CA2152256A1

CA2152256A1 - Vaccines against varicella-zoster virus (vzv)

Info

Publication number: CA2152256A1
Application number: CA002152256A
Authority: CA
Inventors: Paul Jacobs; Marc Massaer; Michele Haumont; Alex Bollen
Original assignee: Individual
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 1992-12-23
Filing date: 1993-12-17
Publication date: 1994-07-07
Also published as: WO1994014962A1; GB9226768D0; EP0675957A1; MX9307998A; ZA939564B; AU672870B2; NZ259362A; KR950704494A; CN1095106A; JPH08504592A; AU5814494A

Abstract

The present invention discloses methods for the production of Varicella-Zoster virus immediate early protein 175 (IEP 175) and derivatives thereof by recombinant technology - DNA and amino acid sequences are provided for IEP 175, structural or functional homologues, as well as fusion proteins and vectors adapted for eukaryotic host cells. Said protein and derivatives thereof are used as vaccines against VZV.

Description

_ NO 94/14962 2 1 ~ 2 ~ S 6 PCT~EPg3/03626 VACCINES AGAINST VARICELLA-ZOSTER VIRUS (VZV) The present invention relates to the ~r~ucLion of Varicella-Zoster virus (VZV) imm~di~te early protein 175 and derivates thereof and vaccines for use in the 5 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 shirlgles (zoster). Varicella results from an initial, or primary infection, usually contracted during chilrlhood which is relatively benign. However, for adults who were not exposed to varicella during childhood, and occ~cion~lly to individuals who are immllnoco~l",lised, VZV can be life~ ingSimilarly, a VZV infection can be life-l},çe~t~- ~ing to ~-e~tcs, for the virus is capable of crossing the placenta. With direct contact, varicella is known to be a highlytr~ncmiccible infectious tlice~ce Like most Herpes-Viruses, VZV has a tendency to infect some cells in which 15 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 es~ te~ S million cases of zoster annually (Plotkin ~ ~L Pos~rad~l 61: 155-63 (1985)). Zoster is ch~ct~rized by infl~mmqtiQn of the cerebral ganglia and pe.i~lh~ l nerves, and it is associated with acute pain. At present, the 20 factors that reactivate the virus are ill ~lefine~
It has been shown that humans vaccin~tlod with ~ttenn~te~ strains of VZV
have received protective ;-------~nity from VZV infections (Arbeter et al., J. Pediatr 100 886-93 (1982) and Brunell et al., Lancet ii: 1069-72 (1982)). While effective, this method has limitations due to the (liffi~lty of prop~gAting the Varicella-Zoster virus.
25 Con~idçrably effort has been ~ nd~l to identify antigenic con~pone~ of the VZ
virus. In order to permit development of improved VZ vaccines, especially subunit v~c~ines, it is il"~)o.~nt to isolate VZV envelope proteins. I~oI~,l.&ni et al. (~ Virol.
52:55-62 (1984)), Okuno et al. (Virol. 129:357-68 (1983)) and Keller et al. (L Virol.
,~:293-7 (1984)) have identifie~ numerous virus-specific glycoplote;ns from VZV-30 infected cells and VZ virions.
To date efforts in producing a recomkin~nt subunit vaccines against VZVhave conce.~ led on the external envelope glycoproteins as the potential glycopl~teins. The present invention departs significantly ~from this approach and relates to the use of lmm~oAi~e early, non-structural proteins of VZV to provide35 protection against subsequent VZV ch~llenge.
Since the ...ech~ m of antigen recognition by Cytotoxic T lymphocytes CTL
involves breakdown of native antigen into peptides, binding of the proteolytic fr~m~nt~ 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 Wo 94/14962 21 ~ 2 ~ ~ B PCT/EW3/03626 glyco~ teins, can be a potentiai targets of T cell m~Ai~te~l responses. However since the VZV genome codes for several non structural proteins and internal virion proteins, in addition to extemal glyco~)roteins, this results in a large number of potential ClL targets and it is not known which protein would be the most relevant.
