The present invention relates to the production of Varicella-Zoster virus (VZV) immediate early protein 175 and derivates thereof and vaccines for use in the prophylaxis and treatment of VZV infections, comprising such proteins.

Varicella-Zoster Virus (VZV) is a human herpes virus which is the etiological agent of chicken pox (varicella) and shingles (zoster). Varicella results from an initial, or primary infection, usually contracted during childhood which is relatively benign. However, for adults who were not exposed to varicella during childhood, and occasionally to individuals who are immunocomprised, VZV can be life-threatening. Similarly, a VZV infection can be life-threatening to neonates, for the virus is capable of crossing the placenta. With direct contact, varicella is known to be a highly transmissible infectious disease.

Like most Herpes- Viruses, VZV has a tendency to infect some cells in which its development is arrested. After a variable latent period, the Varicella-Zoster (VZ) virus can be released to initiate infection in other cells. This reactivation of the VZ virus causes an estimated 5 million cases of zoster annually (Plotkin £l a . Postgrad Med I £1- 155-63 (1985)). Zoster is characterized by inflammation of the cerebral ganglia and peripheral nerves, and it is associated with acute pain. At present, the factors that reactivate the virus are ill defined.

It has been shown that humans vaccinated with attenuated strains of VZV have received protective immunity from VZV infections (Arbeter et al., Pediatr 100 886-93 (1982) and Brunell et al., Lancet ϋi 1069-72 (1982)). While effective, this method has limitations due to the difficulty of propagating the Varicella-Zoster virus. Considerably effort has been expended to identify antigenic components of the VZ virus. In order to permit development of improved VZ vaccines, especially subunit vaccines, it is important to isolate VZV envelope proteins. Forghani et al. (J Virol. 52:55-62 (1984)), Okuno et al. (Virol. 129:357-68 (1983)) and Keller et al. (J. Virol. 52:293-7 (1984)) have identified numerous virus-specific glycoproteins from VZV- infected cells and VZ virions.

To date efforts in producing a recombinant subunit vaccines against VZV have concentrated on the external envelope glycoproteins as the potential glycoproteins. The present invention departs significantly from this approach and relates to the use of Immediate early, non-structural proteins of VZV to provide protection against subsequent VZV challenge.

Since the mechanism of antigen recognition by Cytotoxic T lymphocytes CTL involves breakdown of native antigen into peptides, binding of the proteolytic fragments to MHC molecules and export of the complex to the cell surface, any virus coded polypeptide not just those that are integral membrane proteins like the

glycoproteins, can be a potential targets of T cell mediated responses. However since the VZV genome codes for several non structural proteins and internal virion proteins, in addition to external glycoproteins, this results in a large number of potential CTL targets and it is not known which protein would be the most relevant. VZV infection is characterized by minimal presence of free virus. During latency and reactivation virus is mainly intracellular. Accordingly, recurrent disease is not prevented even by high levels of neutralizing antibodies and virus control depends on cell mediated immunity. In order to obtain protection by vaccination, it is therefore desirable to induce not just an antibody response, but also a CTL response. An effective vaccine should prime CTL capable of acting as early as possible as soon as signs of reactivation of latent virus appear.

In order to identify the most important CTL target antigens for prophylatic or therapeutic vaccine purposes, the present inventors have taken into consideration the VZV replicative cycle. The beginning of viral protein synthesis inside a cell that harbours viral genome will generate viral protein fragments that will be presented by MHC molecules on the surface of the cell, making it a target for CTL of the appropriate specificity. The replication cycle of VZV lasts about 24 hours and involves an ordered expression of α or immediate early (IE) β or early (E) and γ or late (L) gene products. Therefore early CTL attack and consequent lysis of the infected cells prior to late structural gene expression could prevent new virions being made and therefore prevent spread of the virus to neighbouring cells. In order to be most useful, CTL should detect the very first viral proteins that appear inside the cell after infection and reactivation.

The IE protein IEP 175 is encoded by the open reading frame designated ORF62 and the protein itself is sometimes referred to as IE62.

The protein appears to be a phosphoprotein with a relative molecular weight 175kDa (Kinchinton ≤ i J- of Virology Vol 66(1) (1992) p359-366), but a predicated molecular weight of 140kDa. It is recognised by Human T cells (Bergen et al Viral Immunology 4 (3) 1991 p 151) and has been suggested to be an important immune target (Bergen sLSΪ J. Infectious Diseases (1990) 1£2 p 1049).

