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  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention is concerned with an Equine herpesvirus-4 mutant (EHV-4) , a- recombinant DNA molecule comprising EHV-4 DNA, host cell containing said reccmbinant DNA molecule, process for the preparation of said EHV-4 mutant, cell culture infected with the EHV-4 mutant., a vaccine derived from the EHV-4 mutant as well as a process for the preparation of such a vaccine.
• EHV-4 Equine herpesvirus-4 mutant
• Equine herpesvirus-4 (EKV-4) is, like the related Equine herpesvirus-1, an alphaherpesvirus responsible for significant economic losses within the equine industry. EHV-4 is primarily associated with respiratory disease though ⁇ HV-4 induced abortions are occasionally reported.
• EHV-4 has beer, characterized as a double-snranded linear DNA molecule consisting of two ccvalen iy linked segments (L, 109 kbp; S, 35 kbp) the latter being flanked by inverted repeats.
• vaccines comprise chemically inactivated virus vaccines and modified live-virus vaccines.
• inactivated vaccines generally induce only a low level of immunity, requiring additional immunizations, disadvantageously require adjuvants and are expensive to produce. Further, some infectious virus particles may survive the inactivation process and causes disease after administration to the animal.
• Attenuated live virus vaccines are preferred because they evoke a more long-lasring immune response (often both humoral and cellular) and are easier to produce.
• EHV-4 vaccines are available which are based on live EHV-4 viruses attenuated by serial passages of virulent strains in tissue culture.
• uncontrolled mutations are introduced into the viral genome, resulting in a population of virus particles heterogeneous in their virulence and immunizing properties.
• such traditional attenuated live virus vaccines can revert to virulence resulting in disease of the inoculated animals and the possible spread of the pathogen to other animals.
• a positive serological test is obtained for EHV- 4 infection.
• such a mutant EHV-4 is characterized in that it does not produce a functional thymidine kinase (TK ⁇ ) as a result of a deletion and/or insertion in the gene encoding thymidine kinase.
• TK ⁇ thymidine kinase
• the gene encoding thymidine kinase was mapped within the BamHI C fragment of the EHV-4 genome and was further localised to an about 2 kbp EcoRV/XhoI fragment thereof, with a map position of approximately 0,48 (fig.l).
• the nucleic acid sequence of the TK gene was determined and is shown in SEQ ID NO: 1 from which restriction enzyme cleavage sites to be used for the genetic manipulation of the gene can be d -rived.
• the TK gene consists of 1056 nucleotides encoding a 352 amino acid enzyme of predicted molecular weight of 38.300 D.
• the efficiency of the expression of TK is regulated by the presence of expression control sequences.
• promoter sequences are involved in the binding of RNA poly erase to the DNA template and control the site and onset of the mRNA. Such sequences are often found within a 100 bp region before the transcription initiation site.
• Downstream transcriptional control signals are inter alia, the transcription termination codon and a polyadenylation signal.
• the TATA box positioned at base pair 21-25 is the putative promoter TATA box of the EHV-4 TK gene.
• a potential RNA polymerase initiation site is located 22 bp downstream of the TATA box.
• a poly A signal is positioned 42 bp downstream of the termination codon (SEQ ID NO: 1) .
• EHV-4 TK gene natural variations can exist between individual EHV-4 viruses. These variations may result in a change of one or more nucleotides in the TK gene which, however still encodes a functional TK. Moreover, the potential exists to use genetic engineering technology to bring about above-mentioned variations resulting in a DNA sequence related to the sequence shown in SEQ ID NO: 1. It is clear that EHV-4 mutants comprising a deletion and/or insertion in such a related nucleic acid sequence are also included within the scope of the invention.
• the EHV-4 deletion mutants of the present invention comprise a TK gene from which a DNA fragment has been deleted so that no functional TK enzyme is produced upon replication of the virus, e.g. as result of a change of the tertiary structure of the altered TK protein or as a result of a shift of the reading frame.
• deletion in the genome of the EHV-4 mutant may comprise the complete TK gene.
• EHV-4 mutants according to the invention can also be obtained by inserting a nucleic acid sequence into the TK coding region thereby preventing the expression of a functional TK enzyme.
• a nucleic acid sequence can inter alia be an oligonucleotide, for example of about 10-60 bp, preferably also containing one or more translational stop codons, or a gene encoding a polypeptide.
• Said nucleic acid sequence can be derived from any source, e.g. synthetic, viral, prokaryotic or eukaryotic.
• the EHV-4 deletion mutants can contain above-mentioned nucleic acid sequence in place of the deleted EHV-4 DNA.