VZV infection is ch~u~ ized by minim~l 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 epen~1c on cell m~ tç~ .-U,lity. In order to obtain protection by vaccin~tion, it is therefore desirable to induce not just an antibody lc;"~nse, 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 i""x"l~nt 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 lS harbours viral genome will generate viral protein fr~gm~nt~ that will be presented by MHC molecules on the surface of the cell, making it a target for CTL of the app~ iate srecificity. The replic~tion cycle of VZV lasts about 24 hours and involves an ordered eApçession of a or immeAi~te early (E) ~ or early (E) and ~y or late (L) gene products. Thel~,fc,.c early CTL attack and consequent Iysis of theinfected cells prior to late ~lluclul~l gene eA~Iession could prevent new virions being made and therefore prevent spread of the virus to neighbouring cells. In order to be most useful, CI'L should detect the very first viral plolcins that appear inside the cell after infection and reactivation.
The IE protein IEP 175 is encoded by the open reading frame ~lesign~t~d 25 ORF62 and the protein itself is so~ es referred to as IE62.
The protein ap~,eals to be a ~)hos~,hopl~,tein with a relative molecular weight 175kDa (Kinchinton et al J. of Virology Vol ~Ç(l) (1992) p359-366), but a pre-lic~ted molecular weight of 140kDa. It is l~,cognised by Human T cells (Bergen et al Viral lmmllnology 4 (3) 1991 plS1) and has been suggested to be an i,.,~.~nt immune target (Bergen et al J. Infectious Diseases (1990) 162plO49).
There are a nu...ber of systems available to the man skilled in the art to produce ~ ,teins utilising recombinant DNA techniques, however the majority of these have proved nn~ucces~ful in the production of full length IEP175. For example in insect cells, the protein is degradated.
However, the inventors have found that function~lly equivalent to native EP175 and ~l~uclually equivalent (ie the correct size) can be produced by expression in CHO cells.
Accordingly in an embodiment of the present invention their is provided IEP175 free from VZV cont~min~nt~ which is functionally equivalent to the native - 2 -rvo 94/14962 2 1 5 2 2 5 6 PCTIEP93/03626 protein.
The present invention also e~ctends to physiologically functional derivatives ofIEP175.
In one aspect of the present invention there is provided an IEP 175 protein S devoid of VZV cont~minAnts having an amino acid sequence subst~ntiAlly homologous to the sequence depicted in figure 1 ap~ d hereto.
By s~lbst~nti~lly homologous it is meant a the inwntion provides a functionally equivalent E 175 protein which is at least 75% homologous, preferably 80% more preferably at least 90% and most preferably at least 95% homologous to 10 the amino acid se~uence depicttod in figure 1.
A plcf~ d derivative of EP 175 is one which will allow for secretion of the protein on e~,res~ion in E. Coli or CHO cells. In particular there is provided asecretable derivative in which amino acids 226 to 257 and amino acids 648 to 733have been deleteA
Typical of other imrnunogenic derivatives will be a fusion polypeptide contAining additional sequences which can carry one or more ~.pilopf,s from other VZV proteins such as VZV glycoploleins eg gpl, gpII, gpIII, gpIV or gpV, (sG...e~ r,s known as gcI, gcII, gcIlI etc) other VZV antigens, or even other non-VZV antigens eg Hep~titic B surface or core ~nti~nc ~lt~ ely, the 20 hllln~ ogenic derivative of the invention can be fused to a ca~Tier polypeptide which has immunostiml-lAting l,r~p~,l~ies, as in the case of an adjuvant, or which otherwise çnh~nces the i....~ e response to the VZV protein, or which is useful in e~pr,ssi-lg, purifying or form--lAting the VZV protein.
A pler~ d fusion protein c~ ,lises an A~-cllo~less gpII fused to a secretable 25 form of EP 175 as described above.
In a further aspect, the present invention provides an ~ ,ssible DNA
m~xule encofling EP 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 substAn-i~lly homrlogous to the DNA sequence as 30 depicted in figure 1 appended hereto. By subs~ t;~lly ~ mologous 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 enco ling EP 175 or derivatives can be ~ d by the 35 addition, deletion, substitution or rearrange-..f,l-l of the bases, by methods well known in the art. In figure 1, the first ATG codes for a N te- ~..;n~l methionine and the last TGA is a translation termination (ie stop) signal.