There are a number of systems available to the man skilled in the art to produce proteins utilising recombinant DNA techniques, however the majority of these have proved unsuccessful in the production of full length IEP175. For example in insect cells , the protein is degradated. However, the inventors have found that functionally equivalent to native

IEP 175 and structually equivalent (ie the correct size) can be produced by expression in CHO cells.

Accordingly in an embodiment of the present invention their is provided IEP 175 free from VZV contaminants which is functionally equivalent to the native

protein.

The present invention also extends to physiologically functional derivatives of IEP175.

In one aspect of the present invention there is provided an IEP 175 protein devoid of VZV contaminants having an amino acid sequence substantially homologous to the sequence depicted in figure 1 appended hereto.

By substantially homologous it is meant a the invention provides a functionally equivalent IE 175 protein which is at least 75% homologous, preferably 80% more preferably at least 90% and most preferably at least 95% homologous to the amino acid sequence depicted in figure 1.

A preferred derivative of IEP 175 is one which will allow for secretion of the protein on expression in E. Coli or CHO cells. In particular there is provided a secretable derivative in which amino acids 226 to 257 and amino acids 648 to 733 have been deleted. Typical of other immunogenic derivatives will be a fusion polypeptide containing additional sequences which can carry one or more epitopes from other VZV proteins such as VZV glycoproteins eg gpl, gpll, gpIII, gpIV or gpV, (sometimes known as gel, gcll, gcM etc) other VZV antigens, or even other non- VZV antigens eg Hepatitis B surface or core antigens. Alternatively, the immunogenic derivative of the invention can be fused tø a carrier polypeptide which has immunostimulating properties, as in the case of an adjuvant, or which otherwise enhances the immune response to the VZV protein, or which is useful in expressing, purifying or formulating the VZV protein.

A preferred fusion protein comprises an anchorless gpll fused to a secretable form of IEP 175 as described above.

In a further aspect, the present invention provides an expressible DNA molecule encoding IEP 175 or derivatives thereof under the control of a regulatory sequence, which is capable of functioning in a heterologous host. In particular, there is provided a DNA molecule substantially homologous to the DNA sequence as depicted in figure 1 appended hereto. By substantially homologous it is meant a DNA sequence which is at least 75% preferably at least 85% more preferably 90% and most preferably at least 95% homologous to the DNA sequence depicted in figure 1.

DNA sequences encoding IEP 175 or derivatives can be prepared by the addition, deletion, substitution or rearrangement of the bases, by methods well known in the art. In figure 1 , the first ATG codes for a N-teπninal methionine and the last TGA is a translation termination (ie stop) signal.

In a further aspect of this invention there is provided a recombinant DNA molecule or vector comprising a DNA sequence, which codes for Varicella-Zoster

Virus IEP 175 or derivate thereof, operatively linked to a regulatory region which functions in a eukaryotic host cell, most preferably in a CHO cell.

In another aspect of this invention there is provided a process for preparing the Varicella-Zoster Virus IEP175 protein or derivative thereof which process comprises expressing said DNA sequence in a host cell and recovering the protein product.

In related aspects, this invention provides a recombinant CHO cell line transformed with the recombinant DNA molecule.

In a further aspect, the invention provides a process for preparing a VZV IE 175 protein or derivative according to the invention which process comprises expressing a DNA sequence encoding said protein or derivative in a recombinant host cell and recovering the resulting protein product.

The process of the invention may be performed by conventional recombinant techniques such as described in Maniatis £i si, Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, II and III (D.M. Glover ed, IRL Press Ltd).

DNA molecules comprising such coding sequences can be derived from VZV mRNA using known techniques (eg making complementary or cDNAs from a mRNA template) or can be isolated from VZV genomic DNA. See Ecker et al, Proc Natl cad Sci USA 22:156-160 (1982), Straus et al, Prςx? Natf Aςad Sci USA 22:993-7 (1982), Straus et al. J Gen Virol 64:1031-41 (1983) and Davison et al. J Gen Virol £4:1811-1814 (1983). Alternatively the DNA molecules encoding gpl, gpll and gpIII can be synthesized by standard DNA synthesis techniques.

