DE10317008A1 - New recombinant equine herpes (EHV) virus free of heterologous elements, and where protein gM has been deleted, useful as a vaccine for treating or preventing EHV infections - Google Patents

Abstract

A new recombinant Equine Herpes Virus (EHV) where the protein gM is absent, and the EHV is free of heterologous elements. Independent claims are also included for the following: (1) a nucleic acid coding for an EHV defined above; (2) a vaccine preparation comprising the EHV or nucleic acid; (3) obtaining a recombinant EHV; and (4) a cell line for use in the method, where the gene encoding the protein gM is transfected into the cell line, and the cell line expresses gM. ACTIVITY : Virucide. No biological data is given. MECHANISM OF ACTION : Vaccine.

Description

Territory of invention

The The present invention relates to the field of animal health and especially equine herpes viruses (EHV), in which the gene that makes up the protein GM encoded, absent and free of heterologous elements are. Further aspects of the invention relate to pharmaceutical compositions, comprising the viruses, their use and methods for prophylaxis and treatment of EHV infections. The invention also relates to pharmaceutical compositions containing the combination of viruses EHV-1 and EHV-4 contain, in which the gene encoding the protein gM, is absent and free of heterologous elements.

background the invention

Equine herpes virus 1 (EHV-1), a member of the Alphaherpesvirinae, is the main cause for the virus-induced Abortion in horses and causes respiratory and neurological disorders. Equine herpes virus 4 (EHV-4) may also have respiratory symptoms, abortions, or neurological disorders induce. The entire DNA sequence of both species (EHV-1: strain AB4p; EHV-4: strain NS80567) has been determined (Telford, E.A.R. et al., 1992; Telford, E.A.R. et al., 1998). Few genes and However, gene products are relevant to virulence in terms of their relevance and the immunogenic properties of EHV have been characterized.

Herpesvirus glycoproteins are critically involved in early stages of infection, in the release of virions from cells, and in the direct cell-to-cell spread of virions through the fusion of neighboring cells. To date, 11 herpes simplex virus type 1 (HSV-1) encoded glycoproteins have been identified and designated gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL and gM. HSV-1 mutants lacking gC, gE, gG, gI, gJ, and gM are viable, suggesting that these genes are unnecessary for replication in cultured cells. A comparison of the nucleotide sequences of HSV-1 and equine herpes virus 1 showed that all known HSV-1 glycoproteins are conserved in EHV-1. According to the current nomenclature, these glycoproteins are designated by the names of their HSV-1 homologues. It is known that EHV-1 gC, gE and gI are not essential for growth in cell cultures, while gB and gD are essential for virus growth in cultured cells. The contributions of other EHV-1 glycoproteins to replication in cultured cells are unknown (Flowers, CC, et al., 1992). Transcriptional and protein analyzes have shown that the glycoproteins gB, gC, gD, gG, gH and gK are expressed in EHV-1-infected cells. Glycoprotein gM (encoded by the UL10 gene [Baines, JD et al., 1991; Baines, JD et al., 1993]) is the only non-essential glycoprotein reported to be conserved in all herpes viral subfamilies, and it has been described for human and mouse cytomegalovirus and the Gammaherpesvirinae members EHV-2, herpes virus saimiri and Epstein-Barr virus. Like many herpes virus glycoproteins, HSV-1 gM is present in virions and membranes of infected cells. HSV-1 mutants lacking only gM grew to titer in cell culture systems that were approximately 10-fold reduced relative to wild type viruses and showed reduced virulence in a mouse model (Baines, JD et al., 1991; MacLean , CA et al., 1993} The EHV-1 gM homologue (gp21 / 22a; henceforth referred to as EHV-1 gM) was first described by Allen and Yeargan (Allen, GP et al., 1987), and It has been shown to be a major component of the virus envelope, and further studies have shown that gene 52, the gene homologous to HSV-1 UL10, encodes the 450 amino acid EHV-1 gM polypeptide (Pilling, A. et al., 1994 ; Telford, EAR et al., 1992). EHV-1 gM is a multiple hydrophobic protein that contains eight predicted transmembrane domains and has been reported to be present in infected cells and in purified virions as a protein of M _r 45,000 (Pilling, A. et al., 1994; Telford, EAR et al., 1992).

to fight EHV-1 infections follow two different approaches. First modified live vaccines (MLVs) have been developed under Influence of RacH strain (Mayr, A. et al., 1968; Hübert, P.H. et al., 1996), which is widely used in Europe and the United States States is used. Second, inactivated vaccines and subunit vaccines based on recombinant expressed viral glycoproteins, such as the glycoproteins (g) B, C, D and H, which provide partial protection against a subsequent one EHV-1 stress infection induced in a mouse model. Subunit vaccines, comprising the glycoproteins, e.g. gB, gC, gD and gH, only protect bad against re-infection (Awan et al., 1990; Osterrieder et al., 1995, Tewari et al., 1994; Stokes et al., 1996).

The The technical problem on which the invention is based was to provide improved vaccines that better protect against EHV infection than Vaccine from the prior art.

Figure legends

1 : Generation of a gM-negative EHV-1 RacH virus without foreign sequences (HΔgM-w).

This figure shows a map of viruses and plasmids used for the construction of HΔgM-w ver were applied. First generation HΔgM virus was previously constructed by inserting Escherichia coli IacZ (HΔgM-IacZ) or the green fluorescent protein (GFP) expression cassette (HΔgM-GFP). The BamHI map of the EHV-1 strain RacH is shown (A) and the BamHI-HindIII fragment containing the gM ORF is enlarged, showing the genomic organization of the region (B). The gM-negative virus HΔgM-GFP carries a GFP expression cassette which replaces the main part of the EHV-1 gM gene. The GFP-specific probe used in Southern blots is shown (C). Plasmid pBH3.1 carries the EHV-1 BamHI-HindIII fragment of interest and was used to construct plasmid pXuBaxA. After cotransfection of the DNA from HΔgM-GFP with plasmid pXuBaxA, HΔgM-w (D) resulted. Restriction sites: BamHI - B, HindIII - H, Sphl - S, HincII - Hc, Apal - A, Pstl - P.

2 , Southern blot of gM-deleted EHV-1 virus without foreign sequences (HΔgM-w).

DNA from RacH, HΔgM-GFP and HΔgM-w cut with BamHI, HindIII or Pstl and with a GFP-specific Probe (GFP) or the EHV-1 BamHI-HindIII fragment analyzed by pBH3.1 (pBH3.1). DNA hybrids were generated by chemiluminescence detected using CSPD. Molecular weight marker (Biolabs) are given in kbp on the left margin. The arrow points on a barely visible specific hybrid.

3 : Generation of a gM-negative EHV-4 virus without foreign sequences (E4ΔgM-w).

In this figure shows a BamHI map of EHV-4 strain NS80567. The enlarged BamHI-e fragment includes the gM ORF and neighboring ORFs (A). Plasmid constructs and Priming sites are shown (B). Plasmid pgM4GFP + was used for generation from E4ΔgM-GFP, GFP-positive and gM-negative EHV-4, used (B, C). recombination of DNA from E4ΔgM-GFP with plasmid pgM4R (B) containing 3,109 bp of EHV-4 sequences below Inclusion of the gM-ORF resulted in E4RgM, the gM-repaired EHV-4 (A), or with plasmid pgM4w (B) resulted in E4ΔgM-w, the GFP and gM negative EHV-4 (D). Restriction sites: BamHI - B, Pstl - P, EcoRI - E, Sall - Sa, MIuI - M, Asnl - As, EcoRV - EV.

4 : Southern blot of a gM-deleted EHV-4 virus without foreign sequences (E4ΔgM-w).

DNA from EHV-4, E4RgM, E4ΔgM-w and E4ΔgM-GFP was cut with PstI, EcoRV or HindIII as indicated and DNA fragments were transferred to nylon membranes. Parallel membranes were hybridized either with GFP-specific sequences or with a probe called gM3.1, which contained EHV-4-specific sequences, which were taken from the plasmid pgM4R ( 3 ). DNA hybrids were detected by chemiluminescence using CSPD. Molecular weight marker sizes (Biolabs) are given in kbp.

5 : Growth characteristics of the gM-deleted EHV-4 virus E4ΔgM-w.

cell were at a MOI value of 2 with different viruses, as in Box indicated, infected. Kinetics of virus growth are as Virus titer, determined in supernatants from infected cells (extracellular Activity) or within infected cells (intracellular activity), relative to the specified time, designated. The mean values of two individual experiments are shown; Standard deviations are indicated as error bars.

6 : Plaque sizes of E4ΔgM-w.

Vero or Vero-gM cells in six well plates were mixed with 50 PFU infected on either EHV-4, E4RgM, E4ΔgM-GFP or E4ΔgM-w. Maximum diameters of 150 corresponding plaques were determined and the mean plaque size is in Percent, relative to the size of the wild-type plaques, which was set to 100%. Standard deviations are indicated as error bars (A). Plaques were detected by indirect immunofluorescence (anti-gD Mab 20C4, 1: 1000) on day 4 p.i. stained and in an axioscope (x100, Zeiss, Germany). Images were captured and digitally processed (B).