In a further aspect of this invention there is l,lu.ided a l~on-hin~nt DNA
molecule or vector comprising a DNA sequence, which codes for Varicella-Zoster 2 1~ 2 ~ 5 ~ PCT/EP93/03626 Virus IEP175 or derivate thereof, operatively linked to a regulatory region w`hich 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 S comprises e~ s~.ing said DNA sequence in a host cell and recovering the protein product.
In related acpectc7 this invention provides a recombinant CHO cell line tran~rcl.l.cd with the ~co--lbillant 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 ~,Apl~ 7ing a DNA se~u~,nce enco~ling said protein or derivative in a recombinant host cell and recovering the resulting protein product.
The process of the invention may be ~elrc,l-l-ed by convçntior-~l recombinant techniques such as described in Maniatis ~ al, Molecular Cloning - A Labol~toly Manual; Cold Spring Harbor, 1982 and DNA Cloning vols I, Il and III (D.M. Glovered, IRL Press Ltd).
DNA mole~lules comprising such coding sequences can be derived from VZV
mRNA using known techniques (eg making complc--,~,nt~y or cDNAs from a mRNA
templ~e) or can be icol~ted from VZV genomic DNA. See Ecker et al, Proc Ns~tl Acad Sci USA 79:156-160 (1982), Straus et al, Proc Natl Acad Sci USA 79:993-7 (1982), Straus et al, J Gen Virol 64:1031-41 (1983) and Davison et al, J Gen Virol 64:1811-1814 (1983). Alternatively the DNA molecules enco~lin~ gpI, gpII and gpIII
can be synth~ci7ed by standard DNA synthesis techniques.
The invention thus also provides a process for ~,~pa,ing the DNA sequence by the con~enc~tion of ap~).o~,liate mono-, di- or oligomeric nucleotide units.
The ~ al~tion may be carried out chemically, enzym~ic~lly~ or by a cc,.llbination of the two methofls, in vitro or in vivo as ap~,lo~,liate. Thus, the DNA
sequence may be ~ pd~,d by the enzymatic ligation of appr~,iate DNA fr~gmontc, by conve-ntion~l methor1c such as those described by D M Roberts et al in Biochemistry 1985, 24, 5090-5098.
The DNA fr~gm~ntc may be obtained by digestion of DNA cont~inillg the required sequences of nucleotides with ap~ pliate restriction enzymes, by chemic~l synthesic, by enzymatic polymerisation, or by a combination of these methods.
Digestion with restriction enzymes may be performed in an ap~,u~,liate buffer at a te.ll~ ure of 20-70C, generally in a volume of 50~1g or less with 0.1-lO~g DNA.
Enzymatic polymerisation of DNA may be carried out in vitro using a DNA
polymerase such as DNA polymerase I (Klenow fragment) in an a~ liate buffer containing the nucleotide ~liphos~)hates dATP, dCTP, dGTP and dTTP as required at WO 94/14962 21 5 2 2 ~ 6 PCT/EP93/03626 a le,--pe-~ture of 10-37C, generally in a volume of 50~1 or less.
Enzymatic ligation of DNA fragmentc may be carried out using a DNA ligase such as T4 DNA ligase in an ~y~)r~,'iate buffer at a te..,p~.~ture of 4C to ambient, generally in a volume of 50 111 or less.
S The c-h~m;~l synthesis of the DNA sequence or fragment~ may be carried OUt by convention~l ~,ho~,hotriester, phosphite or phosyhc.~..;dite chemistry~ using solid phase techniques such as those described in 'chemic~l and Enzymatic Synthesis of- Gene Fragments - A Laboratory Manual' (ed H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific public~tions, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 1Q, 6243; B.S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771;
M.D. I~ ,cci and M.H. Caruthers, Tetrahedron Letters, 1980, ~, 719; M.D.
M~tte~lcci and M.H. C~UIh~ Journal of the American Chçmir~l Society, 1981, 103, 3185; S.P. Adams ~ al., 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 et al., EMBO Journal. 1984, 3, 801. Preferably an ~u~ t~ DNA ~r..ll.PS;~f,r is employed.
The DNA s~uenre is preferably yl~ d by lig~ting two or more DNA
molec~lles which t~)gell,er comprise a DNA sequence encoAing the protein.