The invention thus also provides a process for preparing the DNA sequence by the condensation of appropriate mono-, di- or oligomeric nucleotide units.

The preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate. Thus, the DNA sequence may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D M Roberts £1 al in Biochemistry 1985, 24, 5090-5098.

The DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.

Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50μg or less with 0.1- lOμg DNA.

Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleotide triphosphates dATP, dCTP, dGTP and dTTP as required at

a temperature of 10°-37°C, generally in a volume of 50μl or less.

Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4°C to ambient, generally in a volume of 50 μl or less. The chemical synthesis of the DNA sequence or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, IQ, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H. Caruthers, Tetrahedron Letters, 1980, 21. 719; M.D. Matteucci and M.H. Caruthers, Journal of the American Chemical Society, 1981. 103. 3185; S.P. Adams £tal. Journal of the American Chemical Society, 1983. 105. 661; N.D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W.D. Matthes ei al., EMBO Journal, 1984, 2, 801. Preferably an automated DNA synthesizer is employed.

The DNA sequence is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the protein. The DNA molecules may be obtained by the digestion with suitable restriction enzymes of vectors carrying the required coding sequences.

The precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired protein product. The design of a suitable strategy for the construction of the DNA molecule coding for the protein is a routine matter for the skilled worker in the art.

The expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the IE175 protein, or derivative under ligating conditions. The ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired. Thus, the DNA sequence may be preformed or formed during the construction of the vector, as desired.

The choice of vector will be determined in part by the host. Most specifically, the preferred host cell of the invention is a CHO cell. Suitable vectors for the host cell of the invention include plasmids, and cosmids.

The preparation of the IE 175 expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis ej L cited above. Polymerisation and ligation may be performed as described above for the preparation

of the DNA polymer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50μl or less with 0.1-10μg DNA.

The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with an expression vector of the invention under transforming conditions. Suitable transforming conditions are conventiional and are described in, for example, Maniatis ei al, cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.

Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells or by electroporation.

Culturing the transformed host cell under conditions permitting expression of the DNA sequence is carried out conventionally, as described in, for example, Maniatis £i ai and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45°C. The VZV IE 175 protein expression product is recovered by conventional methods according to the host cell and whether the product is secreted or released chemically or enzymatically and the protein product isolated from the resulting lysate. Where the product is secretable, the product may generally be isolated from the nutrient medium. The DNA sequence may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the IEP 175 protein; eg bovine papillomavirus vectors or amplified vectors in Chinese hamster ovary cells (DNA cloning Vol.II D.M. ed. IRL Press 1985; Kaufman, R.J. ej al, Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goddel, D.V. et al. European Patent Application No. 0093619, 1983).

In one embodiment of this invention, the VZV IEP 175 protein is expressed in CHO cells. For expression of the IEP 175 protein, the use of the Tdn expression plasmid is preferred. In such system, an expression cassette, comprising the VZV protein coding sequence is operatively linked to the Rous Sarcoma Virus (RSV) promoter. Such vector contains a sufficient amount of bacterial DNA to propagate in E. coli or some other suitable prokaryotic host. Such shuttle vector also contains sufficient amount of eukaryotic DNA flanking the VZV coding sequence so as to permit recombination into the genome of the eukaryotic host and amplification of the integrated DNA using Methotrexate as selective agent.

The promoter of the RSV is preferred because of its high efficiency in promotion of transcription as compared to other promoters.

CHO DHFR cells are preferred because of their sensitivity to methotrexate. The purification of the VZV IEP 175 protein or derivative from cell culture is

carried out by conventional protein isolation techniques, eg selective precipitation, absoφtion chromatography, and affinity chromatography including a monoclonal antibody affinity column.

This invention also relates to a vaccine containing an immunoprotective amount of VZV IEP 175 protein(s) according to the invention. The term

"immunoprotective" refers to a sufficient amount of VZV IEP175 protein(s), when administered to man, which elicits a protective antibody or immune response against a subsequent VZV infection sufficient to avert or mitigate the disease.

Accordingly the present invention provides a vaccine formulation comprising VZV IEP 175 or derivative thereof in admixture with a pharmaceutical carrier, excipient or diluent.

