Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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REDOX REVERSIBLE HCV PROTEINS with NATIVE-LIKE CONFORMATION
FIELD OF THE INVENTION
The present invention relates to HCV proteins in which cysteine residues are
reversibly
protected during purification. Eventually, this purification procedure results
in HCV proteins
with biological activity and a native-like protein conformation, which present
corresponding
epitopes. The present invention pertains also to drug screening methods using
these HCV
proteins, and diagnostic and therapeutic applications, such as vaccines and
drugs.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) infection is a major health problem in both developed
and developing
countries. It is estimated that about 1 to 5 % of the world population is
affected by the virus.
HCV infection appears to be the most important cause of transfusion-associated
hepatitis and
frequently progresses to chronic liver damage. Moreover, there is evidence
implicating HCV
in induction of hepatocellular carcinoma. Consequently, the demand for
reliable diagnostic
methods and effective therapeutic agents is high. Also sensitive and specific
screening
methods of HCV-contaminated blood-products and improved methods to culture HCV
are
needed.
HCV is a positive stranded RNA virus of approximately 9,600 bases which encode
at least
three structural and six non-structural proteins. Based on sequence homology,
the structural
proteins have been functionally assigned as one single core protein and two
envelope
proteins: E1 and E2. The E1 protein consists of 192 amino acids and contains 5
to 6 N-
glycosylation sites, depending on the HCV genotype. The E2 protein consists of
363 to 370
amino acids and contains 9-11 N-glycosylation sites, depending on the HCV
genotype (for
reviews see: Major and Feinstone, 1997; Maertens and Stuyver, 1997). The E1
protein
contains various variable domains (Maertens and Stuyver, 1997), while the E2
protein
contains three hypervariable domains, of which the major domain is located at
the N-terminus
of the protein (Maertens and Stuyver, 1997). These envelope proteins have been
produced
by recombinant techniques in Escherichia coli, insect cells, yeast cells and
mammalian cells.
NS2, NS3, NS4A, NS4B, NS5A and NSSB are non-structural (NS) proteins. NS3 is
about 70
kDa, and has protease and helicase activity. The sequences in NS3 that are
essential for the
helicase activity also have RNA binding, Mg" binding, and ATP binding
properties. Anti-NS3
antibodies often appear first in sero-conversion series. The immuno-reactivity
of the NS3
protein seems to be different in the various commercial assays available
today.
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To date, vaccination against disease has been proven to be the most cost
effective and
efficient method for controlling diseases. Efforts to develop an efficacious
HCV vaccine,
however, have been plagued with difficulties. A conditio sine qua none for
vaccines is the
induction of an immune response in patients. Consequently, HCV antigenic
determinants
should be identified, and administered to patients in a proper setting.
Antigenic determinants
can be divided in at least two forms, i.e. lineair and conformational
epitopes. Conformational
epitopes result from the folding of a molecule in a three-dimensional space.
In general, it is
believed that conformational epitopes will realize the most efficacious
vaccines, since they
represent epitopes which resemble native-like HCV epitopes. However, there are
seemingly
insurmountable problems with culturing HCV, that result in only minute amounts
of virions. In
addition, there are vast problems with the expression and purification of
recombinant proteins,
that result in not properly folded proteins. Therefore, the in vivo structure
of most HCV
proteins is obscure, and hence no solid study on conformational epitopes has
been
conducted.
In addition, the lack of suitable in vitro cultivation systems and small
animal models has
severely impeded the development of new antiviral drugs for hepatitis C
infections. The
chimpanzee is the only available model today for the study of HCV infection,
prophylaxis and
therapy, but the system only allows to study previously selected compounds.
It has been suggested that the E1 envelope protein needs the E2 envelope
protein to reach a
proper folding status (Deleersnyder et al., 1997). In addition, it has been
suggested that E1
and E2 form heterodimers which may form the basic unit of the viral envelope
(Yi et al.,
1997). But, Houghton (1997) reported that repeated immunizations with
recombinant gpE1 E2
(4 x 25 Ng) of 3 chronically HCV-infected chimpanzees did not induce a
significant immune
response. The induction of an anti-envelope immune response in patients with
hepatitis C
would indeed be desirable and beneficial to the patient, since higher levels
of such antibodies
seem to correlate with good response to interteron therapy, and may therefore
help the
patient to clear the virus (PCT/EP 95/03031 to Maertens et al.). The antibody
levels against
E1 in chronic HCV carriers are among the lowest of all HCV antibodies, it may
therefore be
beneficial to raise those antibody levels, and possibly the cellular response,
to induce control
or even clearance of the infection by the host. Also, higher levels of
cellular immunity against
E1 seem to correlate with good response towards interferon therapy (Leroux-
Roels et al.,
1996). Importantly, the above described studies did not rely on native-like E1
peptides.