7 : EHV-4 virus penetration in Vero cells

penetration of EHV-4, E4RgM, E4DgM-w and E4DgM-GFP, produced on non-complementary Vero cells (A) or complementing Vero-gM cells (B), in Vero cells. At specified times, the penetration efficiency was considered to be the Percentage of number of plaques present after citrate treatment relative to that of the plaques that were present after control treatment specified. Average values from two independent experiments are given. Standard rejections are called error bars.

8th : PCR primer sequences and arrangement of amplificates within the EHV-4 genome.

Sequences that represent restriction enzyme recognition sites are printed in bold and the corresponding enzymes are listed. PCR products were inserted into the given vectors, resulting in the plasmids pCgM4, pgM4R, pgM4Del1 or pgM4Del2 as indicated. The arrangement of a fragment within the EHV-4 genome is relative to the sequence described by Telford et al. (1998).

9 : Western blot analysis of horse sera.

lysates of EHV-1 gM expressing cell lines ccgM and of Rk13 cells were either for 2nd min or to 56 ° C (1,2) or 5 min at 95 ° C (3) warmed (GM is highly hydrophobic, and is known to be useful when cooking aggregated so that it no longer enters the separating gel). Identical blots were made with different horse sera (1: 3000) or the anti-gM rabbit serum. Arrows indicate GM-specific Reactivity in cooked and uncooked samples. Neutralizing test titers (NT) are for Sera indicated by a virological diagnostic unit were obtained.

Presentation of the invention

Definitions of expressions that used in the description:

In front the embodiments The present invention should be noted that, as here and in the attached claims uses the singular forms "a / an" and "the / the" plural references unless the context clearly dictates otherwise. So close e.g. the reference to "a EHV virus "a A plurality of such EHV viruses, including all subspecies, like EHV1, 4 and others; the reference to the "cell" is a reference to one or more Cells and equivalents of those known to those skilled in the art, etc. Unless otherwise defined, have all the technical and scientific terms that used here have the same meanings as they are commonly used understood by those skilled in the art to which the invention belongs become. Although any methods and materials are similar or equivalent to those described here for executing or testing the present Invention can be used become the preferred methods, devices and materials now described. All publications, which are mentioned here are by reference for the purpose of description and disclosure of cell lines, vectors and methods as described in the Publications are reported in connection with the present invention can be used made the subject of the present application. There is nothing in it to be interpreted as admitting that the invention is not justified is, due to earlier To date the invention prior to such disclosure.

The Expression "EHV" as used here refers to all equine herpes viruses, such as the EHV-1 species and EHV-4, within the Alphaherpesvirinae family. The expressions EH virus, EH Virus, EHV Virus, EHV Virus and EHV all relate to equine herpes viruses.

Virulence: "Virulence" as used here refers to an EH virus that is used for propagation in target host species (i.e. horses) with the potential to induce subclinical and clinical disease, characterized by respiratory symptoms, abortions or neurological disorders, is able. examples for Virulent EHV are wild-type viruses that have strong clinical symptoms induce. examples for Virulence factors are hemolysins (which lyse red blood cells) and adhesins (proteins by which the Pathogens adhere to the host and initiate colonization or invasion can). More specifically, "virulence" here means that the gM gene product, a major part of the virus envelope, allows the virus to penetrate the host and spread to the cell is involved.

Attenuation: "A weakened EH virus" as used here refers to an infectious EHV that is not an EHV-associated subclinical or clinical disease causes. Such weakened EH viruses are in particular according to the invention EHV that can replicate and do not express gM.

A "functional variant" of the EH virus according to the invention is EHV virus that has a biological activity (either functional or structurally), which is essentially similar to the EHV according to the invention is. The expression “functional variant” also includes “a fragment”, “a functional one Variant "," based on a variant on the degenerative nucleic acid code "or" a chemical derivative ". Such a" functional variant "can, for example one or more nucleic acid exchanges, Wear deletions or insertions. These exchanges, deletions or insertions can Make up 10% of the total sequence. The functional variant at least keeps partially their biological activity, e.g. the function as a infectious clone or a vaccine strain, or even shows improved biological Activity.

A "variant based on the degenerative nature of the genetic code "is a variant that results from the fact that a certain amino acid of several different nucleotide triplets can be encoded. This Variant retains at least partly their biological activity or even shows improved biological activity.

A "fusion molecule" can be a DNA molecule or infectious EHV virus according to the Erfin fusion, for example, to a reporter, such as a radiolabel, a chemical molecule, such as a fluorescent label, or any other molecule known in the art.

How used here is a "chemical Derivative "accordingly the invention a DNA molecule or an infectious EHV clone according to the invention, chemically modified or containing additional chemical components that are not normally part of the molecule. Such shares can the solubility Improve absorption, biological half-life and the like of the molecule.

On molecule is "essentially similar" to another Molecule, if both molecules essentially similar Have nucleotide sequences or biological activity. So if two molecules a similar activity then they are considered variants because of this expression is used here if the nucleotide sequence is not identical, and two molecules, which has a similar nucleotide sequence are considered variants because this expression here is used even if its biological activity is not is identical.

The Expression "Vaccin" refers to how used here on a pharmaceutical composition that at least an immunologically active component that has an immunological response induced in an animal, and possibly, however, not necessary one or more additional components that the immunological activity reinforce the active component, includes. A vaccine can be added other components for pharmaceutical compositions are typical. The immunologically active component of a vaccine can either be whole virus particles in original form or as weakened particles in a so-called modified live vaccine (MLV) or particles that are suitable for Processes were inactivated in a so-called killed vaccine (kV) include. In another form, the immunologically active Component of a vaccine include suitable elements of the organism (Subunit vaccine), these elements being generated by destruction of whole particles or breeding cultures containing such particles, and optionally subsequent Cleaning levels that the desired Structures), or by synthetic methods including inclusion appropriate manipulation by using a suitable system e.g. based on bacteria, insects, mammals or other species plus if necessary subsequent Isolation and cleaning processes or by induction of the synthetic Processes in the animal that a vaccine needs through direct ingestion of the genetic material using appropriate pharmaceutical Compositions (PolyNucleotide Vaccination). A vaccine can one or more than one of those described above Include items.

The Expression "Vaccin" as understood here is a vaccine for veterinary use, comprehensive antigenic substances, and is administered for the purpose of inducing one specific and active immunity against a disease caused by EHV. The EHV vaccine according to the invention imparts active immunity which is passive over antibodies of the Mother against the immunogens it contains, and occasionally against antigen related organisms can be.

additional Components to reinforce the Immune responses are components commonly called adjuvants be referred to, e.g. Aluminum hydroxide, mineral or other oils or auxiliary molecules that added to the vaccine or from the body after the corresponding induction by such additional Components are generated, such as, but not limited to, interferons, Interleukins or growth factors.

A "vaccine composition or pharmaceutical composition "consists essentially of an or several components that are used to modify physiological, e.g. immunological functions, the organism to which it is administered or from organisms that live in or on the organism, are suitable. The term includes, but is not limited to, antibiotics and antiparasitics, as well as other ingredients commonly used used to achieve certain other purposes, such as, however without limitation, Processing characteristics, sterility, Stability, simplicity administration of the composition on enteral or parenteral, like oral, intravenous, intranasal, intramuscular, subcutaneous, intradermal or other suitable route, tolerance after administration, controlled release properties.

presentation the invention

The solution The technical problem mentioned above is explained by the description and the embodiments the in the claims are characterized.

The invention overcomes the difficulties and the prejudice in this area that an equine herpes virus cannot be generated free from foreign sequences. Using the methods of the invention, EH viruses of superior quality are provided for use in vaccines. The central coding sequence for the Protein gM is eliminated in such a way that the remaining gM-carboxy-terminal sequences are in a different reading frame than the amino-terminal sequences. The adjacent gene for the UL9 essential protein homolog (gene 53), its orientation and overlap with the gene encoding the gM protein, requires that a minimal nucleotide sequence for the gM gene be left to express the gene 53 to allow and thus to achieve viability of the virus. An EHV according to the invention therefore relates to EHVs which are characterized in that the gene which codes for the protein gM is deleted in such a way that the expression of the gene which codes for the UL9 homologue (gene 53) is not is influenced. The term "unaffected" does not refer to certain quantitative or qualitative properties of UL9, but simply means that the expression of the gene is not affected as long as the protein is expressed by the virus and in an essentially sufficient amount for the viability of the Virus is present.

The long-standing need in this field for a vaccine, the recombinant Equine herpes virus 4 was satisfied by the inventors, overcome the major difficulties in this area. The viruses according to the invention EHV-1 and EHV-4 can advantageously e.g. For to be used in a vaccine.

A first important embodiment the invention therefore relates to an equine herpes virus (EHV), in which the gene encoding the protein gM, and thus gM itself, is absent is characterized in that it is free of heterologous elements is. "Free of heterologous Elements "means that no foreign sequence, i.e. no non-EHV sequence like one IacZ or GFP-coding cassette, in the coding sequence for the virus according to the invention is present (a so-called "white clone"). The EHV according to the invention so completely encoded by EHV sequences. The EHV according to the invention is free from bacterial elements or nucleic acids, that encode such bacterial elements. Furthermore, almost that entire coding sequence for the gM protein and thus the encoded gM protein mentioned above eliminated. The EHV according to the invention is therefore preferably characterized in that that the protein gM due to a deletion of the gene responsible for the protein GM encoded, is absent. As stated above, "the gene that the Protein gM encodes but is absent " also that a minimal gM sequence remains, so that at least the overlapping Gene 53 sequence yet is present, the remaining gM sequences being deleted can (see below). This can be achieved through molecular biological techniques (see below) so that the recombinant EHV is generated.