The vaccine of the present invention may additionally contain other antigenic components such as VZV gpl, gpll, gpIII, gpIV or gpV or their truncated derivatives. In particular truncated gpl, gpll or gpIII as disclosed in European Patent application published under No. 0405867.

In a preferred embodiment of the present invention there is provided a vaccine composition comprising an anchorless gpll in combination with IEP 175 or IEP 175 derivative.

By anchorless it is meant, a VZV glycoprotein derivative which is devoid of substantially all of the C-terminal anchor region and which allows for secretion on when expressed in mammalian cells. Such proteins are described in EP-A-0405867. In an alternative embodiment there is provided a vaccine comprising a fusion protein comprising an amino acid sequence embodying IEP 175 or derivative and amino acid sequence embodying one of gpl, gpll, gpIII, gpIV or gpV or derivative thereof.

In a further embodiment there is the use of VZV IEP 175 or derivative thereof for the manufacture of a vaccine for the treatment or prophylaxis of VZV infections.

The present invention also provides VZV IEP 175 or derivative thereof for use in medicine. In a further aspect of the invention there is provided a method of treating a human susceptible to or suffering from VZV infection, which comprises administering a safe and effective amount of a vaccine according to the invention.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 2-100 μg, most preferably 4-40 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisation

adequately spaced.

In addition to vaccination of persons susceptible to VZV infections, the pharmaceutical compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from VZV infections, in order to prevent or significantly decrease recurrent disease, frequency, severity or duration of shingles episodes.

In the vaccine of the invention, an aqueous solution of the VZV IEP 175 protein(s), can be used directly. Alternatively, the VZV IEP 175 protein(s), with or without prior lyophilization, can be mixed together or with any of the various known adjuvants. Such adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A, in particular QS21 or 3 Deacylated monophosphoryl lipid A (3D-MPL). As a further exemplary alternative, the protein can be encapsulated within microparticles such as liposomes. In yet another exemplary alternative, the VZV IEP175 protein(s) can be conjugated to an immunostimulating macromolecule, such as killed Bordetella or a tetanus toxoid.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds), University Park Press, Baltimore, Maryland, 1978. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,954 and Armor et al., US Patent 4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand. 18:349 (1977). 3D-MPL is available from Ribi immunochem, USA, and is disclosed in British Patent Application No. 2,220211 and US Patent 4912094. QS21 is disclosed in US patent No. 5057540.

The examples which follow are illustrative but not limiting of the invention. Restriction enzymes and other reagents were used substantially in accordance with the vendors' instructions.

Example 1

Expression of IEP 175 in cells infected with a vaccinia virus recombinant.

VZV geomic DNA was extracted from viruses recovered from a patient suffering from Varicella (Material provided by Dr. Rentier, Institut de Pathologie, Universitέ de Liege, Sart-Tilman,Liege, Belgium).

The viral DNA was digested with EcoRl and a ~ 16.6 kb-pair fragment, corresponding to bases 100441 to 117034 (Davison et al, J. Gen Virology 67, 1759- 1816, 1986) was isolated then cloned into the EcoiRl site of plasmit pUC9, a standard E. coli cloning vector. From this plasmid, a ~7 Kb Sspl fragment (102241 to 109293) was isolated and cloned into the incil site of pUC19 to create plasmid pNIV2017. This plasmid encodes the entire IEP 175 protein plus 5' and 3' untranslated DNA.

Plasmid pNIV2017 was then digested with a set of restriction enzymes to generate three fragments : a BstXI-BamHI fragment of 2199 bp coding for the N- terminal part of the protein; a BamHI-PpumI fragment of 1002 bp and a Ppuml- Maelll 728 bp fragment coding for the C-terminal part of the protein. Synthetic oligonucleotides were added to these fragments to generate a coding cassette flanked by unique restriction sites. The coding cassette was introduced into pUC19 for preservation (plasmid pNIV2020). The sequence of the oligonucleotides and their junction were confirmed. The IEP 175 coding cassette was recovered from pNIV2020 and then inserted into the transfer vector pULB5213, a derivative of plasmid pSCl 1 described in Chakrabarti et al (Molecular and Cellular Biology 5, 3403-3409, 1985). The final construct, ρNIV2026, is represented in Figure 2.