The most crucial epitopes in NS3 for detection of HCV positive sera are
related to
conformational epitopes. Apparently, NS3 epitopes are scattered all over the
NS3 protein
(see also Leroux-Roels et al. 1996; Rehermann et al., 1996, 1997; Diepolder et
al., 1995,
1997). In assays foremost the NS3 protein has been employed instead of
peptides.
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3
Advances in molecular biology and genetic engineering have made it possible to
produce
large amounts of protein products using heterologous expression systems. The
use of
heterologous hosts, however, can lead to differences in the biological and/or
structural
properties of the recombinant product. Amongst the biochemical modifications
that can occur
to proteins during or following the synthesis in the cell and the subsequent
purification, the
formation and sustainment of disulphide bonds is of importance. Cysteine redox
status is
intricately linked to the correct folding or assembly of disulphide-bonded
proteins. Moreover,
very often the biological function of a protein is regulated or at least
influenced by the state of
oxidation of its sulfhydryl groups. This is the case for some enzymatic
activities where the
reversibility and timing of oxidation of sulfhydryl groups has been proposed
as a physiological
control mechanism (see also Thomas et al., 1995; Nakamura et al., 1997; Aslund
and
Beckwith, 1999).
Several protein factors that catalyze the cysteinyl redox status (thiol versus
disulphide bond
formation) have been characterised (Mossner et al, 1998; Prinz et al, 1997;
Loferer &
Hennecke,1994). Predominantly, these protein factors belong to the
"thioredoxin protein
superfamily", of which the members contain 2 redox-active cysteines in the Cys-
X-X-Cys
consensus sequence (X = any amino acid). This superfamily can be divided in
different
classes on the basis of the redox potential of the active site, substrate
specificity or biological
activity. Another classification relies on the consensus sequence of the redox-
active centre,
namely:
(i) One class, commonly represented by Thioredoxin (TRX), consists of small
ubiquitous
proteins. The redox-active centre has the consensus sequence Cys-X-Pro-Cys,
that is highly
conserved in many species, ranging from bacteria to plants and mammals (X =
any amino
acid). Oxidised TRXox is regenerated to its reduced form in a complex with TRX-
reductase,
FAD and NADPH.
(ii) Glutharedoxine (GRX) is a common representative of a second class of the
thioredoxine superfamily. The redox-active centre has the consensus sequence
Cys-Pro-X-
Cys, in which X is preferentially an aromatic amino acid, ie Tyr or Phe. GRX
as well TRX act
both as reductants with disulfides, but GRX would be a specific glutathion
(GSH)-mixed
disulfide reductase, e.g. in the reduction of thiolated proteins.
It has been demonstrated that the CXXC motif may also be involved in various
intra- and
extracellular biochemical and biological functions, eg thiol/disulfide
exchange reactions,
binding of transition metals, lipid incorporation site, and regulatory
activities, such as, for
example, control of gene transcription, regulation of signal transduction,
including functioning
as a cytokine, and the like, and control of the (de)thiolation status of
proteins. Importantly, the
CXXC motif can function in tertiary as well as quaternary protein structures
(see also Thomas
et al., 1995; Pinter et al., 1997; Aslund and Beckwith, 1999; Nakamura et al.,
1997).
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HCV proteins contain CXXC motifs. However, to date there is no suggestion nor
indication in
the prior art, that the reversible redox status of these CXXC motifs is of
importance to HCV.
Purification protocol described to date do not account for a reversible -S-S-
bridge in the
CXXC motif. As a consequence, the conformation of purified HCV proteins as
well as their
biological activity are impaired.
There have been numerous attempts to study native HCV proteins. The problem
encountered
was the inability to purify HCV proteins with the correct or native-like
conformation.