The DNA mo!e~ules may be obtained by the digestion with suitable restriction enzymes of vectors carrying the lequi-~d coding sequences.
The precise ~I-uclu.e of the DNA mole~ es and the way in which they are oblaih~d depends upon the SlluClul~ of the desired protein product. The design of a suit~ble strategy for the construction of the DNA molecule coding for the protein is a routine matter for the skilled worker in the art.
The eA~ ion vector may be p.el)~ in acco-dance with the invention, by cleaving a vector ~o...~ ible with the host cell to provide a linear DNA seg~ent and co...bining said linear seglllenl with one or more DNA molecules which, togetherwith said linear seg.~.ent encode the IE175 protein, or derivative under lig~ting 30 cQnAitions. The ligation of the linear seg.~ t and more than one DNA molec lle may be ca~ied out s~ neously or seqsenti~lly as desired. Thus, the DNA sequence may be ~JIefc.llll~ or formed during the construction of the vector, as desired.The choice of vector will be detel,...ned in part by the host. Most specifi~lly,the p.~,fel.ed host cell of the invention is a CHO cell. Suitable vectors for the host 35 cell of the invention include pl~cmi(ls~ and cosmi-1~
The ~ tion of the IE175 eA~ ssion vector may be carried out convention~lly with ~plol,-iate en~...es for restriction, polymerisation and ligation of the DNA, by ~ cedul~,s described in, for example, Maniatis ~ al., cited above.
Pol~...elisalion and ligation may be p~,rc, ..-ed as described above for the preparation WO 94il4962 2 15 ~ ~ 5 ~ PCT/EP93/03626 of the DNA polymer. Digestion with restriction enzymes may be ~Ç~I"ed in an al)~.u~fiate buffer at a h..-pc.ature of 20-70C, generally in a volume of 50~11 or less with 0.1~ Lg DNA.
The ,cccs..-binant host cell is y,~,parcd, in accor~ance with the invention, by S transforming a host cell with an expression vector of the invention under transforming con-lition.c. Suitable transforming con~ nc are conven~ r ql and are described in, for example, Mqni~tic et ~, cited above, or "DNA ~loning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.
~qmmqliqn cells in culture may be transr(""-ed by calcium co-~,icci~i~tion of the vector DNA onto the cells or by clecl~u~, a~ion.
~ultnring the transformed host cell under cQn-litiQn~ ~.",iuing ~,A~,~,ssion of the DNA se~u~,nce is carried out conventiQIlqlly, as described in, for exarnple,Maniatis ~ al and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutrient and cultured at a le.,Jpclalu,c below 45C.
The VZV IE175 protein ex~,,cs~ion ~r~ucl is recovered by conventional methods according to the host cell and whether the product is s~x,eted or released chemic~lly or enzym~tic~lly and the protein product i~ol~tsA from the resulting lysate.
Where the product is secretable, the product may generally be isol~ted from the nutrient ...~.1;.....
The DNA sequence may be r--e~-'-'ed into vectors designed for ico!atioll of stable transformed m~mm~ n cell lines cA~ ssing the EP 175 ~lulcin; eg bovine papillomavirus vectors or amplified vectors in Chinese h~ t~,~ ovary cells (DNA
cloning Vol.II D.M. ed. IRL Press 1985; K~--fm~n, R.J. ~ al, M-:>lr ~ r and Cellular Biology 5, 175~1759, 1985; Pavlakis G.N. and Hamer, D.H., I~uceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goddel, D.V. et al, Eur~)peall Patent Applicatiûn No. 0093619, 1983).
In one e-m~im~ t of this invention, the VZV IEP175 protein is expressed in CHO cells. For eAIJr~,s~iûn of the EP 175 prûtein~ the use of the Tdn eAp,~ssionplasmid is pl.,f~.lcd. In such system, an cA~"~s~ion c~csetre, comprising the VZV
protein coding se.lu~,nce is operatively linked to the Rous Sarcoma Virus (RSV) ~lo.l~otcl. Such vector contains a sufficient amount of bacterial DNA to propag~te in E. cûli or some other suitable prokaryotic host. Such shuttle vector also Cûnt~in~
sllffi~ient amount of eukaryûtic DNA flanking the VZV coding sequence so as to permit reco~ ion into the genome of the eukaryotic host and arnplification of the integrated DNA using Methotrexate as selective agent.
The ~ ,-,oter of the RSV is preferred because of its high efficiency in "~",olion of transcription as cou-~J~ucd to other plolllolcl~.