The Use of IacZ as a brand for the successful deletion of the gM gene of EHV-1 or 4 not for the successful generation of viruses according to the invention (cf. Examples 1, 2). The inventors have therefore developed a method according to the invention in order to to get the virus. An EH virus was constructed in which the gM gene by insertion a cassette containing the GFP marker was deleted. This approach allowed surprisingly the distinction between parent virus (green fluorescent plaques) and new recombinant plaques (non-fluorescent plaques).

• a) Isolierung eines Wildtyp-EHV;
• b) Etablierung eines Plasmids, das für das EHV-gM-Gen kodiert, gegebenenfalls mit flankierenden Sequenzen;
• c) Erzeugung einer komplementierenden Zelllinie, die gM oder Teile davon exprimiert;
• d) Etablierung eines EH-Virus, das ein GFP-kodierendes Kassetten-Insert in seiner gM kodierender Sequenz trägt, durch Cotransfektion der komplementierenden Zelllinie von Stufe b) mit EHV-Nucleinsäure und einem Plasmid, das gM kodiert, das durch ein GFP-kodierendes Kassetten-Insert unterbrochen ist;
• e) Deletion der GFP-kodierenden Kassette; und
• f) Selektion auf die EHV-Klone, bei denen die GFP-kodierende Kassette erfolgreich deletiert ist.

An EHV which is obtainable by a process which comprises the following stages is preferred:

• a) isolation of a wild-type EHV;
• b) establishment of a plasmid which codes for the EHV gM gene, optionally with flanking sequences;
• c) creating a complementing cell line expressing gM or parts thereof;
• d) Establishing an EH virus carrying a GFP-coding cassette insert in its gM coding sequence by cotransfection of the complementing cell line from step b) with EHV nucleic acid and a plasmid coding for gM encoded by a GFP coding Cassette insert is interrupted;
• e) deletion of the GFP-encoding cassette; and
• f) Selection for the EHV clones in which the GFP-coding cassette has been successfully deleted.

"IacZ" is the specialist than the gene, the β-galactosidase encoded, known. According to the invention "GFP" on green fluorescent Protein (GFP) produced by a bioluminescent jellyfish (Chalfie et al., 1994).

"Complementing Cell line " on a cell line that contains a gene that is normally in that Cell line genome does not exist, is introduced, being constitutive is expressed. Suitable cell lines include, but are not limited to, Rabbit kidney cell line Rk13, cell line cc (Seyboldt et al., 2000) and the Vero cell lines (ATCC Catalog # CRL-1586), such as clone 1008, as also disclosed in Examples 1 and 2, as well as any other cell lines, that are known to the person skilled in the art. Usually it can be selected on cell clones that have this additional Express protein. This cell line expresses the gene found in the virus is deleted, this deficiency is complemented, and enables the growth of the virus after gene deletion.

Standard molecular biology methods using restriction enzymes, ligation, PCR, transfection and the like are known in this field (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Preferably is such an EHV according to the invention characterized in that it is EHV-1. More preferred is the EHV-1 according to the invention characterized in that 850 to 1,100 bp of the 1,332 bp open reading framework for gM are deleted. Even stronger the EHV-4 according to the invention is preferred characterized in that 900 to 1,000 bp of the open reading frame for gM are deleted. Also stronger preferred is the EHV-1 according to the invention characterized that 960 to 970 bp of the open reading frame for gM is deleted (960, 961, 962, 963, 964, 965, 966, 967, 968, 969 or 970 bp). The strongest the EHV-1 according to the invention is preferred characterized by deleting 962 bp of the open reading frame for gM are.

More preferred is the EHV-1 according to the invention characterized in that the coding sequence for gM with Exception of 150 to 200 base pairs (bp) of the coding sequence, encoding the C-terminal portion of gM, and 150 to 250 bp the coding sequence encoding the N-terminal portion of gM, are deleted. According to this more preferred embodiment the coding sequence of gM is deleted; nucleotides 93118-93267 only to 93118-93317 of the sequence that the C-terminal portion of gM encoded, and nucleotides 94223-94472 to 94323-94472 of the coding sequence that the N-terminal portion encoded by GM, remain. Stronger an EHV-1 according to the invention is preferred, which is characterized in that Nucleotides 93268-93318 to 94222-94322 (encoding the core section from gM) are deleted (numbering according to Telford, 1992). Within the specified ranges can be any number of nucleotides be deleted. According to the Deletions at a position not lower than the nucleotide position 93268 must start however, start at position 93318. The deletion can already Item 94222, but not later as position 94322. A preferred EHV-1 according to the invention is characterized in that the nucleotides 93268 to 94322 the gM coding sequence corresponding to the Telford positions (1992) are deleted. Any combinations are within the scope the invention, such as 93272 to 94312, 93300 to 94300, etc.

Yet stronger the EHV-1 according to the invention is preferred characterized in that the coding sequence for gM with Exception from 160 to 190 bp of the coding sequence encoding the C-terminal Section of gM, and 190 to 220 bp of the coding sequence, coding the N-terminal portion of gM is deleted. According to this more preferred embodiment the coding sequence of gM is deleted; nucleotides 93118-93277 only to 93118-93307 of the sequence that the C-terminal portion in gM encodes, and nucleotides for 94253-94472 to 94273-94472 of the coding sequence which is the N-terminal Section encoded by GM remains. A is therefore more preferred EHV-1 according to the invention, which is characterized in that nucleotides 93278-93308 to 94252-94282 (coding the core section of gM) have been deleted (Telford numbering, 1992).

Likewise stronger the EHV-1 according to the invention is preferred characterized in that the coding sequence deletes for gM with the exception of 180 to 190 (180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190) bp of the coding sequence which is the C-terminal Section of the gM coding sequence, and 200 to 210 (200, 201, 202, 203, 204, 205, 206, 207, 208, 209 or 210) bp of the coding Sequence encoding the N-terminal portion of gM. According to this stronger preferred embodiment the coding sequence of gM is deleted; nucleotides 93118-93297 only to 93118-93307 (93297, 93298, 93299, 93300, 93301, 93302, 93303, 93304, 93305, 93306, 93307) of the sequence comprising the C-terminal section encoded by gM, and nucleotides 94263-94472 to 94273-94472 (94263, 94264, 94265, 94266, 94267, 94268, 94269, 94270, 94271, 94272, 94273) the coding sequence encoding the N-terminal portion of gM, remain. Stronger an EHV-1 according to the invention is preferred, which is characterized in that that nucleotides 94298-94308 to 94262-94272 (encoding the core section from gM) are deleted (numbering according to Telford, 1992). This means, that any nucleotides remain within the above Nucleotides deleted according to the invention can be e.g. Nucleotides 94299-94263 or 94299-94264 or 94300-94272 or any combination of these.

Most preferably, the EHV-1 according to the invention is characterized in that the coding sequence for gM with the exception of 184 bp of the coding sequence coding for the C-terminal section of the gM coding sequence and 209 bp of the coding sequence coding for the N-terminal Section of gM that is deleted. According to this most preferred embodiment, the coding sequence of gM is deleted; only nucleotides 93118-93301 of the sequence encoding the C-terminal portion of gM and nucleotides 94264-94472 of the coding sequence encoding the N-terminal portion of gM remain. Most preferred is therefore an EHV-1 according to the invention, which is characterized in that nucleotides 94263 to 93302 (encoding the nucleus section of GM) are deleted (numbering according to Telford, 1992). In accordance with this most preferred embodiment, 962 nucleotides of the sequence encoding gM are deleted. This is exemplified in a non-limiting manner in Example 1.

Likewise stronger an EHV-1 is preferred, which is characterized in that gM is deleted and that it is free of heterologous elements and that it is a recombinant variant based on a strain acts, selected from the group AB69 (ATCC VR2581), EHV-1 Ts mutant ECACC V99061001, KyA, KyD, Ab1, Ab4, RacH, RacL11 or RacM of EHV-1, and that none heterologous elements such as GFP or lac elements are present. Also stronger an EHV-1 according to the invention is preferred characterized in that gM is deleted and that it is free of heterologous elements, such as GFP or lac-Z elements, and that it is a recombinant variant based on RacH strain is about EHV-1. The strongest an EHV-1 according to the invention is preferred characterized in that gM is deleted and that it is free of heterologous elements, such as GFP or IacZ elements, is and that it is the RacH-based recombinant variant isolation HΔgM-w, such as disclosed in Example 1. Said EHV-1 according to the invention HΔgM-w is at the "Center for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures (ECACC) ", Salisbury, Wiltshire SP4 0JG, UK Budapest contract deposited. The filing date was 10/16/2002, the provisional Designation H-delta-gM-w, and the deposit number 02101663. Also preferred are EHV-1, which all have characterizing properties of said deposited EHV-1.

All of the aforementioned EHV-1 have superior properties over viruses with heterologous elements such as GFP. The EHV-1 according to the invention has an advantageously higher extracellular infectivity than that which still comprises heterologous elements. This is exemplified in 5 illustrated (e.g. between 4 and 12 hours).

