletion gM950EHV S'-AAAACCACAGCGTGGTCGATGGAGTGTGGATGCGGCAG Kan^r950 gene for gM rev A TA GCTGGTGGACGATTTAlTCAACAAAGCCACG-'i ' deletion gD-950 for S'-CGCCCACTCAACTTCCAACTTCGCTTTAGTGGCTGCGACC Kan^r950 gene for gD

ACGCTAACAGCGATTTAmCAACAAAGCCACG-y deletion gD-950-1 rev y-TTCTTCCGACGCAAGCAGACGTATAGAATGACGCCCACC Kan^r950 gene for gD

AA TA TA (?GCCAGTGTTACAACAAATTAACC-3 ' deletion

Mutagenesis of EHV-1 BACs For mutagenesis of RacH-BAC DNA in Escherichia coli, recE- and recT-catalyzed reactions promoting homologous recombination between linear DNA fragments, also refened to as E/T cloning, was performed (Muyrers et al., 1999; Zhang et al., 1999). Plasmid pGETrec (kindly provided by Dr. Panos Ioannou, Murdoch Institute, Melbourne, Australia) harboring the recE, recT and bacteriophage λ gam gene (Narayanan et al., 1999) was transformed into RacH-BAC- containing DH10B cells. After induction of recE, recT and gam by addition of 0.2 % arabinose, electrocompetent cells were prepared essentially as described (Muyrers et al., 1999). To delete the gD and gM gene in RACH-BAC, the kanamycin resistance gene (kan^R) of plasmid pACYC177 (Stratagene) was amplified by PCR. The designed primers contained 50 nucleotide homology arms bordering the desired deletion within gD or gM and 20 nucleotides for amplification of kan^R (Table 1). The resulting 0.95 kbp fragment was purified from an agarose gel (Qiagen) and electroporated into pGETrec-containing RacH-BAC cells. Colonies harboring the cam^R and kan^R genes were identified on plates containing both antibiotics. H-BACΔgD and H-BACΔgM DNA were isolated from Escherichia coli by chromatography and subjected to restriction enzyme digestion and Southern blot analysis (Fig. 4) transfection studies were performed. Whereas RacH-BAC and H-BACΔgM were able to induce viral plaques on RK13 cells, H-BACΔgD was able to induce plaques on cells expressing gD in trans only. The gD cells transiently expressed EHV-1 gD after transfection of a recombinant plasmid in which gD is under control of the HCMV immediate early promoter/enhancer. These observations indicated that EHV-1 gD is essential for viras growth in vitro.

References

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Borst, E.M., G. Hahn, U.H. Koszinowski, and M. Messerle. 1999. Cloning of the human cytomegaloviras (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J. Virol. 73: 8320-8329

Flowers, C.C. and O'Callaghan, D J., 1992. The equine herpesviras type 1 (EHV-1) homolog of herpes simplex viras type 1 US9 and the nature of a major deletion wethin the unique short segment of the EHV-1 KyA strain genome. Virology 190, 307-315. Hubert, P.H., Birkenmaier, S., Rziha, HJ. and Ostemeder, N., 1996. Alterations in the equine herpesviras type-1 (EHV-1) strain RacH during attenuation. J. Vet. Med. B 43, 1-14.

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Mumford, J.A., Hannant, D.A., Jessett, D.M., O'Neill, T., Smith, K.C. and Ostlund, E.N., 1995. Abortigenic and neurological disease caused by experimental infection with liquid herpesviras- 1. In proceedings 7^th International Conference of Equine Infectious Disease"(H. Nakajima and W. Plowright, Eds.) pp. 261-175. R&W Publ., Newmarket, U.K. United Kingdom.

Muyrers, J.P., Y. Zhang, G. Testa, and A.F. Stewart. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27: 1555-1557.

Narayanan, K., R. Williamson, Y. Zhang, A.F. Stewart, and P . Ioannou. 1999. Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther. 6: 442-447.

Neubauer, A., Beer, M., Brandmϋller, C, Kaaden, O.-R. and Ostemeder, N., 1997. Equine herpesviras 1 mutants devoid of glycoprotein B or M are apathohenic for mice but induce protection against challenge infection. Virology 239, 36-45. Ostemeder, N., Neubauer, A., Brandmϋller, C, Braun, B., Kaaden, O.-R. and Baines, J.D., 1996. The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions. Journal of virology, June 1996, p. 4110-4115.

Ostemeder, N., Wagner, R., Brandmϋller, C, Schmidt, P., Wolf, H. and Kaaden, O.-R., 1995. Protection against EHV-1 challenge infection in the murine model after vaccination with various formulations of recombinant glycoprotein gpl4 (gB). Virology 208, 500-510.

Peeters, B., Biendowska-Szewcyk, K., Hulst, M., Giellens, A. and Kimman, T., 1997. Biologically safe, non-transmissible pseudorabies viras vector vaccine protects pigs against both Aujeszky's disease and classical swine fever. J. Gen. Virol. 78, 3311-3315.

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Sun Y, MacLean AR, Dargan D, Brown SM. Identification and characterization of the protein product of gene 71 in equine herpesvirus 1. J Gen Virol. 1994 Nov;75 ( Pt 11):3117-26. Telford, E.A.R., Watson, M.S., McBride, K. and Davison, A.J., 1992. The DNA sequence of equine herpes viras- 1. Virology 189, 304-316.

Tewari, D., Whalley, J.M., Love, D.N. and Field, H.J., 1994. Characterisation of immune responses to baculoviras expressed equine herpesvirus type 1 glycoproteins D and H in a murine model. J. Gen. Virol. 75, 1735-1741.