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 `~0 94/14962 ~1~ 2~6 PCT/EP93/03626 carried out by conventional protein isolation techniques, eg selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
This invention also relates to a vaccine containing an i,l"llunoplot~i~e S amount of VZV IEP175 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 illllllune response against a subsequent VZV infection sufficient to avert or mitigate the ~ice~ce Accordingly the present invention provides a vaccine formulation comprising 10 VZV IEP 175 or derivative thereof in admixture with a pharm~ceutic~l carrier, excipient or diluent.
The vaccine of the present invention may additionally contain other 3ntigenic components such as VZV gpl, gpll, gpIII, gpIV or gpV or their tnlnc~ted derivatives.
In particular truncate(i gpl, gpll or gpIII as disclosed in European Patent application lS published under No. 0405867.
ln a pl~;fell~d embodiment of the present invention there is provided a vaccine composition comprising an anchorless gpll in combination with IEP 175 or EP 175 derivative.
By anchorless it is meant, a VZV glycoplotein derivative which is devoid of 20 su~st~nti~lly all of the C-terminal anchor region and which allows for secretion on when e~plessed in m~mm~ n cells. Such proteins are described in EP-A-0405867.
In an alternative embodiment there is provided a vaccine com~ illg a fusion protein comprising an amino acid sequence embodying IEP 175 or derivative and amino acid sequence embodying one of gpI, gpII, gpIIl, gpIV or gpV or derivative25 thereof.
In a funher embo~liment there is the use of VZV IEP 175 or derivative thereof for the m~nl-factllre of a vaccine for the treatmsnt or prophylaxis of VZV infections.
The present invention also provides VZV IEP 175 or derivative thereof for use in medicine. In a funher aspect of the invention there is provided a method of 30 treating a human susceptible to or suffering from VZV infection, which colll~"ises ~mini~t~rjng 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 immunogen35 is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 ~g of protein, preferably 2-100 ~g, most preferably 4-40 llg. Anoptimal amount for a panicular vaccine can be ascenained by standard studies involving observation of a~lu~fiate immune responses in subjects. Following an initial vaccin~tion, subjects may receive one or several booster i-....---.-i~tion Wo 94/14962 PCT/EP93/03626 21522~S
adequately spaced.
In addition to va~cin~tion of persons susceptible to VZV infections, the pharrnaceutical co,-,po~itions of the present invention may be used to treat, immunotherapeutically, patients suffering from VZV infections, in order to prevent or S significantly decrease recurrent riise~ce~ frequency, severity or duration of shingles episodes.
In the vaccine of the invention, an aqueous soluuon of the VZV IEP175 protein(s), can be used directly. Alternatively, the VZV IEP175 protein(s), with or without prior Iyophilization, can be mixed together or with any of the various known adjuvants. Such adjuvants include, but are not limited to, ~luminium hydroxide, alllyl dipeptide and saponins such as Quil A, in particular QS21 or 3 Deacylatedmonuphos~horyl lipid A (3D-MPL). As a further exemplary altemative, the protein can be enc~psul~ted within microparticles such as liposomes. In yet another exemplary altemative, the VZV IEP175 protein(s) can be conjugated to an immunostim~ ting ",a~,v",olecule, such as killed Bordetella or a tetanus toxoid.Vaccine pl~palation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds), University Park Press, Baltimore, Maryland, 1978.
Fncars~ tion within liposomes is described by Fullerton, US Patent 4,235,877.
Conjugation of pmvlcins to ",ac.o",olecules is disclosed, for example, by ~ ikhite, 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 ;-- -- ~nOChf~ , USA, and is disclosed in British Patent Application No. 2,220211 and US Patent 4912094. QS21 is ~ close~ 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.
.