• a vector vaccine based on a safe live attenuated EHV-4 mutant offers the possibility to immunize against other pathogens by the expression of antigens of said pathogens within infected cells of the immunized host and can be obtained by inserting a heterologous nucleic acid sequence encoding a polypeptide heterologous to EHV-4 in an insertion-region of the EHV-4 genome.
• the prerequisite for a useful EHV-4 vector is that the heterologous nucleic acid sequence is incorporated in a permissive position or -region of the geno ic EHV-4 sequence, i.e. a position or region which can be used for the incorporation of a heterologous sequence v:ithout disrupting essential functions of EHV-4 such as those necessary for infection or replication.
• a region is called an insertion-region.
• insertion-region Prior to the present invention no insertion-region in the EHV-4 genome has been described.
• EHV-4 mutants which can be used as a viral vector, characterized in that said mutants do not produce a functional TK as a result of an insertion of a heterologous nucleic acid sequence encoding a polypeptide m the gene encoding TK.
• EHV-4 insertion mutants as described above having a heterologous nucleic acid sequence inserted in place of deleted TK DNA are also within the scope of the present invention.
• EHV-4 insertion mutants comprises inter alia infective viruses which have been genetically modified by the incorporation into the virus genome of a heterologous nucleic acid sequence, i.e. a gene which codes for a protein or part thereof said gene being different of a gene naturally present in EHV-4.
• polypeptide refers to a molecular chain of a ino acids with a biological activity, does not refer to a specific length of the product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation thus inter alia peptides, oligopeptides and proteins are included within the definition of polypeptide.
• the heterologous nucleic acid sequence to be incorporated into the EHV-4 genome according to the present invention can be derived from any source, e.g. viral, prokaryotic, eukaryotic or synthetic.
• Said nucleic acid sequence can be derived from a pathogen, preferably an equine pathogen, which after insertion into the EHV-4 genome can be applied to induce immunity against disease.
• nucleic acid sequences derived from EHV-1, equine influenza virus, -rotavirus, -infectious anemia virus, arteritis virus, -encephalitis virus, Borna disease virus of horses, Berue virus of horses, E.coli or Streptococcus equi are contemplated of for incorporation into the insertion-region of the EHV-4 genome.
• nucleic acid sequences encoding polypeptides for pharmaceutical or diagnostic application in particular immune modulators such as lymphokines, interferons or cytokines, may be incorporated into said insertion-region.
• an essential requirement for the expression of the heterologous nucleic acid sequence in a EHV-4 mutant is an adequate promoter operably linked to the heterologous nucleic acid sequence.
• a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells infected by the EHV-4 mutant, such as the SV-40 promoter (Science 222. 524-527, 1983) or, e.g., the metallothionein promoter (Nature 296, 39-42, 1982) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA 82., 4949-53, 1985) or the human cytomegalovirus IE promoter or promoters present in EHV-4, e.g. the TK promoter.
• Such a recombinant DNA molecule may be derived from any suitable plasmid, cosmid, virus or phage, plasmids being most preferred, and contains EHV-4 DNA possibly having a nucleic acid sequence inserted therein if desired operably linked to a promoter.
• suitable cloning vectors are plasmid vectors such as pBR322, the various pUC and Bluescript plasmids, bacteriophages, e.g. ⁇ gt-WES- ⁇ B, charon 28 and the M13mp phages or viral vectors such as SV40, Bovine papillomavirus, Polyoma and Adeno viruses.
• Vectors to be used in the present invention are further outlined in the art, e.g. Rodriguez, R.L. and D.T. Denhardt, edit., Vectors: A survey of molecular cloning vectors and their uses, Butterworth ⁇ , 1988.
• an EHV-4 DNA fragment comprising the insertion region, i.e. the TK gene, is inserted into the cloning vector according to recDNA techniques.
• Said DNA fragment may comprise part of the TK gene or substantially the complete TK gene, and if desired flanking sequences thereof.
• an EHV-4 TK deletion mutant is to be obtained at least part of TK gene is deleted from the recombinant DNA molecule obtained from the first step. This can be achieved for example by appropriate exonuclease III digestion or restriction enzyme treatment of the recombinant DNA molecule from the first step.
• the nucleic acid sequence is inserted into the TK gene present in the recombinant DNA molecule of the first step or in place of the TK DNA deleted from said recombinant DNA molecule.
• the EHV-4 DNA sequences which flank the deleted TK D? T A or the inserted nucleic acid sequence should be of appropriate length as to allow homologous recombination with the viral EHV-4 genome to occur.