Wagner, M., S. Jonjic, U.H. Koszinowski, and M. Messerle. 1999. Systematic excision of vector sequences from the BAC-cloned herpesviras genome during virus reconstitution. J. Virol. 73:7056-7060.

Zhang, Y., F. Buchholz, J.P. Muyrers, and A.F. Stewart. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nature Genet. 20:123-128. APPENDIX 3

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BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS ₁ FOR THE PURPOSES OF PATENT PROCEDURE O

Dr N Ostemeder INTERNATIONAL FORM

Federal Researfch Centre for Virus Diseases of Animals

Institue of Molecular Biology

Bodden Buck 5A

Insel Riens

D-17498

Germany

NAME AND ADDRESS OF DEPOSITOR

Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired

FormBP/4 (solepage) 1991 APPENDIX 3

Page ₂4

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

VIABILITY STATEMENT

Issued pursant to Rule 10.2 by the for Virus Diseases of Animals INTERNATIONAL DEPOSITARY AUTHORITY

Institue of Molecular Biology identified on the following page

Bodden Blick 5A

Insel Riens

D-17498

Germany

NAME AND ADDRESS OF THE PARTY

TO WHOM THE VIABILITY OF STATEMENT

IS ISSUED

I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

Dr N Osterrieder me : Accession number given by the

Federal Research Centre INTERNATIONAL DEPOSITORY AUTHORITY: for Virus Diseases of Animals

Institue of Molecular Biology 01032704

Address: Bodden Blick 5A

Insel Riens Date of the deposit or of the transfer:

D-17498

27 March 2001

Germany

II. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested ^on 27 March 2001 On that date, the said microorganism was

X ³ viable

³ no longer viable

Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the most relevant date (date of the new deposit or date of the transfer) .

In the cases referred to in Rule 10.2 (a) (ii) and (iii), refer to the most recent viability test.

Mark with a cross the applicable box. Form BP/4 (first page) Appendix 3 Page 25

4 Fill in if the information has been requested and if the results of the test were negative. Form BP/9 (second and last page)

Claims

I . Bacterial artificial chromosome vector characterized in that it comprises essentially the entire genome of an EHN strain. s 2. Artificial chromosome vector according to claim 1 , characterized in that the EHN is EHN-1.

3. Artificial chromosome vector according to claim 1 or 2, characterized in that the EHN is EHN-4.

4. Artificial chromosome vector according to any one of claim 1 to 3, characterized in that the EHN strain is RacH. o 5. Artificial chromosome vector according to claim 4, chraterized in that it is the vector with the accession No. ECACC 01032704.

6. Artificial chromosome vector according to any one of claim 1 to 5, characterized in that the EHN strain is lacking the glycoprotein gB.

7. Artificial chromosome vector according to any one of claim 1 to 6, characterized in that the s EHN strain is lacking the glycoprotein gC.

8. Artificial chromosome vector according to any one of claim 1 to 7, characterized in that the EHN strain is lacking the glycoprotein gD.

9. Artificial chromosome vector according to any one of claim 1 to 8, characterized in that the EHN strain is lacking the glycoprotein gE. 10. Artificial chromosome vector according to any one of claim 1 to 9, characterized in that the EHN strain is lacking the glycoprotein gG.

I I . Artificial chromosome vector according to any one of claim 1 to 10, characterized in that the EHN strain is lacking the glycoprotein gH.

12. Artificial chromosome vector according to any one of claim 1 to 11, characterized in that the 5 EHV strain is lacking the glycoprotein gl.

13. Artificial chromosome vector according to any one of claim 1 to 12, characterized in that the EHN strain is lacking the glycoprotein gK.

14. Artificial chromosome vector according to any one of claim 1 to 13, characterized in that the EHN strain is lacking the glycoprotein gL. 0 15. Artificial chromosome vector according to any one of claim 1 to 14, characterized in that the EHN strain is lacking the glycoprotein gM.

16. Artificial chromosome vector according to any one of claim 1 to 15, characterized in that the EHV strain is lacking the glycoprotein gpl/2.

17. Artificial chromosome vector according to any one of claim 1 to 16, characterized in that the artificial chromosome as deposited under accession number Q4297 at the ECACC.

18. Polynucleotide encoding an an artificial chromosome vector or EHN contained therein according to any one of claims 1 to 17.

19. Use of an artificial chromosome vector or a polynucleotide according to any one of claims 1 to 18 for the generation of infectious EHN.

20. Method for the generation of replicating EHN, characterized in that an artificial chromosome vector according to any one of claims 1 to 19 is used to infect a suitable cell line and the shedded virus is collected and purified.

21. Method for the generation of an attenuated EHV, characterized in that the EHV sequence contained in an artificial chromosome vector according to any one of claims 1 to 17 is specifically modified by molecular biology techniques.

22. Method for the cloning and generation of an attenuated EHV, characterized in that the EHV sequence contained in an artificial chromosome vector according to any one of claims 1 to 17 is specifically modified by molecular biology techniques and contains a foreign sequence of another viral, bacterial or parasitic pathogen.

23. Method for the cloning and generation of a virulent EHV, characterized in that the EHV sequence contained in an artificial chromosome vector according to any one of claims 1 to 17 is specifically modified by molecular biology techniques.

24. EHV obtainable by a method according to any one of claims 20 to 23.

25. Pharmaceutical composition comprising a polynucleotide according to claim 18 and pharmaceutically acceptable carriers and/or excipients.

26. Pharmaceutical composition comprising an EHV according to claim 24 and pharmaceutically acceptable carriers and/or excipients.

27. Use of a polynucleotide according to claim 18 in the manufacture of a vaccine for the prevention and/or treatment of EHV infections.

28. Use of an EHV according to claim 24 in the manufacture of a vaccine for the prevention and/or treatment of EHN infections.