Consequently, conformational epitopes as well as other biochemical and
biological functions
and activities dependent on the native-like conformation remain enigmatic. In
addition, drug
targets for liver diseases and viral hepatitis suffer from the same
shortcoming, and drug
screening programs are bound to fail.
SUMMARY OF THE INVENTION/AIMS
It thus appears that due to the lack of or inefficient expression and
purification systems the
correct folding or assembly of proteins is impaired. Such purified proteins
are often not
biologically active and/or have an incorrect protein structure. As a
consequence, native anti-
HCV antibodies fail to recognize an important subset of antigenic determinants
on these
proteins, see for example Houghton (1997).
The present invention overcomes these problems, since it describes and makes
for the first
time HCV proteins with a native-like conformation, due to a reversible redox
status of
cysteinyl residues. Thus, new structures of HCV proteins are disclosed. In
particular, the
present invention allows for the purification of HCV proteins that are
biologically active and/or
have a native-like conformation. The native-like HCV proteins result in new
conformational
and oligomerisation-dependent epitopes.
The direct or indirect (mediated) in vitro and in vivo activities of the
native-like HCV proteins
create the possibility to study biochemical and biological pathways and
cascades, eg.
metabolic, enzymatic, signal-transduction, immuno-reactivity. The
identification of active
centres, binding sites and interaction domains (protein-protein, protein-
sugar, protein-nucleic
acid and protein-small molecule) allow for the development of drugs, that
interfere with the
cellular and viral processes involved in hepatitis.
-the purification and folding method of the present invention, in which a
cysteinyl shielding
group is removed, followed by refolding and reoxidation of the cysteine
residues in the HCV
protein, allows to restore the native-like conformation of HCV proteins;
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AIMS
The present invention aims at an HCV protein, or any functionally equivalent
part thereof,
comprising a Cys-amino acid, which has a reversible redox status. In
particular, the present
invention pertains to an HCV protein, which comprises at least two Cys-amino
acids with a
reversible redox status. The latter Cys-amino acids can be spaced by other
amino acids.
Preferentially said Cys amino acids are comprised in the amino acid sequence
Cys-X~-X2-
Cys, in which amino acid X, denotes any amino acid, and amino acid X2 denotes
any amino
acid. More preferentially, amino acid X~ denotes either amino acid Val, Leu or
Ile, and amino
acid X2 denotes amino acid Pro.
Moreover, the present invention aims at providing an HCV protein, or any
functionally
equivalent part thereof, comprising at least two Cys-amino acids, with a
reversible redox
status, according to above, obtainable by the following process:
(a) purifying an HCV protein, or any functionally equivalent part thereof, in
which the
cysteine residues are chemically and/or enzymatically reversibly protected,
(b) removal of the reversibly protection state of the cysteine residues,
(c) obtaining an HCV protein, or any functionally equivalent part thereof, in
which the
cysteine residues have a reversible redox status.
Moreover, the present invention aims at providing the HCV protein, or any
fuctionally
equivalent part thereof, as defined above, for use as a medicament.
Moreover, the present invention aims at the use of the HCV protein, or any
functionally
equivalent part thereof, as defined above, for the manufacture of an HCV
vaccine
composition, in particular a therapeutic vaccine or a prophylactic vaccine.
Moreover, the present invention aims at providing the HCV protein, or any
functionally
equivalent part thereof, as defined above, for raising specific antibodies.
In addition, the present invention aims at providing an immunoassay for
detecting HCV antibody by
determining formation of an HCV antibody-HCV protein complex.
Finally, the present invention aims at providing a bioassay for identifying
compounds that
modulate the activity of HCV proteins as defined above, by monitoring changes
in oxido-
reductase activity.
All the aims of the present invention are considered to have been met by the
embodiments as
set below.
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FIGURE LEGENDS
Figure 1: Size exclusion of reversibly protected and irreversibly blocked Vero
samples after
lysis in the presence of L-ascorbate.
Vero Cells were lysed with Triton X-100 in the presence of 1 mM L-ascorbate.