• a construct can be made which contains two or more different inserted (heterologous) nucleic acid sequences derived from e.g. the same or different pathogens said sequences being flanked by insertion- region sequences of EHV-4 defined herein.
• Such a recombinant DNA molecule can be employed to produce an EHV-4 mutant which expresses two or more different antigenic polypeptides to provide a ultivalent vaccine.
• cells for example rabbit cells, TK + or TK " phenotype, or equine cells, e.g. equine dermal cells
• EHV-4 DNA in the presence of the recombinant DNA molecule containing the deletion and/or insertion of (heterologous) nucleic acid sequence flanked by appropriate EHV-4 sequences whereby recombination occurs between the corresponding regions in the recombinant DNA molecule and the EHV-4 genome.
• Recombination can also be brought about by transfecting EHV-4 genomic DNA containing host cells with a DNA containing the (heterologous) nucleic acid sequence flanked by appropriate flanking insertion-region sequences without vector DNA sequences.
• Recombinant viral progeny is thereafter produced in cell culture and can be selected for example genotypically or phenotypically, e.g. by hybridization, detecting enzyme activity encoded by a gene co-integrated along with the (heterologous) nucleic acid sequence, screening for EHV-4 mutants which do not produce functional TK (Roiz an, B. and Jenkins, F.J. (1985), ibid)) or detecting the antigenic heterologous polypeptide expressed by the EHV-4 mutant immunologically.
• the selected EHV-4 mutant can be cultured on a large scale in cell culture whereafter EHV- 4 mutant containing material or heterologous polypefcides expressed by said EHV-4 can be collected therefrom.
• mutant EHV-4 could be generated by cotransfection of several cosmids, containing between them the entire EHV-4 genome, where an insertion and/or deletion has been engineered into the cosmid possessing EHV-4 TK DNA.
• a live attenuated EHV-4 mutant which does not produce a functional TK, and if desired expresses one or more different heterologous polypeptides of specific pathogens can be used to vaccinate horses, susceptible to EHV-4 and these pathogens.
• Vaccination with such a live vaccine is preferably followed by replication of the EHV-4 mutant within the inoculated host, expressing in vivo EHV-4 polypeptides, and if desired heterologous polypeptides.
• An immune response will subsequently be elicited against EHV-4 and the heterologous polypeptides.
• An animal vaccinated with such an EHV-4 mutant will be immune for a certain period to subsequent infection of EHV-4 and above-mentioned pathogen(s) .
• An EHV-4 mutant according to the invention optionally containing and expressing one or more different heterologous polypeptides can serve as a monovalent or multivalent vaccine.
• An EHV-4 mutant according to the invention can also be used to prepare an inactivated vaccine.
• the EHV-4 mutant according to the presentation can be given inter alia by aerosol, spray, drinking water, orally, intrader ally, subcutaneously or intramuscularly.
• Ingredients such as skimmed milk or glycerol can be used to stabilise the virus. It is preferred to vaccinate horses by intranasal administration. A dose of 10 3 to 10 8 TCID 50 of the EHV-4 mutant per horse is recommended in general
• This can be achieved by culturing cells infected with said EHV-4 mutant under conditions that promote expression of the heterologous polypeptide.
• the heterologous polypeptide may then be purified with conventional techniques to a certain extent depending on its intended use and processed further into a preparation with immunizing therapeutic or diagnostic activity.
• roller bottles of slightly sub-confluent onolayers of equine dermal cells (NBL-6) grown in Earle's Minimum Essential Medium (Flow) supplemented with 0,2% sodium bicarbonate, 1% non-essential amino acids, 1% gluta ine, 100 units/ml penicillin, 100 mg/ml streptomycin and 10% foetal calf serum were infected with virus of the EHV-4 strain 1942 at a .o.i. of 0,003 and allowed to adsorb for 60 min at 37 °C. They were incubated at 31 °C until extensive c.p.e. was evident and the majority of cells had detached from the bottle surface (2-6 days) . The infected cell medium was centrifuged at 5.000 r.p.m. for
• the pelleted virus was resuspended in 10 ml NTE
• a chloroform extraction was followed by ethanol precipitation of the DNA as described above.
• the DNA was pelleted, washed with 70% ethanol, resuspended in 10 ml of 100 mM NaCl and 10 ⁇ g/ml RNase and left overnight at room temperature. Further purification was achieved by treatment with 1 mg/ml proteinase K for 2 hours at 31 °C.
