Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
_ 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
- 1 -
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
- 3 -
WO 94/14962 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
- 4 -
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
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`~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
- 7 -
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.
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- 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
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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).
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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.
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