To the present invention was made, no one was on this Able to produce a recombinant EHV-4 virus that can be used as a vaccine. EHV-1 and EHV-4 are homologous and cross-reacting to a certain extent. However, there was one long-standing need in this area for weakened EHV-4 viruses, since EHV-1 is obviously not for provides adequate protection against EHV-4 infection. On EHV according to the invention is therefore preferably characterized in that it is an EHV-4 is. Stronger the EHV-4 according to the invention is preferred characterized by 900 to 1150 bp of the open reading frame for gM deleted from 1332 bp. The EHV-4 according to the invention is even more preferred characterized in that 1000 to 1150 bp of the open reading frame for gM are deleted. Also stronger the EHV-1 according to the invention is preferred characterized in that 1110 to 1115 bp of the open reading frame for gM are deleted (1110, 1111, 1112, 1113, 1114 or 1115 bp). Most preferred is the EHV-1 according to the invention characterized in that 1110 bp deletes the open reading frame for gM are.

More preferred is the EHV-4 according to the invention characterized in that the coding sequence for gM with Exception of 0 to 50 base pairs (bp) of the coding sequence, which encodes the C-terminal portion of gM, and 150 to 250 bp of coding sequence encoding the N-terminal portion of gM, is deleted. According to this more preferred embodiment the coding sequence of gM is deleted; only nucleotides 92681-92680 through 92681-92730 of the sequence that the C-terminal portion of gM encoded, and nucleotides 93766-94033 to 93866-94033 of the coding sequence that includes the N-terminal portion encoded by GM, remain. Stronger an EHV-4 according to the invention is preferred, which is characterized in that Nucleotides 92681-92731 to 93765-93865 (encoding the core section from gM) are deleted (numbering according to Telford, 1998). Within any number of nucleotides can be present in the given ranges be deleted. According to the So deletions are not at a position lower than the nucleotide position 92681 must start however, start at position 92731. The deletion can already End position 93765, but not later than position 93865. Preferably is an EHV-4 according to the invention thus characterized in that nucleotides 92681 to 93865 of gM coding sequence according to the Telford positions (1998) deleted are. Any combination is within the scope of the invention, such as 92672 to 93801, 92700 to 93800 and the like.

Even more preferably, the EHV-4 according to the invention is characterized in that the coding sequence for gM with the exception of 10 to 40 bp of the coding sequence, coding for the C-terminal portion of gM, and 190 to 220 bp of the coding sequence, coding for the N -terminal section of gM, is deleted. According to this more preferred embodiment, the coding sequence is deleted from gM; only nucleotides 92681-92690 to 92681-92720 of the sequence encoding the C-terminal portion of gM, and nucleotides from 93806-94033 to 93836-94033 of the coding sequence encoding the N-terminal portion of gM remain. An EHV-4 according to the invention, which is characterized thereby, is more preferred is that nucleotides 92691-92721 to 93805-93835 (encoding the core section of gM) are deleted (numbering according to Telford, 1998).

Likewise stronger the EHV-4 according to the invention is preferred characterized in that the coding sequence deletes for gM is with the exception of 30 to 40 (30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) bp of the coding sequence, coding the C-terminal Section of the gM coding sequence, and 200 to 210 (200, 201, 202, 203, 204, 205, 206, 207, 208, 209 or 210) bp of the coding Sequence encoding the N-terminal portion of gM. According to this stronger preferred embodiment the coding sequence of gM is deleted; only nucleotides 92681-92710 to 92681-92720 (92710, 92711, 92712, 92713, 92714, 92715, 92716, 92717, 92718, 92719, 92720) of the sequence encoding the C-terminal section from gM, and nucleotides 93816-94033 to 93826-94033 (93824, 93825, 93826, 93827, 93828, 93829, 93830, 93831, 93832, 93833, 93834) of the coding Sequence encoding the N-terminal portion of gM remain. Stronger an EHV-4 according to the invention is preferred, which is characterized in that is that nucleotides 92711-92721 to 93823-93833 (encoding the Core section of gM) are deleted (Telford numbering, 1998). This means that any nucleotides within the above the remaining nucleotides mentioned can be deleted according to the invention, e.g. Nucleotides 94299-94257 or 94299-94256 or 94300-94257 or any Combinations of these.

At the most the EHV-4 according to the invention is preferred characterized in that the coding sequence for gM with with the exception of 34 bp of the coding sequence encoding the C-terminal Section of the gM coding sequence, and 209 bp of the coding Sequence encoding the N-terminal portion of gM deleted is. According to this the strongest preferred embodiment the coding sequence of gM is deleted; only nucleotides 92681-92714 the sequence encoding the C-terminal portion of gM and nucleotides 93825-94033 of the coding sequence encoding the N-terminal Section of GM, remain. So most preferred is one EHV-4 according to the invention, which is characterized in that nucleotides 92715-93824 (coding the core section of gM) have been deleted (Telford numbering, 1998). According to this the strongest preferred embodiment 1110 nucleotides of the gM coding sequence have been deleted. This is exemplified in a non-limiting manner in Example 2 illustrated.

Likewise stronger an EHV-4 according to the invention is preferred, which is characterized in that gM is deleted and that it is free of heterologous elements, such as GFP or IacZ elements, and that it is a recombinant variant, based on the MSV strain Lot 071398 from EHV-4, is. The strongest an EHV-4 according to the invention is preferred characterized in that gM is deleted and that it is free of heterologous elements, such as GFP or IacZ elements, is and that it on strain MSV Lot 071398 and isolation E4ΔgM-4 as disclosed in Example 2, based. Said EHV-4 according to the invention is at the “Center for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures (ECACC) ", Salisbury, Wiltshire SP4 0JG, UK Budapest contract deposited. The filing date was January 14, 2003, the preliminary Designation EHV-4, and the deposit number 03011401. Also preferred are EHV-4, which have all the characterizing properties of said deposited EHV-1 have.

All of the aforementioned EHV-4 have superior properties to viruses known in the prior art, since no recombinant EHV-4 are available in the prior art. Furthermore, the EHV-4 according to the invention has an advantageously higher extracellular infectivity than those which still comprise heterologous elements, such as GFP. This is exemplified in 10 illustrated (e.g. after 24 hours).

On Another important element of the invention is a nucleic acid which for a EHV encoded as disclosed above. The expert can easily corresponding sequence using standard molecular biological methods, who are known in this field.

There was a particular difficulty in this area in obtaining the EHV of the invention. The inventors constructed gM-negative EHV viruses by introducing a marker gene (IacZ) into the gM gene. When attempting to remove this cassette, in both EHV-1 and EHV-4 mutants generated by IacZ insertion, all clones that were phenotypically IacZ negative still contained the lacZ cassette. The inventors therefore developed an inventive method to obtain the viruses. An EH virus was constructed in which the gM gene was deleted by inserting a cassette containing the GFP marker. This approach surprisingly allowed a distinction to be made between parent virus (green fluorescent plaques) and new recombinant plaques (non-fluorescent plaques). In addition, a Vero cell line (based on Vero cell clone 1008) that constitutively expresses EHV4-gM was created by the inventors to overcome the difficulties in this field. The cell line was determined by transfection of the corresponding gM gene and subsequent selection for Vero cells expressing gM generated. Only these cells enabled the inventors to replicate EHV4 gM negative viruses. Said gM-complementing Vero cell line is deposited with the Center for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire SP4 0JG, UK, as a patent deposit under the Budapest Treaty. The filing date was the January 28, 2003, the provisional name VERO GM, and the accession number 03012801. Also preferred are cell lines which have all the characterizing properties of the said stored VERO GM cell line.

• a) Isolierung eines Wildtyp-EHV;
• b) Etablierung eines Plasmids, das das EHV gM-Gen kodiert, gegebenenfalls mit flankierenden Sequenzen;
• c) Erzeugung einer komplementierenden Zelllinie, die gM oder Teile davon exprimiert;
• d) Etablierung eines EH-Virus, das ein GFP-kodierendes Kassetten-Insert in seiner gM kodierenden Sequenz trägt, durch Cotransfektion der komplementierenden Zelllinie von Stufe b) mit der EHV-Nucleinsäure und einem Plasmid, das gM kodiert, das durch ein GFP-kodierendes Kassetten-Insert unterbrochen ist;
• e) Deletion der GPF-kodierenden Kassette; und
• f) Selektion auf die EHV-Klone, bei denen die GFP-kodierende Kassette erfolgreich deletiert ist.

Preferred is a method for obtaining a recombinant EHV, which comprises the following stages:

• a) isolation of a wild-type EHV;
• b) establishment of a plasmid which encodes the EHV gM gene, optionally with flanking sequences;
• c) creating a complementing cell line expressing gM or parts thereof;
• d) Establishing an EH virus carrying a GFP-coding cassette insert in its gM coding sequence by cotransfecting the complementing cell line from step b) with the EHV nucleic acid and a plasmid which codes for gM by a GFP- coding cassette insert is interrupted;
• e) deletion of the GPF-encoding cassette; and
• f) Selection for the EHV clones in which the GFP-coding cassette has been successfully deleted.