• the DNA was extracted once with phenol:chloroform (1:1 vol/vol) , once with chloroform, ethanol precipitated, drained well and resuspended in 0.1 X SSC.
• EHV-4 BamHI DNA fragments were ligated into the vector pUC9, a plasmid which includes the ampicillin-resistance gene from pBR322 and the polylinker region from M13mp9 (Vieira, J. and Messing, J. (1982), Gene 19_, 259). 5 ⁇ g of EHV-4 DNA and 5 ⁇ g pUC9 DNA were separately digested with BamHI.
• E.coli DHI cells (Hanahan, D. (1983) , J. Mol. Biol. 166, 557) were transformed with the recombinant plasmids essentially described by Cohen et al. (Proc. Natl. Acad. Sci., USA 69., 2110, 1972). Additional clones were derived by restriction digestion of recombinant plasmid pUC9 containing BamHI C fragment (fig. lb) , followed by recovering of the specific E tT -4 restriction fragments and sub-cloning thereof within the multi-cloning site of the Bluescript M13 + plasmid vector (Stratagen ; Maniatis, T. et al. ibid) .
• TK "1" colonies were selected in HAT supplemented medium (Hypoxanthine l ⁇ 4 M, A inopterin 4 x 10 ⁇ 5 M, thymidine
• TK transforming activity was thus localised to a 2 kbp EcoRV/XhoI fragment (RX2) , cloned in construct pBSRX2, with a map position of approximately 0,48 (fig. lb).
• the nucleotide sequence of both. trands of fragment RX2 was determined by using single stranded plasmid DNA as template and Bluescript-derived custom-made oligonucleotides as primers in a Sanger dideoxy sequencing strategy (Sanger et al., Proc. Natl. Acad. Sci: Z L ,5463,1977) (fig.lc). The exact localisation, nucleic acid sequence and corresponding amino acid sequence of the TK gene is shown in the SEQ ID NO: 1.
• the linear plasmid was then self ligated to produce a plasmid containing RS3 fragment deleted from the Smal-BstXI site (fig. 2b) .
• a 0,52kbp deletion within the TK gene was achieved by cloning EHV-4 RX2 into a Bluescript vector (at the Smal and Xhol sites within the multicloning site) and deleting from the Smal-BstEII sites within the TK gene by restriction digestion with these enzymes.
• the larger vector fragment was separated from the 0,52 kbp EHV-4 fragment, the overhang filled in and the plasmid religated.
• the resultant plasmid possesses the 5' and 3 1 coding regions of the EHV-4 TK gene but is deleted from the Smal-BstEII sites (figure 2c) .
• the two plasmid constructs preferred in Example 2 contain EHV-4 DNA with distinct deletions within the TK gene.
• the positions of these deletions are dictated by the availability of restriction endonuclease sites within the TK gene which could be utilised in the deletion strategy.
• Both deletions span the N-terminal coding region of the TK gene. Given that this region is likely to contain the promoter for the adjacent UL24-type gene, incorporation of these DNAs into the EHV genome could possibly result in the altered expression of both the UL24-type gene and of the TK gene.
• DNAs were therefore constructed with deletions within the C-terminal coding region of the TK gene in order to ultimately produce EHV recombinants affected solely in TK expression.
• PCR polymerase chain reaction
• the 3' terminal sequences of the following primers were derived from the published sequence information on the TK genes of EHV-1 and EHV-4 (sequences underlined below) .
• pr.imers 2 and 3 the 5' regions of the primers are complementary in order to facilitate annealing of denatured first round amplification products at step 2.
• the PCR technique is carried out as follows. First primers 1 and 2 are hybridized onto the single stranded EHV genome. Then the second strand is extended along the first strand starting from primer 1 using a DNA polymerase until the primer 2 is encountered, when DNA synthesis stops. Similarly a second DNA oligonucleotide strand is synthesised from primer 3 up to primer 4. The strands are then dehybridised into single DNA strands by heating. If necessary the process can be repeated using further quantities of primer in order to amplify the amount of PCR product. 1 ?
• Figure 1 Strategy for the localisation and sequencing of the EHV-4 thymidine kinase gene.
• Figure 3 shows the strategy for deletion of a region of the TK gene using a polymerase chain reaction (PCR) technique according to steps 1 to 3 of Example 4;
• Figure 4. shows the strategy for cloning of the TK- DNA PCR product obtained from Example 4 into a suitable plasmid vector (step 4) .
• Sequence type nucleotide with corresponding protein
• Sequence length 1260 base pairs; 352 amino acids.
• Organism Equine herpesvirus -4..

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