The lysate was
loaded on Lentil Lectin and reduced with 7.5 mM DTT at pH 7.2 as described in
PCT
EP95/03031 to Maertens et al. The reduced Ei s was either (1 ) sulfonated with
sodium
tetrathionate, (2) irreversibly blocked with N-ethylmaleimide or (3) left
untreated but the pH of
the solution was decreased to 6.
A SEC profile following the protocol by PCT EP95/03031 to Maertens et al. is
included as
reference.
The gel filtrations on Superdex 6200 10/30 (Pharmacia) were run in PBS, pH
7.2, 3%
Empigen, except for condition (3). This gel filtration was run at 10 mM
phosphate, 150 mM
NaCI, pH 6Ø
SEC profiles:
A: lysis in presence of ascorbate and sulfonation after reduction with DTT
B: lysis in presence of ascorbate and irreversibly blocking after reduction
with DTT
C: lysis in presence of ascorbate and without further treatment, but SEC was
run at pH 6.0
D: reference: blocking with NEM/ NEM.bio in lysate and after DTT reduction
(PCT
EP95/03031 to Maertens et al.)
The bars indicate the pools for analysis by silver staining and Western
blotting
The histogram gives the sandwich ELISA results: Mab 14H11 B2 (IGH 207) was
used for
coating and the detection was performed with HRP labeled 25C3 (IGH 200).
Fig. 2: Size exclusion chromatography of reversibly protected and irreversibly
blocked Vero
E1 s after lysis in the presence of sulfonation agents
Vero cells were lysed as described in PCT EP95/03031 to Maertens et al., but
sodium
tetrathionate was added instead of NEM/ NEM.bio.
The purification on lentil and reduction were performed as described in PCT
EP95/03031 to
Maertens et aLThe reduced material was either ( 1 ) sulfonated by sodium
tetrathionate either
(2) treated with IAA (=irreversibly blocked).
The material obtained by the method as described in PCT EP95/03031 to Maertens
et al. is
included as reference.
The 3 different E1s samples were separated on a Superdex 6200 10/30 column,
which had
been equilibrated with PBS, 3% Empigen, pH 7.2.
A: Sulfonation of the Vero cell lysate and sulfonation after reduction with
DTT
B: Sulfonation of the Vero cell lysate and irreversible blocking with lodo-
acetamide
C: Vero E1s obtained after irreversible blocking with NEMMEM.bio as described
in PCT
EP95/03031 to Maertens et al.
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D: overlay of the SEC profiles
The results of the sandwich ELISA are presented in the histograms.
The E1s-fractions were pooled as indicated with the bars and analysed by
silver staining and
Western blot.
Fig. 3: Fraction analysis of the SEC in 3% Empigen by SDS-PAGE and Western
blotting.
(A): SEC Fractions obtained after the different conditions of reversible
protection and
irreversible blocking were analysed by SDS-PAGE and silver staining.
SDS-PAGE analysis of Fractions obtained after lysis in ascorbate and gel
filtration at pH 6
(see fig 1.c) or lysis in ascorbate and sulfonation (fig. 1.a) are given as
examples in Fig. 3A.1.
Fig 3A.2 shows the fraction screening by Western blot with 11 B7D8 for the
conditions
described as in Fig 1.C (lysis in ascorbate and SEC at pH 6 after DTT
reduction).
(B) Western blots of the SEC-pools were performed with anti-E1s MAbs 5E1A10,
which
recognizes the amino- and carboxy-terminal epitope respectively.
The pools were made as indicated in Fig 1 and Fig. 2.
Lane 1 and 6: Molecular weight markers
Lane 2 and 7: reference material as prepared by PCT EP95/03031 to Maertens et
al.
Lane 3 and 8: reference material prepared with irreversibly blocked cysteines
(Treatment with
lodo-acetamide)
Lane 4 and 9: material obtained after sulfonation of lysate and sulfonation
after reduction
Lane 5 and 10: material obtained after lysis in the presence of ascorbate and
sulfonation after
reduction.
Fig. 4 E. coli expressed (his)s- tagged NS3 fusion protein
Purification on metal affinity after reversibly protection as well as sample
preparation for
ELISA is schematized.
+/- AO: in the presence or absence of reversible protecting agent (AO)
Fig. 5 ELISA reactivity of the mTNF(His)6 NS3 fusion proteins after different
coating
conditions.