The cells referred to above are an important embodiment of the present invention. The invention therefore relates to a cell line for the use in a method according to the invention, thereby is characterized in that the gene encoding the protein gM in the cell line is transfected and that the cell line expresses gM. The The invention preferably relates to a cell line according to the invention, characterized in that it is a cell line, those from the group Vero cells (Vero-gM cells), RK-13 and cc (cc-gM) selected is.

How above for EHV-1 disclosed the Use of IacZ as a marker instead of GFP also in EHV-4 not for the successful generation of viruses according to the invention (cf. in non-limiting Wise example 2). "LacZ positive" cells became common stained less intensely with Vero cells than with Rk13 cells and were therefore more difficult to identify and the EHV-4 system showed replication slower than EHV-1, resulting in less time between plaque identification and isolation of viable Progeny virus. The use of GFP was therefore the only one Way to get the EHV-4 virus. The procedure was carried out as above for EHV-1 described, and surprisingly performed also for the successful identification of EHV-4 gM-deleted virus based on the identification of fluorescent plaques.

The Wild-type EHV is isolated by extracting lung tissue in the case of necropsy of suspected animals with EHV to be, and isolation of EHV from tissue cells, like on this one Area known. The complete one EHV 1 genome sequence is from Telford et al. (1992) Service. The complete genome sequence for EHV-4 is corresponding by Telford et al. (1998) Service. PCR amplification of DNA sequences by use of specific primers attached to complementary strands of target DNA, the DNA sections flanking, binding, of interest represents a molecular biological Standard procedures are. Procedures for ligation of suitable DNA sequences in plasmids for the intended constructions are suitable for DNA transfection into eukaryotic Cells, for Southern blot and western blot analyzes, for directional cutting of DNA fragments over Restriction enzymes and for the selection of cell lines, the desired heterologous genes or Plasmids that the desired Have a gene or virus in which a particular gene has been deleted, are known to the person skilled in the art. Standard molecular biological methods, like the techniques mentioned above, are known to those skilled in the art and can be found e.g. also in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and Bertram, S. and Gassen, H.G .: Genetic engineering methods, G. Fischer Verlag, Stuttgart, New York, 1,991th

"Deletion" means removal one or more nucleotides or amino acids.

A another important embodiment The invention is a pharmaceutical composition or a Vaccin, comprising an EHV according to the invention, optionally in Combination with a pharmaceutically acceptable carrier or Excipient.

Likewise an important part of the present invention is a pharmaceutical A composition comprising a nucleic acid of the invention as disclosed above includes.

Preferably relates to a vaccine according to the invention to a vaccine as defined above. The term "live vaccine" refers to a Vaccin, which is a particle capable of replication, in particular a replication active viral component.

Preferably comprises a vaccine according to the invention a GM-deleted EHV-1 according to the invention, as disclosed above, in combination with a gM deleted according to the invention EHV-4 as disclosed above, or optionally any other antigenic group and optionally a pharmaceutical acceptable carrier or Excipient. The vaccine can be used as a combination vaccine at the same time Time to be administered. The attenuated EHV-1 according to the invention can most preferably to be administered first, followed by administration of a attenuated EHV-4 according to the invention three or four weeks later. Also the strongest the attenuated EHV-1 according to the invention can preferably in combination with an attenuated EHV-4 according to the invention administered in a typical vaccination schedule, two or three basic vaccinations are given. A typical vaccination schedule for one Vaccin is two vaccinations separated for four weeks (basic vaccination), followed by regular Refreshes every six months. The vaccines according to the invention, as above disclosed however at different time intervals, e.g. every three months, be administered.

The Those skilled in the art may choose to administer in two or more Split applications that are administered in quick succession, or at any other specified intervals. Preferably can such intervals are: first immunization, second immunization about 4 Weeks later and optionally third immunization 5 to 6 months later. Depending on the desired one The duration and effectiveness of the treatment can be taken once or can be administered several times, including intermittently. The vaccines according to the invention can to a mare before conception and again during pregnancy administered to prevent EHV-associated abortions. Other Horses can vaccinated, e.g. once a year. Foals can be vaccinated shortly after birth become.

The Vaccine according to the invention can by various routes of administration known to the person skilled in the art are, especially intravenous injection or direct injection into target tissue. For the systemic Administration are intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral or intramucosal (e.g. nasal or respiratory spray or injection) preferred routes. More local application can be subcutaneous, intracutaneous, intrapulmonary or direct into or near the tissue to be treated (connective tissue, Bone tissue, muscle tissue, nerve tissue, epithelial tissue) become. A vaccine composition according to the invention can also about an implant or administered orally. Most preferred is the intramuscular Administration.

Those skilled in the art can use known injectable, physiologically acceptable sterile solutions to prepare suitable vaccine preparations for the applications described above. For the preparation of a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, for example saline or corresponding plasma protein solutions, are readily available. The vaccine preparations can be in the form of lyophilisates or dry preparations which can be reconstituted with a known injectable solution directly before use under sterile conditions, for example as a kit of parts. The final preparations of the vaccine preparations according to the invention are produced for injection, infusion or perfusion by mixing purified virus according to the invention with a sterile, physiologically acceptable solution, which can be supplemented with known carrier substances and / or excipients. The applied dose of the individual EH viruses according to the invention, which is contained in the vaccine formulation, is preferably between 10 ⁴ and 10 ⁸ TCID ₅₀ / animal, between 10 ⁵ and 10 ⁷ TCID ₅₀ / animal and in particular 10 ⁶ TCID ₅₀ / per animal.

The The invention further relates to the use of EHV according to the invention in the manufacture of a medicament for prophylaxis and / or treatment from EHV-associated States.

The The invention further relates to the use of a nucleic acid according to the invention the manufacture of a medicament for prophylaxis and / or treatment of EHV-associated conditions.

The The invention further relates to a method for prophylaxis and / or Treatment of an animal that is characterized by a pharmaceutical according to the invention The composition is administered to the animal and the therapeutic success is monitored.

The invention relates to a method for treating an EHV-infected horse animal with a gM-deleted EHV according to the invention, as described above, the attenuated EHV or the vaccine composition, as described above, providing the horse animal in need in a suitable dose, which is known to the person skilled in the art, is administered and the reduction in EHV symptoms such as viraemia and leukopenia and / or cough and / or pyrexia and / or nasal secretion and / or diarrhea and / or depression and / or abortion is monitored. The treatment can preferably be repeated. The invention thus relates to a method for prophylaxis and / or Treatment of an animal, which is characterized in that a pharmaceutical composition according to the invention is administered to the animal and the therapeutic success is monitored. The treatment can be carried out as described above for the vaccine composition.

The The invention preferably relates to a method for the detection of antibodies against specific structures of infecting EHV-1 or EHV-4 and one Procedure for distinguishing wild-type infections from their presence of gM deleted EHV-1 or EHV-4 as described above an immunological procedure. Immunological procedures are the Those skilled in the art are known and include, but are limited to, ELISAs (enzyme-linked immunosorbent assay) or sandwich ELISAs, dot blots, immunoblots, radioimmunoassays (radioimmunoassay, RIA), Diffusion-based ouchterlony tests, rocket immunofluorescence assays or western blots. examples for immunological methods are e.g. described in: An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et., Techniques in Immunocytochemistry, Academic Press, Orlando, FL, Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

at the ELISA immobilized gM gene product or part of a gM gene product or any other EH-Virus 1 or EH-Virus 4 gene products on one Plastic surface, those for ELISA analysis is suitable to be used; however, the ELISA is not on it limited.

• a) Immobilisierung eines gM-Genprodukts oder eines Fragments davon auf einem Kunststoffträger;
• b) Spülen der Kunststoffoberfläche mit einem geeigneten Waschpuffer (z.B. PBS-Tween);
• c) Zugabe der Proben zu ausgewählten Vertiefungen und Inkubation der ELISA-Platte nach Standardverfahren;
• d) Waschen der Vertiefungen der ELISA-Platte und Zugabe eines geeigneten Antikörpers, gekoppelt an ein Enzym, wie HRP (Meerrettichperoxidase);

An ELISA according to the invention comprises the following stages, but is not limited to this:

• a) immobilization of a GM gene product or a fragment thereof on a plastic carrier;
• b) rinsing the plastic surface with a suitable washing buffer (eg PBS-Tween);
• c) adding the samples to selected wells and incubating the ELISA plate according to standard procedures;
• d) washing the wells of the ELISA plate and adding a suitable antibody coupled to an enzyme such as HRP (horseradish peroxidase);

proof of bound antibody / HRP conjugate by adding a suitable substrate, followed by the photometric Reading the optical density of individual wells. suitable Antibody, e.g. Rabbit anti-horse Ig are known in the art.

The The following examples serve to further explain the invention; the However, examples should not be construed to limit the scope limit the invention disclosed herein.

EXAMPLES

Example 1: GM deleted EHV-1 isolations

The GM negative EHV-1 was constructed by either the Escherichia coli IacZ- (HΔgM-IacZ) or the green one fluorescent protein (GFP) expression cassette (HΔgM-GFP) inserted was replaced with 74.5% of the GM gene sequences. The expression a marker protein makes identification and subsequent purification easier of a recombinant virus. The presence of any "non-EHV-1 sequences" within the vaccine virus to avoid it was decided to remove the marker gene sequences and another second generation gM negative EHV-1, a "white" HΔgM (HΔgM-w) to construct.