The recombinant transfer plasmid, pNIV2026, was transfected into vaccinia- infected CV-1 cells and recombinant viruses were isolated after Bromo-Uridine slection and plaque purification on the basis of their blue colour in the presence of X- gal. It will be referred to as VV2026. The human H143 fibroblast TK- strain was used preferably to the RAT2 cells for plaque assays. The vaccinia virus used to infect cells was of the WR type (origin Borysiewicz L.K.). The procedure follows that one previously described for the obtention of vaccinia virus recombinants (Mackett, M. and Smith, G.L., J. Gen. Virology 67, 2067-2082, 1986; Mackett, M., Smith, G.L. and Moss, B., J. Virology 49, 857-864, 1984).

The recombinant vaccinia virus, VV2026, was used to infect CV-1 cells in culture at a multiplicity of infection of 1 (moi 1). Infected cells (about 3 10*5 per assay) and spent culture medium (about 2 ml) were collected between 16 and 17 hours post infection. The presence of the IEP 175 protein was identified by Western blotting experiments. Proteins were resolved onto 12% SDS-polyacrylamide gels, transferred onto nitrocellulose filters and probed with a mouse serus raised against a synthetic peptide derived from the amino acid sequence of the IEP 175 protein (aa

1299 to 1310). Complexes were detected using a goat anti-mouse IgG conjugated to alkaline phosphatase and the appropriate chromogenic substrate, according to standard procedures.

The results show that cells infected with VV2026 effectively accumulate the IEP 175 protein in the cytoplasm but are not able to export it in the medium. The size of the recombinant protein was about 150 K

a) Structure of the DNA insert of pNIV2026 is depicted in figure 2.

Example 2

Expression of IEP 175 in cells insect cells infected with a recombinant Baculovirus.

In view of producing large amounts of the recombinant IEP 175 protein, we used the expression system based on the Baculovirus and insect cells in culture. Starting from plasmid pVIV2020, the cassette coding for IEP 175 was recovered by digestion with EcoRI and Xbal and inserted blunt-ended into the Baculovirus transfer plasmid pAcYMl, previously cut with BamHI and blunted. The resulting recombinant plasmid pNIV2038 thus carries, under the control of the polyhedrin promoter and in the correct orientation, the sequence coding for IEP 175 (Fig.3.)

Plasmid pAcYMl is a Baculovirus shuttle vector containing sequences from the AcMNPV genome which includes the polyhedrin gene promoter, but not the polyhedrin gene, and sequences from a high copy bacterial plasmid, pUC8. See Matauura et al, J. Gen. Virol. 68, 1233-1250 (1987). The recombinant baculovirus transfer plasmid pNTV2038 was introduced by contransfection with the wild type DNA Baculovirus into Spodoptera frugiperda (Sf9) insect cells at a respective ratio of 50 to 1 μg, following published protocols (Summers et al, TAES Bulletin NR 1555, May 1987; Texas Agricultural Experimental Station). Spodoptera frugiperda cells (Sf9) are available from the ATCC (Rockville, Md, USA).

Resulting virus particles were obtained by collecting the supernatants. The virus-containing media was then used to infect Sf9 cells in a plaque assay. Several recombinant Baculoviruses were isolated and purified. They were then used to infect Sf9 cells in Culture. Total proteins of infected cells were recovered at different times post-infection and assayed by Western blotting for the presence of IEP 175, using the mouse antipeptide serum specific for IEP 175 (see supra). In no cases, were we able to demonstrate the expression of a complete IEP 175 in this sytem. We observed however the expression of multiple truncated forms of the protein, indicating that extensive proteolytic degradation occurred intracellularly.

The recombinant baculovirus transfer plasmid pNIV2038 is depicted in figure 3.

Example 3 Expression of IEP 175 in CHO cells

In order to test an additional expression system to obtain the massive expression of IEP 175, we turned to the CHO system.