'1VO 94/14962 21 5 2 2 ~ 6 PCT/EP93/03626 Example 1 Expression of IEP 175 in cells infected with a vaccinia virus recombinant.
VZV geomic DNA was ~AIIaeled from viruses recovered from a patient suffering from Varicella (Material provided by Dr. Rentier, Institut de Pathologie, 5 Université de Liège, Sart-Tilm~n~l iège, Belgium).
- The viral DNA was ~ e-stçd with EcoR1 and a - 16.6 kb-pair fT~m~nt, co~ ,onding to bases 100441 to 117034 (Davison et al, J. Gen Virology 67, 1759-1816, 1986) was icol~ted then cloned into the EcoiR1 site of plasmit pUC9, a standard E. coli cloning vector. From this plasmid, a ~7 Kb Sspl fragment (102241 10 to 109293) was isolated and cloned into the incil site of pUC19 to create pl~cmi~l pNIV2017. This plasmid enco~les the entire IEP 175 protein plus 5' and 3' untr~n~l~te~ DNA.
Plasmid pNIV2017 was then digested with a set of restliction enL~nleS to generate three f.dg-"ents: a BstXI-BamHI fragment of 2199 bp coding for the N-15 terminal part of the protein; a BamHI-PpumI fragment of 1002 bp and a Ppuml-MaeIII 728 bp fragment coding for the C-terminal part of the protein. Synthetic oligorn1cleotides were added to these fra~mentc to gel-~late a coding c~c-~el~e fl~nk~d by unique restriction sites. The coding c~csette was int~duced into pUC19 for preservation (pl~cmid pNIV2020). The sequence of the oligonuc~ ;dçs and their 20 junction were conr..l..ed. The IEP 175 coding c~csette was recovered from pNIV2020 and then inserted into the transfer vector pULB5213, a derivative of plasmid pSC11 described in Chakrabarti et al (Molecular and C'ell~ Biology 5, 3403-3409, 1985). The final construct, pNIV2026, is leplcsel-ted in Figure 2.
The reco.~.binant transfer pl~cmirl. pNlV2026, was tl~1sÇe~,t~d into vaccinia-25 infected CV- 1 cells and recombin~nt 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 ~,r~cedu.~, follows that one 30 previously described for the obtention of vaccinia virus lecol~lh;l~ntc (Macken, 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 ,~co--,binant vaccinia virus, VV2026, was used to infect CV-1 cells in culture at a multiplicity of infection of 1 (moi 1). Infected cells (about 3 105 per 35 assay) and spent culture m~inm (about 2 ml) were collected ~I.. ~n 16 and 17 hours post infection. The presence of the IEP 175 protein was i~lentifie~l by Western blotting e~h,.,.,lents. 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 g Wo 94/14962 ~ 1 5 2 ~ PCT/EW3/03626 1299 to 1310). Complexes were detected using a goat anti-mouse IgG conjugated toalkaline phosphatase and the appropriate chromogenic substrate, according to standard procedures.
The results show that cells infected with VV2026 effectively acc~mul~te the S IEP 175 protein in the cytoplasm but are not able to export it in the merlillm The size of the l~co",binant protein was about 150 K