For this purpose the plasmid pXuBaxA was constructed ( 1 ). First, a recombination of pXuBaxA sequences into the IacZ-labeled virus HΔgM-IacZ was attempted. In a first step, DNA transfections mediated by the calcium phosphate method were optimized so that several white plaques resulted from transfection supernatants after plating from 100 to 1000 PFU. As a result, several plaques were selected for the purification of progeny virus and four to five rounds of isolation of individual plaques were carried out.

Several independently isolated phenotypically IacZ-negative virus populations were genotypically analyzed by Southern blot and found to still have sequences from the IacZ cassette. Difficulties in isolating truly IacZ negative virus populations due to "IacZ silencing" had been anticipated, and therefore a large number of phenotypically IacZ negative virus populations were cleaned and analyzed, but to no avail. Therefore, the strategy of generating a "white" gM-negative RacH virus was changed by cotransfecting with the gM-negative EHV-1, which was constructed by inserting a GFP cassette. The use of the GFP-expressing virus made it easier to distinguish between the original virus (green fluorescent plaques) and new recombinant viruses (non-fluorescent plaques) and thus increased the effectiveness of isolating phenotypically GFP-negative plaques. It was not expected to change the gM-ne negative "output" RacH would affect the genotype of the expected recombinant virus because (i) the two first generation HΔgM viruses, apart from the marker, are genetically identical and (ii) the eventual genotype in the region of interest by the recombination plasmid (pXuBaxA) is determined.

To construct plasmid pXuBaxA (construct required to obtain the "white" gM negative EHV-1), the 962 bp Apal HincII fragment was within the 1352 bp open reading frame of EHV-1 gM from the Plasmid pBH3.1 removed ( 1D ). Plasmid pBH3.1 carries the EHV-1 BamHI-HindIII fragment that surrounds the gM gene (Seyboldt et al., 2000). In order to prevent the expression of a truncated gM product, the restriction endonucleases Apal and HincII were chosen so that after the blunt ends and ligation were set, the remaining C-terminal gM sequences (183 bp) were not in the frame with the remaining N-terminal sequences (208 bp).

The EHV-1 gM expressing cell line ccgM (Seyboldt et al., 2000; obtained from Dr. N. Osterrieder) was kept in minimally essential medium enriched with 5-10% fetal calf serum (biochrome, γ-irradiated). The homologous recombination in EHV-1 was achieved by calcium phosphate-mediated cotransfection of ccgM cells with 5-10 μg of the plasmid pXuBaxA ( 1D ) and 2 μg DNA from HΔgM-IacZ or HΔgM-GFP.

• (i) ein 2.757 bp- und ein 9.043 bp-Fragment bei BamHI,
• (ii) ein 10.006 bp- und ein 825 bp-Fragment bei HindIII,
• (iii) und ein 5.415 bp- und ein 4.474 bp-Fragment bei Pstl-verdauter DNA.

The subsequent analysis of the DNA of HΔgM-GFP with a digoxigenin-labeled probe, specific for the BamHI-HindIII fragment of the plasmid pBH3.1 ( 2 ), showed

• (i) a 2,757 bp and a 9,043 bp fragment at BamHI,
• (ii) a 10,006 bp and an 825 bp fragment in HindIII,
• (iii) and a 5,415 bp and a 4,474 bp fragment with PstI digested DNA.

The cut restriction enzymes used (BamHI, HindIII, Pstl) not within sequences of the GFP marker cassette, causing the fragment pattern is changed relative to the corresponding GFP marker cassette-free DNA.

The GFP probe bound to the corresponding first fragments (i-iii) and detected no GFP-specific sequences on RacH or HΔgM-w DNA.

For DNA from RacH, the expected BamHI (11,166 bp), HindIII (10,199 bp) and Pstl (9,257 bp) fragments were detected, which were reduced in size after removal of 962 bp from gM sequences (to 10,204 bp, 9,237 bp and 8,279 bp; 3B ).

Single stage growth kinetics of gM negative viruses (HΔgM-w or HΔgM-GFP) that had been constructed as in the legend to 1 and RacH were performed. Rk13 cells in 24-well plates were infected with an MOI of 2 from the corresponding viruses. Supernatants and infected cell pellets were harvested separately at different times pi (0, 4, 8, 12, 16, 20, 24 h pi). Supernatants were cleared from cellular debris by low speed centrifugation and the cells subjected to freeze-thaw before detecting cell-associated infectivity. All virus titers were determined individually on Rk13 or ccgM cells in 24-well plates. The results (data not shown) can be summarized as follows: Cell-associated infectivity of both HΔgM viruses was a factor between 1.6 (4 h pi) and 45 (20 h pi) on Rk13 cells in comparison with titers of Cells infected with RacH decreased (intracellular infectivity). Extracellular virus titers of both HΔgM viruses were maximally 186-fold (HΔgM-w) or 650-fold (HΔgM-GFP) (12 h pi) reduced compared to those of RacH (extracellular infectivity), which also indicated a role of gM in RacH virus leak indicates.

Example 2: GM deleted EHV-4 insulators

In order to proceed in parallel with the construction of gM-negative EHV-1, an IacZ selection was chosen for the selection of EHV-4. To enable isolation of a gM-negative EHV-4, a Vero cell line that constitutively expressed EHV-4 gM was constructed. Vero cell clone C1008 (ATCC number CRL-1586) was kept in minimally essential medium enriched with 5-10% fetal calf serum (biochrome, γ-irradiated). The Vero-gM recombinant cell line was mediated by Effectene ^TM (Qiagen) -mediated transfection of 1 μg plasmid pCgM4 ( 3B ) and 0.1 μg of plasmid pSV2neo (which confers resistance to G418; Neubauer et al., 1997) in Vero cells. Cell clones were first selected for resistance to G418 (Calbiochem), then for trans-complementation of a gM-negative EHV-4. G418 was added to the medium of every 5th passage of the recombinant cell lines (500 μg / ml). All cells were regularly analyzed for mycoplasma by PCR and for viral bovine diarrhea virus (BVDV) antigen by FACS analysis. The selected cell clone was named Vero-gM and used in the experiments below.

EHV-4 DNA was extracted with plasmid pgM4β + ( 3B ) cotransfected in Vero-gM cells. The recombination resulted in several "IacZ positive" Plaques that were isolated and replated. Then, however, it was found that the purification of a deletion mutant was more difficult and slower with EHV-4 than with EHV-1, because: (i) "IacZ positive" plaques generally stained less intensely with Vero cells than with Rk13 Cells and were therefore more difficult to identify, and because (ii) the EHV-4 system was slower to replicate than EHV-1 and therefore left less time between plaque identification and isolation of viable virus progeny.

All IacZ positive plaques that were isolated were lost in the first round of cleaning, making it necessary to look for another solution. An attempt was therefore made to use the GFP marker for EHV-4 as well. In a first step, the plasmid pgM4GFP + ( 3B ) constructed and used for recombination in EHV-4 DNA. The resulting GFP positive plaques were purified in three rounds of single plaque isolation on the Vero-gM cell line. A homogeneous GFP-positive virus population was chosen and viral DNA was subjected to Southern blot analysis ( 4 ). DNA of the resulting virus E4ΔgM-GFP ( 3C ) was then co-transfected with the plasmid pgM4R or pgM4w ( 3B ). Plasmid pgM4R was used to construct a gM-repaired EHV-4, designated E4RgM ( 3A ). Plasmid pgM4w was the basis for the generation of the gM and marker gene negative EHV-4, E4ΔgM-w ( 3D ). E4RgM and E4ΔgM-w virus populations were isolated to a GFP-negative phenotype, purified on Vero or Vero-gM cells and finally analyzed by Southern blot ( 4 ). The plasmid pCgM4 was generated to express EHV-4 gM in eukaryotic cells ( 3B ). The full ORF of EHV-4 gM was determined by PCR using primers that are in 8th are specified, and the Taq polymerase (MBI fermentas) amplified. The resulting PCR product was inserted into the vector pcDNAI / Amp (Invitrogen).

The plasmid pgM4R resulted from the PCR amplification of nucleotides 91,699 to 94,808 (Telford et al., 1988) of EHV-4 and the insertion of the amplification product into the vector pGEM3Zf + (Promega) ( 3B ; 8th ). This plasmid was used in the construction of the gM-repaired EHV-4, E4RGM ( 3A ).

To construct the plasmid pgM4β + ( 3B ), which was developed to initially delete 1110 bp from the 1352 bp of EHV-4 gM, a multi-stage strategy was chosen. In the first step, the two flanking sequences required for DNA recombination were independently amplified by PCR using the PFU polymerase (Strategene). Restriction sites that were required for the gradual cloning were supplemented by primer sequences ( 8th ). PCR products were inserted into the vector pTZ18R (Pharmacia), resulting in the plasmids pgM4Del1 and pgM4Del2 ( 8th ) led.

In a next step, the 3.9 kbp E, coli IacZ expression cassette from the plasmid ptt264A + (Osterrieder et al., 1996) was released by Sall and BamHI digestion and inserted into the plasmid pgM4Del1, resulting in the plasmid pgM4Del1β + led. Subsequently, EHV-4-specific sequences, taken from pgM4Del2, were introduced into pgM4Del1 β + using Pstl and Sall, so that the marker gene cassette was flanked by EHV-4-specific sequences in the resulting plasmid pgM4β + ( 3B ).