Fig 5A: 90% pure mTNF(His)6NS3B9 fusion protein was desalted to 25 mM citrate,
1 mM
EDTA, pH 4 after reduction with 200 mM DTT. The fusion protein was diluted
till 500 Ng/ mL
in desalting buffer and stored at -70°C in the presence or absence of
thiol protective agents
(antioxidant group 1, group 2).
The samples were diluted to 0.5 Ng/mL in ELISA coating buffer (50 mM
bicarbonate buffer,
pH 9.6) with or without thiol protecting agents (anti-oxidant).The wells were
blocked with PBS
in presence or absence of protecting agent.
Serum sample incubation was performed in presence or absence of 10 mM DTT and
the
ELISA was developed with HRP conjugated rabbit anti human antibodies (Dako,
Denmark)
after washing. The reaction was stopped by addition of 2N H2S04.
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Sera (17790, 17826, 17832, 17838) were tested. Sera 17790 and 17832 are
considered as
difficult sera, because they are only detected as HCV positive sera after
treatment with 200
mM DTT (positive control). The 10 mM DTT treatment is included as negative
control for
these sera. Sera 17826 and 17838 are sera, that react with the NS3B9 protein
after 10 mM
DTT treatment (and are considered as easily detectable HCV sera).
Antioxidant group 1: 1 mM EDTA, 1 mM L-ascorbic acid, 1 mM reduced glutathion.
1mM tocopherol was supplementary added to these thiol protecting agents during
the ELISA
process, if the blocking was performed in the presence of protective agent.
Antioxidant group 2: 1 mM thiodiethyleneglycol (TEG), 1 mM thiophenecarboxylic
acid (TPCB),
1 mM pyrrolidone dithiocarbamate (PDTC), 1 mM diethyl dithiocarbamate (DETC).
Fig 5B: Thiol Compounds and NS3 B9 reactivity
The ELISA was pertormed as described in Fig. 5A, except that the effect (type
and
concentration) of mono- and dithio compounds as reversible protection group
was
investigated in more detail.
The sample diluent was incubated in this ELISA always in the presence of 3 mM
DTT.
Antioxidant 1 = 1 mM EDTA, 1 mM L-ascorbate
Antioxidant 2 = 1 mM thiophenecarboxylic (TPBC) acid, 1 mM thioethyleneglycol
(TEG), 1 mM
Diethyl dithiocarbamate (DETC), imM pyrrolidone dithiocarbamate (PDTC).
4mM DTC = 2 mM DETC, 2 mM PDTC
4 mM Mono-SH = 2 mM TPBC, 2mM TEG.
GSH and Cys are reduced glutathion and cysteine respectively.
Fig. 6 SDS-PAGE analysis of purified reversible protected (his)s-tagged HCV
proteins after
metal affinity chromatography
6A: E. coli expressed mTNF( His)sNS3B9 (batch NS3B9 B96092511).
Western blot with anti mTNF and silver stained SDS-PAGE under non reducing
conditions.
(1 Ng protein/ lane).
6B: Saccharomyces cerevesiae (Yeast) expressed (his)s- tagged E1s
The proteins were visualised by (a) silver staining; (b) Western blotting anti
E1s or (c) GNA
blotting.
Vaccinia expressed Els, purified as described by Maertens et al (PCT
EP95/03031) was
included as reference.
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DETAILED DESCRIPTION OF THE INVENTION
The invention described herein draws on previously published work and pending
patent
applications. By way of example, such work consists of scientific papers,
patents or pending
patent applications. All these publications and applications, cited previously
or below are
hereby incorporated by reference.
The present invention relates to HCV proteins with specific conformations. For
the first time
HCV proteins with a native-like conformation are generated, in particular HCV
E1 protein.
Specific cysteine bonds involved in the conformation of these HCV proteins
were found to be
important. As a way of example, a new and inventive purification protocol is
disclosed that
enables to purify HCV proteins with a native-like conformation. These new HCV
proteins are
able to not only present conformational epitopes but also display biological
activity. These
new HCV proteins can be used for various studies, such as, for example,
studies on drug
screening, biological activities, signal-transduction pathways, intra- and
extracellular
processing, interactions and binding between HCV and/or non-HCV molecules,
oligomerisation, conformational epitopes, antibody screening, metabolism and
enzymatic
activity, immuno-reactivity. Apparently, these studies can be placed in a
context for an
eventually diagnostic and/or therapeutic application.