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

10 Example 2 Expression of IEP 175 in cells insect cells infected with a recolnbir~nt Baculovirus.
In view of producing large amounts of the ,~,co,llbinant IEP 175 protein, we used the e~,lcssion system based on the Baculovirus and insect cells in culture.Starting from plasmid pVIV2020, the c~ccette coding for IEP 175 was recovered by digestion with EcoRI and XbaI and inserted blunt-ended into the Baculovirus transfer plasmid pAcYM I, previously cut with BamHI and blunted. Theresulting l~."hin~nt plasmid pNIV2038 thus carries, under the control of the polyhedrin promoter and in the correct cfier,tation, the sequence coding for EP 175 (Fig.3.) Plasmid pAcYM I is a Baculovirus shuttle vector containing sequences from the AcMNPV geno,lle which includes the polyhedrin gene pn ,llotc., but not the polyhedrin gene, and sequences from a high copy bacterial pl~cmid~ pUC8. See Matauura et al, J. Gen. Virol. 68, 1233-1250 (1987).
The recombinant baculovirus transfer pl~cmid pNIV2038 was introduced by contransfection with the wild type DNA Baculovirus into Spodoptera frugiperda (Sf9) insect cells at a l~s~ ive ratio of 50 to 1 yg, following published plolocols (Su~ w~ et al, TAES Bulletin NR 1555, May 1987; Texas Agricultural E~ ,.imental Station). Spodoptera frugiperda cells (Sf9) are available from the ATCC (Rockville, Md, USA).
Resulting virus particles were obtained by collecting the supern~t~ntc. The virus-co~ ining media was then used to infect Sf9 cells in a plaque assay. Several eco,llbinant 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 Westem 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 d~,n~olw.ale the expression of a complete IEP 175 in this sytem. We observed ho~ e~ the expression of multiple tmnc~t~d fomms of the protein, indicating thatextensive proteolytic degradation occull~,d intracellularly.

Wo 94/14g62 215 2 2 5 g PCT/EP93/03626 - The recombinant baculovirus transfer plasmid PNIV2038jS depicted in figure 3.

F~Y~r~PIe 3 S Expression of IEP 175 in CHO cells - In order to test an additional eA~Iession system to obtain the massive eA~,ression of EP 175, we turned to the CHO system.
Starting from plasmid pNlV2020, a PstI (blunted)-PvuII 3946 bp fragment was isolated and inserted between the BgIII (blunted) and EcoRV sites of plasmid10 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 c~csette of amplification. pNVI2042 codes for the signal peptide of the tissue plasminogen activator (tPA) followed by 4 amino acid residues co~ onding to the N-terminal amino acid residues of mature tPA, themselves followed by the naturally 15 initi~ting methionine of IEP 175 and the complete sequence of this protein (Fig.l).
Plasmid pNIV2042 was introduced by ele~ Jpolation into CHO dhfr~ cells.
Selection of recombinant cell lines was done using geneticin (G418) and ~rnrlifir~tiQn was perforrned using methotrexate. All ~loccdul~,5 used follow those described in Moguilevsky et al. (Eur. J. Biochem 197, 605-614, 1991).
G41 8R clones were obtained and assayed for the production of the full size E P 175 protein using the system described supra. Clones shown to produce the recombinant protein were ~mplified with methotrexate at different conce,.tlations (from 5 to 50 nM) and retested for production. The results show that EP 175iS
produced efficiently in CHO cells, that it accl-mul~tçs in the cytoplasm and that its 25 a~,pdrent molecular weight is around 175 kDalton. No proteolytic degradation was observed in the CHO eA~I~,ssion 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 ..o~ o-~d using an ELISA
involving two mouse antipeptide sera specific for the protein (peptide 1299 to 1310 30 and peptide 175-436). Western blot analysis, performed as described above, conrl~ ed the structural integrity of the recombinant EP 175. UncA~,ec~edly, thereCOnIbiI1ant E P 175 was not secreted into the culture medium of CHO cells despite the presence of a signal peptide sequence on the DNA for EP 175.
The expression plasmid pNIV2042 for CHO cells is depicted in figure 4.
In order to verify that the recombinant EP protein is not only structurally apl,lol,liate but also that it exhibits the known regulatory function of the natural protein, we ~ rolll.ed the following eA~ illlents (ref~l~,nces: Jackers et al, 1992;
Liny et al, 1992).
CHO cells expressing the E P 175 protein, clone 18-5-22, and control CHO

.