The plasmid pgM4β + was then digested with SalI and BamHI, releasing the IacZ cassette again. The resulting 5 'overhangs were filled in with Klenow polymerase and the construct resulting from the re-ligation was designated pgM4w ( 3B ). Another frame shift was carried out between the N-terminal 208 nt and the remaining C-terminal 33 nt of the gM sequence.

To generate the plasmid pgM4GFP + ( 3B ), the IacZ cassette was made from pgM4β + ( 3B ) using the Sall and BamHI sites and replaced with the GFP cassette (including the CMV promoter and SV40-polyA) which had been removed from the vector pEGFP-C1 (Clontech) via AsnI and MIuI digestion. Overhangs generated by restriction enzymes were blunt-ended by Klenow polymerase.

The correct amplification of all PCR products was performed by cyclo-sequencing (MWG Biotech) and comparison with the published sequence of EHV-4 Strain NS80567 (Telford et al., 1998).

Recombination in EHV-4 was done using the Vero-gM cell line, and plasmid pgM4β + or pgM4GFP + ( 3B ) was co-transfected with EHV-4 DNA to produce a first generation gM negative EHV-4 ( 3C ). Plasmid pgM4w ( 3B ) in combination with DNA of the gM-negative EHV-4 resulted in E4ΔgM-w ( 3D ). Finally, the gM-repaired EHV-4 E4RgM ( 3A ) after cotransfection of plasmid pgM4R ( 3B ) isolated with DNA from E4ΔgM-GFP in Vero cells.

DNA from EHV-4, E4RgM, E4ΔgM-w and E4ΔgM-GFP was cut with PstI, EcoRV or HindIII and DNA fragments were transferred to nylon membranes. Parallel membranes were either with GFP specific sequences (identical to 2 ) or hybridized with a probe, designated gM3.1, which contained the EHV-4 specific sequences taken from the plasmid pgM4R ( 3B ).

• (i) Bei DNA von E4ΔgM-GFP erkannte die GFP-Sonde Fragmente bei 5.531 bp, wenn mit Pstl geschnitten wurde, bei 8.383 bp nach EcoRV-Verdau und bei 4.528 bp nach HindIII-Verdau. Identische Fragmente plus Fragmente bei 1.792 bp (Pstl), 1.801 bp (EcoRV) und 826 plus 5.487 bp (HindIII) reagierten mit der Sonde gM3.1.
• (ii) Weder Eltern-EHV-4 noch repariertes Virus E4RgM oder E4ΔgM-w trugen irgendwelche GFP-spezifischen Sequenzen.
• (iii) Die gM3.1-reaktiven DNA-Fragmente in EHV-4 und E4RgM wurden bei 6.806 bp (Pstl), bei 7.874 bp plus 1.801 bp (EcoRV) und bei 4.837 plus 5.487 bp (HindIII) nachgewiesen.
• (iv) Das gM- und GFP-Kassette-negative Virus E4ΔgM-w hybridisierte nicht mit GFP-Sequenzen, jedoch mit den entsprechenden EHV-4-spezifischen Sequenzen (gM3.1). Die letztgenannte Sonde wies Fragmente nach, denen 1110 bp an gM- Sequenzen im Vergleich mit Wildtypvirus fehlten, d.h. bei 5.696 bp (Pstl), bei 6.764 bp plus 1.801 bp (EcoRV) bzw. bei 3.727 bp plus 5.487 bp (HindIII).

The observed results of digestion ( 4 ) were as follows:

• (i) For DNA from E4ΔgM-GFP, the GFP probe recognized fragments at 5,531 bp when cut with Pstl, at 8,383 bp after EcoRV digestion and at 4,528 bp after HindIII digestion. Identical fragments plus fragments at 1,792 bp (Pstl), 1,801 bp (EcoRV) and 826 plus 5,487 bp (HindIII) reacted with the probe gM3.1.
• (ii) Neither parent EHV-4 nor repaired virus E4RgM or E4ΔgM-w carried any GFP-specific sequences.
• (iii) The gM3.1-reactive DNA fragments in EHV-4 and E4RgM were detected at 6,806 bp (Pstl), at 7,874 bp plus 1,801 bp (EcoRV) and at 4,837 plus 5,487 bp (HindIII).
• (iv) The gM and GFP cassette negative virus E4ΔgM-w did not hybridize with GFP sequences, but with the corresponding EHV-4-specific sequences (gM3.1). The latter probe detected fragments lacking 1110 bp gM sequences compared to wild type virus, ie at 5,696 bp (Pstl), at 6,764 bp plus 1,801 bp (EcoRV) or at 3,727 bp plus 5,487 bp (HindIII).

In the case of HindIII-cut DNA from all of these viruses, there is a further gM3.1 probe-specific fragment at 126 bp ( 2C ); however, this fragment is too small to be shown on this Southern blot.

The Viruses E4HΔgM-GFP and E4HΔgM-w lost their assets for expression of gM, and E4HΔgM-w also lost the marker gene sequence.

• a) Virus growth on culture cells. Virus growth properties of the various mutant viruses, as detailed above, were compared on Vero and Vero-gM cells. Cells inoculated into 24-well plates were infected at an MOI of 1-2, and extracellular (extracellular infectivity) and intracellular (intracellular infectivity) virus titers were determined at different times pi ( 5 ). While the growth properties of the reconstituted E4RgM virus corresponded well to those of EHV-4, there was a surprising inhibition of the formation of extracellular virus titers in E4ΔgM-w and E4ΔgM-GFP on non-complementing cells. In this series of experiments (mean of two independent experiments), extracellular infectivity could never be detected before 24 h pi. Even at 30 h pi only very low titers were observed extracellularly (maximum 1.5 plaques / ml at the lowest dilution 10 ^-1 ), although cells show a severe cytopathic effect. However, differences in intracellular infectivity never reach the factor 100 and reached a maximum value at 24 h pi (84-fold between EHV-4 and E4ΔgM-w). There was only one delay in the detection of intracellular infectivity (12 h vs. 15 h pi). Taken together, it was surprisingly shown that the deletion of gM sequences from the EHV-4 background had a massive influence on virus replication in vitro, but that the expression of gM is not essential for replication. In particular, extracellularly infectious virus decreased and the ability to directly infect neighboring cells was reduced, which is reflected by the plaque sizes.
• b) Plaque size. Diameters of 150 plaques after infection of Vero or Vero-gM cells with EHV-4, E4RgM, E4ΔgM-w or E4ΔgM-GFP were measured, and mean plaque sizes were relative to the sizes of wild-type plaques that were 100 % was determined. It was clearly shown that the gM-negative viruses were able to infect and replicate Vero cells, but the maximum plaque diameter was markedly reduced to less than 20% of the wild-type plaque. Diameter ( 6 ). Infection with parental or recovered virus resulted in a wild type-like appearance of the plaques, indicating that the observed phenotype was indeed induced by the gM deletion. This was further confirmed by the fact that plaque formation of E4ΔgM-w and E4ΔgM-GFP was completely restored in the Vero-gM cell line (data not shown).
• c) penetration experiments. In this experiment, the influence of EHV-4-gM on the entry kinetics of EHV-4 was assessed. 100 PFU of the different viruses: starting EHV-4, gM-repaired virus E4RgM as well as gM deletion mutants and E4ΔgM-GFP (cf. 3 ) was allowed to adsorb on Vero cells in 6-well plates at 4 ° C. After 90 min, the appropriate vaccinations were replaced with fresh medium and the penetration was initiated by shifting the incubation temperature to 37 ° C. At various times after the temperature shift - starting immediately (= 0 min) - the extracellular infectivity was pH-inactivated by treating the cells with citrate buffer (pH 3.0). Parallel samples were washed accordingly, the Cit However, rat buffer was replaced by PBS, so that the "adsorbed infectivity" could be compared with the "penetrated infectivity" at any time. The numbers of the plaques were determined by incubating cells for 4 days under a methocel overlay.
• c) Several sets of experiments were carried out: In 7A results for genotypically and phenotypically gM-negative viruses are shown after proliferation in non-complementing Vero cells 7B shows the kinetics of phenotypically complemented E4ΔgM-w and E4ΔgM-GFP, the viruses being grown on Vero-gM cells expressing gM.

An average of 52.8% (56.7%) of the initial infectious EHV-4 (E4RgM) was 40 min after extracellular acid treatment after initiation of the penetration ( 7A , open circles), while only 33.7% (E4ΔgM-w - filled rectangles) and 38.5% (E4ΔgM-GFP, filled triangles) of the gM-negative viruses were protected. At later times of entry kinetics, the graphs begin to overlap in various ways, and a maximum penetration efficiency between 61.7% and 78.9% is reached after 150 min penetration time, indicating that a certain variability of the assay for the minor differences observed may be responsible.

( 7A ). When gM-negative viruses were generated on complementing Vero-gM cells, no difference in their penetration efficiency was observed ( 7B ). In contrast to the delay in the entry kinetics of gM-negative EHV-1 from about 20% (strain RacL11; Osterrieder et al., 1996) to 40% (strain KyA; Seyboldt et al., 2000), the effect of the deletion of gM on EHV-4 should be seen with caution. Nevertheless, the following conclusions can be drawn: (i) A difference was observed in the kinetics of phenotypically complemented versus uncomplemented gM-negative viruses ( 7A - B ), (ii) the influence of deletion of gM on EHV-4 penetration is, however, practically negligible.