The present invention is based on the finding that HCV proteins have specific,
native-like
conformations and biological activity, due to reversible redox status of
cysteinyl residues.
The present invention pertains therefore to an HCV protein, or any
functionally equivalent part
thereof, comprising a Cys-amino acid, which has a reversible redox status. In
particular, the
present invention pertains to an HCV protein, which comprises at least two Cys-
amino acids
with a reversible redox status. The latter Cys-amino acids can be spaced by
other amino
acids. Preferentially said Cys amino acids are comprised in the amino acid
sequence Cys-X,-
XZ-Cys, in which amino acid X, denotes any amino acid, and amino acid X2
denotes any
amino acid. More preferentially, amino acid X~ denotes either amino acid Val,
Leu or Ile, and
amino acid X2 denotes amino acid Pro.
HCV
In this regard, the present invention relates to HCV, and other members of the
genus
Flaviviridae, such as, for example, Hepatitis G virus, Dengue virus, Yellow
Fever Virus. Thus,
the term "HCV" contemplates all members of the genus Flaviviridae.
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PROTEIN
t0
The term "protein" as used herein, refers to an HCV protein, or any
functionally equivalent
part thereof, containing in its amino acid sequence at least one cysteine, the
redox status of
which is variable (see below). Also, protein "domains" containing at least one
cysteine in its
amino acid sequence are contemplated in the term "protein". The term
"functionally equivalent
part thereof" as used herein refers to a part or fragment of said HCV protein
that contains in
its amino acid sequence at least one cysteine, the redox status of which is
variable. In
particular, the terms "protein" and "functionally equivalent part thereof"
refers to HCV proteins
and fragments thereof comprising a redox active center, such as, for example,
HCV E1
protein. More particularly, the present invention relates to HCV Els, and HCV
El p. In this
regard, the term "redox active center" as used herein connotates a protein
motif with the
consensus sequence CXXC.
The term "a peptide" refers to a polymer of amino acids (aa's) derived from
the well-known
HCV proteins (Linnen et al., 1996; Maertens and Stuyver, 1997). The term "HCV
E1" is a
well-known protein by a person skilled in the art (Wengler, 1991). HCV E1,
together with HCV
E2 , which was previously called non-structural protein 1 (NS1) or E2/NS1,
constitute the
envelope region of HCV.
HCV E1 s (192-326)
YEVRNVSGMY HVTNDCSNSS IVYEAADMIM HTPGCVPCVR ENNSSRCWVA
LTPTLAARNA SVPTTTIRRH VDLLVGAAAF CSAMYVGDLC GSVFLVSQLF
TISPRRHETV QDCNCSIYPG HITGHRMAWD MMNIrIW
HCV E1 p (192-237)
YEVRNVSGMY HVTNDCSNSS IVYEAADMIM HTPGCVPCVR ENNSSR
The term "peptide" refers to a polymer of amino acids and does not refer to a
specific length
of the product. The terms "peptide", "polypeptide", "polyprotein" and
"protein" are thus
included within the definition of "peptide", and are used interchangeably
herein. The term
"peptide" does not refer to or exclude post-expression modifications of the
peptide, for
example, glycosylations, acetylations, phosphorylations, and the like.
Included within the
definition of peptide are, for example, polypeptides containing one or more
analogues of an
amino acid (including, for example, unnatural amino acids, PNA (Nielsen et
al., 1991, 1993),
etc.), peptides with substituted linkages, as well as other modifications
known within the art,
both naturally occurring and non-naturally occurring. Hence, peptides may be
linear, circular
or constrained (cyclised or stabilised by 'S-S' bridges, other than according
to the present
invention), consisting of D- or L-amino acids; peptides may be multimeric,
branched,
presented on phages or immobilised covalently or non-covalently on polymers
from different
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a
nature, such as, for example, organic, lipid, carbohydrate, protein, nucleic
acid polymers; or
peptides may be present in a scaffold. It is thus to be understood that
peptidomimitics or
mimotopes are inherent in the terms "polypeptide", "peptide" and "protein".