Starting from plasmid pNIV2020, a PstI (blunted)-PvuII 3946 bp fragment was isolated and inserted between the BgHI (blunted) and EcoRV sites of plasmid TDN to create pNIV2042. Plasmid TDN is described in Connors et al, DNA 7, 651 - 661 (1988). It carries the RSV LTR promoter, the G418 selection marker and the DHFR cassette of amplification. pNVI2042 codes for the signal peptide of the tissue plasminogen activator (tPA) followed by 4 amino acid residues corresponding to the N-terminal amino acid residues of mature tPA, themselves followed by the naturally initiating methionine of EEP 175 and the complete sequence of this protein (Fig.l). Plasmid pNIV2042 was introduced by electroporation into CHO dhfr ^" cells. Selection of recombinant cell lines was done using geneticin (G418) and amplification was performed using methotrexate. All procedures used follow those described in Moguilevsky et al. (Eur. J. Biochem 197, 605-614, 1991). G418- clones were obtained and assayed for the production of the full size

IEP 175 protein using the system described supra. Clones shown to produce the recombinant protein were amplified with methotrexate at different concentrations (from 5 to 50 nM) and retested for production. The results show that IEP 175 is produced efficiently in CHO cells, that it accumulates in the cytoplasm and that its apparent molecular weight is around 175 kDalton. No proteolytic degradation was observed in the CHO expression system in contrast to what was observed in the insect cell sytem. The best producing clone, 18.5.22, was obtained after amplification with 50 nM methotrexate. The production of IEP 175 was monitored using an ELISA involving two mouse antipeptide sera specific for the protein (peptide 1299 to 1310 and peptide 175-436). Western blot analysis, performed as described above, confirmed the structural integrity of the recombinant IEP 175. Unexpectedly, the recombinant IEP 175 was not secreted into the culture medium of CHO cells despite the presence of a signal peptide sequence on the DNA for IEP 175.

The expression plasmid pNIV2042 for CHO cells is depicted in figure 4. In order to verify that the recombinant IEP protein is not only structurally appropriate but also that it exhibits the known regulatory function of the natural protein, we performed the following experiments (references : Jackers et al, 1992; Liny et al, 1992).

CHO. cells expressing the IEP 175 protein, clone 18-5-22, and control CHO

cells were electroporated with a set of plasmids carrying various VZV promotor DNA sequences upstream to the coding sequence for the reporter gene chloramphenicol acyl transferase (CAT). (These plasmids were obtained from Professeus B. Rentier, Institut de Pathologie, Universitέ de Liege, Sart-Tilman, Liege, Belgium). The resulting transfected cell lines were then assayed for the enzymatic activity (CAT assay) which measures potential activation effects of IEP 175 on the function of VZV promotors.

Table 1 summarizes the results of these experiments. It can be seen that the IEP 175 protein, produced in CHO cells, is able to stimulate the promotor activity in several cases. This shows that the recombinant IEP 175 protein behaves in this respect as its natural counterpart.

Table 1: Functional activation of VZV promotor elements by rec IEP 175 (as measured by CAT activity).

VZV promotor element CHO cell line derived from gene Control Clone 18-5-22 Conclusion

no promotor CMV promotor +-H- Constitutive Gene 29 (MDBP) Activation ORF4 +++ No activation

+++ strong CAT activity no CAT activity MDBP major DNA binding protein Pol RNA polymerase

Example 4

Construction of a DNA coding for a secretable IEP 175 protein lacking karyophilic motifs

4a. Plasmid pNIV2020 (see example 1) carries the sequence coding for the complete IEP 175 protein, including the two karyophilic motifs. These have the following amino acid sequences. Motif 1 is comprised between aa residues 226 and 254 and contains the nucleophilic amino acid stretch KSPKKKTLKVK; motif 2 is comprised between aa residues 648 and 733 and contains the nucleophilic amino acid stretch PRKRKS. Digestion of pNIV2020 with a) Aatll and SphI; b) PpumI and BstEII; appropriate blunting, are ligated together to reconstitute a plasmid lacking the DNA sequences containing the karyophilic motifs (Figure 5).

4b. The cassette encoding the resulting truncated IEP 175 is recovered and is inserted into a plasmid downstream to the sequence specifying the tPA signal in the manner described in Example 3.

Transformation of CHO cell with the resulting plasmid will allow for expression of IEP 175 in a secreted form.

Example 5

Construction of IEP 175 gcll fusion

The fragment described in 4b, is inserted in a plasmid as a fusion downstream to the sequence encoding gcll (EP-A-405 867). The resulting plasmid is used to transform CHO cells to enable expression of an IEP 175 truncate gcll truncate fusion protein.