Example 3: Analysis of Horse sera on anti-gM antibodies using a GM specific serological test

Around determine whether gM as a serological marker for differentiation a wild type infection from vaccination with a GM negative vaccine multiple assumptions had to be tested. First had to be assessed whether sera from horses infected with field virus gM-specific antibodies contain. For the initial Analysis, a Western blot test was chosen because this system allowed a specific reaction to a background reactivity to identify. When using highly neutralizing Sera (EHV-1 and / or -4 neutralizing titer between 1: 128 and 256) was found (Data not shown) that lysates of the EHV-1 gM expressing cell line ccgM enabled the detection of a specific signal by horse sera and that a thinning from sera to 1: 3,000 seemed to work best.

Accordingly the sera of all 12 foals (6 vaccinated and 6 controls), who participated in the EHV-1 gM vaccine trial for gM reactivity in one Wester blot analyzed. There were three different horses from each Sera tested: taken before entering the test (pre), 4 weeks after the second vaccination (V2) and two weeks after the stress infection (C).

Was summarized by Western blot analyzes ( 9 ) showed that (i) sera from horses showing EHV-1 neutralization activity all gave positive test results for gM, that (ii) gM reactivity was never detected in a sample prior to known contact with EHV-1 or was analyzed after vaccination with gM-negative EHV-1 and that (iii) after infection of vaccine test horses with the gM-positive strain virus, gM was clearly detectable in 10 of 12 cases. Finally (iv) it was observed that anti-EHV-1 antibody titer and the intensity of the gM reactivity did not appear to be directly correlated.

Due to the high background reactivity of horse sera, the establishment of serological tests is difficult. Based on indirect immunofluorescence (IIF) data obtained in horse sera, it was confirmed that an indirect or blocking test system needs to be established or that highly purified gM polypeptides have to be used in an ELISA test. For this purpose, an ELISA was established as follows. Purified gM polypeptides or complete gM were immobilized on a solid support of a 96-well plate, which was coated to ensure good adhesion of the capturing protein. For the assay, non-specific binding sites with milk powder or similar substances were blocked to prevent non-specific binding. The plastic surface was then washed with a suitable washing buffer (eg PBS-Tween) in order to remove excess blocking agent. The test samples were then added to selected wells and the ELISA plate was incubated at 37 ° C according to standardized procedures, which allowed the binding of antibodies in the test sample to the immobilizing capture protein. In the next stage, the vertie The ELISA plate was washed thoroughly several times with a rinse with wash buffer, followed by the addition of a suitable horse antibody coupled to an enzyme such as HRP (horseradish peroxidase). Bound antibody / HRP conjugate was finally detected by adding a suitable substrate, followed by photometric reading of the optical density of the individual wells. The value obtained was compared with positive and negative control batches in the same assay.

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Claims (34)

Equine Herpes Virus (EHV), which is the protein gM is absent, characterized in that the EHV is free of heterologous elements. EHV according to claim 1, characterized in that the gene for the protein encodes gM is deleted. EHV according to claim 1 or 2, obtainable by a process, which includes the following levels: a) isolation of a wild-type EHV; b) Establish a plasmid encoding the EHV gM gene, if necessary with flanking sequences; c) generating a complementary Cell line expressing gM or parts thereof; d) establishment an EH virus, which contains a GFP-encoding cassette insert in its gM coding sequence carries, by cotransfecting the complementing cell line from step b) with EHV nucleic acid and a plasmid encoding gM interrupted by a GFP-encoding cassette insert is; e) deletion of the GFP-encoding cassette; and f) Selection for the EHV clones in which the GFP-encoding cassette is successful is deleted. EHV according to one of claims 1 to 3, characterized in that that it is EHV-1. EHV-1 according to one of claims 1 to 4, characterized in that that 850 to 1100 bp of the open reading frame are deleted for gM. EHV-1 according to one of claims 1 to 5, characterized in that the entire gM coding sequence is deleted, with the exception from 150 to 200 bp of the coding sequence for the C-terminal section and with the exception of 150 to 250 bp of the coding sequence for the N-terminal section. EHV-1 according to one of claims 1 to 6, characterized in that nucleotides 93268-93318 to 94222-94322 of the gM coding sequence, as they correspond to the Telford positions (1992) are deleted. EHV-1 according to one of claims 1 to 7, characterized in that nucleotides 93268 to 94322 of the gM coding sequence, as they correspond to the Telford positions (1992) are deleted. EHV-1 according to one of claims 1 to 8, characterized in that the entire gM coding sequence with the exception of 184 bp the coding sequence for the C-terminal section and with the exception of 209 bp the coding Sequence for the N-terminal section is deleted. EHV-1 according to one of claims 1 to 9, characterized in that that nucleotides 94263 to 93302 of the gM coding sequence, as they correspond to the Telford positions (1992} are deleted. EHV-1 according to one of claims 1 to 10, characterized in that it is a recombinant variant based on the RacH strain is about EHV-1. EHV-1 according to one of claims 1 to 11, characterized in that it is the RacH-based recombinant variant isolation HΔ-gM-w, at the ECACC on October 16, 2002 under the deposit number 02101663 was deposited. EHV according to one of claims 1 to 3, characterized in that that it is EHV-4. EHV-4 according to one of claims 1 to 3 or 13, characterized characterized that 900 to 1150 bp deletes the open reading frame for gM are. EHV-4 according to one of claims 1 to 3 or 13 to 14, characterized in that the entire gM coding sequence is deleted except for 0 to 50 bp of the coding sequence for the C-terminal Section and with the exception of 150 to 250 bp of the coding sequence for the N-terminal Section. EHV-4 according to one of claims 1 to 3 or 13 to 15, characterized in that the nucleotides 92681-92731 to 93765-93865 the gM coding sequence as used in the Telford positions (1998) correspond, are deleted. EHV-4 according to one of claims 1 to 13 to 13 to 16, characterized in that nucleotides 92681 to 93865 of gM coding sequence as they correspond to the Telford positions (1998), are deleted. EHV-4 according to one of claims 1 to 3 or 13 to 17, characterized in that the entire gM coding sequence with Exception of 34 bp of the coding sequence for the C-terminal section and with the exception of 209 bp of the coding sequence for the N-terminal Section is deleted. EHV-4 according to one of claims 1 to 3 or 13 to 18, characterized in that nucleotides 92715 to 93824 of gM coding sequence as they correspond to the Telford positions (1998), are deleted. EHV-4 according to one of claims 1 to 3 or 13 to 19, characterized in that it is a recombinant variant based on MSV batch 071398 from EHV-4. EHV-4 according to one of claims 1 to 3 or 13 to 20, characterized in that it is on MSV batch 071398 and insulation E4ΔgM-w based and that it is the EHV-4, which at the ECACC on 16.10.02 under the deposit number 02101663 was deposited. nucleic acid coding for an EHV according to one of the claims 1 to 21. Vaccine preparation, comprising an EHV according to one of claims 1 to 21. Vaccine preparation, comprising a nucleic acid according to claim 22. Use of EHV according to one of claims 1 to 21 in the manufacture of a medicament for prophylaxis and / or treatment of EHV-associated conditions. Use of a nucleic acid according to claim 22 in the Manufacture of a drug for the Prophylaxis and / or treatment of EHV-associated conditions. Procedure for prophylaxis and / or treatment of an animal, characterized in that that a vaccine preparation according to one of the claims 23 or 24 administered to the animal and monitored therapeutic success becomes. Vaccine preparation, comprising at least one EHV-1 according to any one of claims 1 to 12 and an EHV-4 according to one of claims 1 to 3 or 13 to 21. Use of at least one EHV-1 according to one of the Expectations 1 to 12 and an EHV-4 according to one of claims 1 to 3 or 13 to 21 in the manufacture of a medication for the treatment of EHV-associated States. Method for prophylaxis and / or treatment of a Tiers, characterized in that a vaccine preparation after Claim 28 administered to the animal and monitored the therapeutic success becomes. Method to obtain a recombinant EHV which includes the following levels: a) isolation of a wild-type EHV; b) If appropriate, establishment of a plasmid which codes for the EHV gM gene with flanking sequences; c) generating a complementary Cell line expressing gM or parts thereof; d) establishment of an EH virus that contains a GFP-encoding cassette insert in its gM coding sequence carries, by cotransfecting the complementing cell line from step b) with EHV nucleic acid and a plasmid encoding gM interrupted by a GFP-encoding cassette insert is; e) deletion of the GFP-encoding cassette; and f) Selection for the EHV clones in which the GFP-encoding cassette is successful is deleted. Cell line for the use in a method according to claim 31, characterized in that the gene encoding the gM protein transfected into the cell line and that the cell line expresses gM. Cell line according to claim 32, characterized in that it is a cell line that belongs to the group Vero cells, RK-13 and cc selected is. Cell line according to one of claims 31 to 33, characterized in that that it is the VERO GM cell complement cell line that the ECACC on January 28, 2003 under the deposit number 03012801 has been.