Immobilisation on polymers can be realised by residues of the HCV peptide self
or by HCV
peptide fused or coupled to other molecules such as, for example via a his-tag
(Dietrich et al.,
1996) or lipid chelators (Dietrich et al., 1995)
The term "mimotopes" refers to polypeptides which mimic the polypeptides as
defined herein
immunologically. Since sequence variability has been observed for HCV, it may
be desirable
to vary one or more amino acids as to better mimic the epitopes of different
strains. It should
be understood that such mimiotopes need not be identical to any particular HCV
sequence as
long as the subject compounds are capable of providing for immunological
competition with at
least one strain of HCV.
The term "peptidomimitics" refers to molecules that do not need to be composed
solely of
amino acids, but mimic the polypeptides as defined herein immunologically.
The present invention specifically refers to peptides prepared by classical
chemical synthesis.
The synthesis can be carried out in homogeneous solution or on solid phase.
For instance,
the synthesis technique in homogeneous solution which can be used is the one
described by
Houbenweyl (1974). The peptides of the present invention can also be prepared
by solid
phase according to the methodes described by Atherton and Shepard (1989). In
addition,
HCV peptides, peptidometics and mimotopes synthesized by dendrimer (Zhang &
Tam,
1997), polyketide (Carreras & Santi, 1998) or intein technology (Southworth et
al, 1999) are
also included in the present invention.
The peptides according to the present invention can also be prepared by means
of
recombinant DNA techniques, such as described in Sambrook et al. (1989), in
prokaryotes or
lower or higher eukaryotes. The term 'lower eukaryote' refers to host cells
such as yeast,
fungi and the like. Lower eukaryotes are generally (but not necessarily)
unicellular. The term
'prokaryotes' refers to hosts such as E.coli, Lactobacillus, Lactococcus,
Salmonella,
Streptococcus, Bacillus subtilis or Streptomyces. Also these hosts are
contemplated within
the present invention. Preferred lower eukaryotes are yeasts, particularly
species within
Schizosaccharomyces, Saccharomyces, Kluiveromyces, Pichia (e.g. Pichia
astoris),
Hansenula (e.g. Hansenula polvmorpha), Schwaniomvces, Schizosaccharomyces,
Yarowia,
Zygosaccharom~ and the like. Saccharomyces cerevisiae, S. carlsber eq nsis and
K. lactis
are the most commonly used yeast hosts, and are convenient fungal hosts. The
term 'higher
eukaryote' refers to host cells derived from higher animals, such as mammals,
reptiles,
insects, and the like. Presently preferred higher eukaryote host cells are
derived from Chinese
hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney
(BHK), pig
kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line
143 B, the
human cell line HeLa and human hepatoma cell lines like Hep G2, and insect
cell lines (e.g.
Spodoptera fru4iperda). The host cells may be provided in suspension or flask
cultures, tissue
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cultures, organ cultures and the like. Alternatively the host cells may also
be transgenic
animals.
The proteins according to the present invention can also be isolated from
mammalian hosts,
in particular mice or primates, e.g. humans as well as non-humans.
It is well known in the art that amino acids can be denoted by their full
name, three-letter
abbreviation, and one-letter symbol (see eg Stryer, 1981 ).
Furthermore, the present invention pertains to an HCV protein or part thereof
as defined
above, which specifically binds intra- or intercellular host molecules (host-
derived molecules),
such as, for example,
(i) receptor proteins, eg. annexin V, apolipoprotein B, tubulin, 24 kDa plasma
membrane
protein (Abrigani WO 97/09349), mannose receptor, asialoglycoprotein receptor;
(ii) molecules (protein or non-protein compounds) involved in redox
regulation, eg.
Gluthathion, TRX and GRX;
(iii) chaperone proteins, eg calnexin;
(iv) various glycoseaminoglycans (peptide and/or sugar core);
(v) nucleic acids or lipids.
Furthermore, the present invention pertains to an HCV protein or part thereof
as defined
above, which specifically binds another HCV protein or HCV nucleic acid (HCV-
derived
molecules), or parts thereof, resulting in homo- and/or hetero-oligomeric
complexes.