Wo 94/14962 21~ 2 2 S 6 PCT/EW3103626 cells were elecL.u,)cl~ted with a set of pl~cmi~ls carrying various VZV promotor DNA
sequences u~)sl~calll to the coding s~uence for the reporter gene chloramphenicol acyl transferase (CAT). (These p1~cmi~ls were obtained from Professeus B. Rentier, Institut de Pathologie, Université de Liège, Sart-Tilman, Liège, Belgium). The 5 res~ ing transrectcd cell lines were then assayed for the enzymatic activity (CAT
assay) which l~,~sur-,s potential activation effects of IEP 175 on the function of VZV
promotors.
Table 1 s~ izes the results of these eA~ "ents. It can be seen that the EP 175 protein, produced in CHO cells, is able to stimlll~te the promotor activity in 10 several cases. This shows that the ~ --bi~nt IEP 175 protein behaves in this respect as its natural Coulllu~
Table 1: Functional activation of VZV ~,onlolor ek...~nt~ by rec IEP 175 (as measured by CAT activity).

VZV p,u,notor elementCHO cell line derivedfrom gene Control Clone 18-5-22 Conclusion no ~.r~",otor CMV ~)~U~olol ~++ +++ Co~ e Gene 29 (MDBP) - l l I Activation ORF4 +++ +++ Noactivation +++ strong CAT activity - no CAT activity MDBP major DNA binding protein Pol RNA polymerase Example 4 Construction of a DNA coding for a secretable ~P 175 protein lacking karyophilic motifs 4a. Plasmid pNlV2020 (see eY~mrle 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 and254 and contains the nucleophilic amino acid stretch KSPKKK l LKVK; motif 2 is comprised between aa residues 648 and 733 and con~ins the nucleophilic amino acid stretch PRKRKS.
Digestion of pNIV2020 with a) AatII and SphI; b) PpumI and BstEII;
ap~,l~,iate bhlnting, are ligated together to l~or,s~i~ut~ a plasmid lacking the DNA
sequences cont~ining the karyophilic motifs (Figure 5).

WO 94/l4g62 2~ 1 5 2 2 5 6 PCT/EP93/03626 4b. The c~cset~e encoding the resulting truncated IEP 175 is recovered and is inserted into a plasmid do~n~l,call, to the sequence specifying the tPA signal in the manner described in Ex~mple 3.
Transformation of CHO cell with the resulting plasmid will allow for 5 expression of IEP 175 in a secreted form.

Example 5 Construction of EP 175 gcll fusion The fragment described in 4b, is inserted in a pl~cmi~ as a fusion downstream to the sequence encoding gcII (EP-A-405 867). The res~lting plasmid is used to transform CHO cells to enable e~,lession of an IEP 175 tluncate gcII truncate fusion protein.

Claims

1. An isolated recombinantly produced VZV IEP 175 free from VZV
contaminants which is functionally equivalent to native VZV IEP 175 and physiologically functional derivatives thereof.

2. A VZV IEP 175 according to claim 1, having an amino acid sequence substantially homologous to the sequence depicted in figure 1.

3. A physiologically functional derivative of VZV IEP 175 according to claim 1, which is secretable on expression from a suitable host.

4. A derivative according to claim 3 wherein amino acids 226 to 257 and 648 to 733 have been deleted.

5. A fusion protein wherein a portion of the fusion protein comprises a VZV IEP
175 or physiologically functionally derivative according to claims 1 to 4.

6. A fusion protein according to claim 5 a portion of which comprises an anchorless gpII protein derivative from VZV.

7. A vector comprising a DNA sequence encoding a protein according to any one of claims 1 to 6 operatively linked to a regulatory region which functions in a eukaryotic host cell.

8. A process for producing a protein according to any one of claims 1 to 6 comprising transforming a host with a vector according to claim 7, and recovering the resulting protein produced.

9. A vaccine composition comprising a protein according to any one of claims 1 to 6 inadmixture with a pharmaceutically acceptable diluent, excipient or carrier.

10. VZV IEP 175 or physiologically functional derivative thereof according to any of claims 1 to 6 for use in medicine.

11. Use of VZV IEP 175 or physiologically functional derivative thereof according to any of claims 1 to 6 for the manufacture of a vaccine for prophylatically treating a subject susceptible to VZV infections.

12. A method of prophylatically treating a subject susceptible to VZV infectionscomprising admixing a non-toxic efficacious dose of a protein according to claims 1 to 6.