The complexes resulting from HCV proteins, or parts thereof, as defined above
bound to
other HCV-derived molecules or host-derived molecules are colloquially denoted
"HCV
derived complex". Thus, an "HCV-derived complex" consists of at least an HCV
protein as
defined above connected to another molecule, ie (HCV-protein)-X, in which X is
a host-
derived molecule or an HCV-derived molecule.
PURE
The term "purified" as applied herein refers to a composition wherein the
desired
components, such as, for example, HCV envelope proteins, comprise at least 35%
of the total
components in the composition. The desired components preferably comprises at
least about
40%, more preferably at least about 50%, still more preferably at least about
60%, still more
preferably at least about 70%, even more preferably at least about 80%, even
more
preferably at least about 90%, even more preferably at least about 95%, and
most preferably
at least about 98% of the total component fraction of the composition. The
composition may
contain other compounds, such as, for example, carbohydrates, salts, lipids,
solvents, and the
like, without affecting the determination of the percentage purity as used
herein. An "isolated"
HCV protein intends an HCV protein composition that is at least 35% pure. In
this regard it
should be clear that the term "a purified HCV protein" as used herein, refers
to isolated HCV
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proteins in essentially pure form. The term "essentially purified HCV
proteins" as used herein
refers to HCV proteins such that they can be used for in vitro diagnostic
methods and
therapeutics. These HCV proteins are substantially free from cellular
proteins, vector-derived
proteins or other HCV viral components. Usually, these proteins are purified
to homogeneity,
at least 80% pure, preferably 85%, more preferably 90%, more preferably 95%,
more
preferably 97%, more preferably 98%, more preferably 99%, even more preferably
99.5%,
and most preferably the contaminating proteins should be undetectable by
conventional
methods such as SDS-PAGE and silver staining.
ANTIBODIES
The present invention relates also to an HCV-antibody that can recognise an
HCV-peptide as
described above.
Furthermore, the present invention relates to an HCV protein or a functionally
equivalent part
thereof as defined supra, for raising anti-HCV antibodies, that specifically
recognise said HCV
protein or a functionally equivalent part thereof.
The term an "HCV-antibody" refers to any polyclonal or monoclonal antibody
binding to an
HCV-protein of the present invention or an HCV-derived complex.
Moreover, the term "HCV-antibodies" also connotates specific HCV-antibodies
that are raised
against epitopes which result from the conformation in HCV proteins due to the
presence of
S-S-bridges in these HCV proteins. Notably, said S-S-bridges can be an
intrinsic be part of
the epitope. But the oxido-reduction status of the cysteines (reduced or
oxidised; in a thiolated
or S-conjugated form) may change or stabilise the protein conformation (in the
vicinity or not
of these cysteine residues) which result in new epitopes, that may or may not
contain these
cysteine residues. These new epitopes are also part of the invention. In
addition, the term an
"HCV antibody" refers also to any polyclonal or monoclonal antibody binding to
mimitopes, as
defined above.
In addition, the term "HCV-antibody" thus also pertains to antibodies that
bind antigenic
determinants resulting from the specific conformation of HCV-derived
complexes, ie
antibodies that bind antigenic determinants which are not present on either
the HCV-peptide
of the present invention or the molecule said HCV-peptide is bound to, such
as, for example,
epitopes that find their origin from the interaction between the HCV peptide
of the present
invention and non-protein compounds like glycosaminoglycans (GAGs), heparine,
nucleic
acids, lipids, cofactors like metal-ions, and the like. Moreover, antigenic
determinants may be
formed by conformational changes, such as for example introduced by protein
processing,
cleaving or pH changes.
The term "epitope" refers to that portion of the antigen-antibody complex that
is specifically
bound by an antibody-combining site. Epitopes may be determined by any of the
techniques
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known in the art, or may be predicted by a variety of computer prediction
models known in the
art.
The expressions "recognising", "binding", or "formation of an antibody-protein
complex" as
used in the present invention is to be interpreted that binding, i.e.
interaction, between the
antigen and the antibody occurs under all conditions that respect the
immunological
properties of the antibody and the antigen.
Moreover, there are various other procedures known to produce HCV peptides,
that differ
from the procedure of the present invention. These other procedures might
result in HCV
l0 peptides capable of presenting epitopes. It is conceivable that the HCV
peptides, obtained by
these various and different procedures, are capable of presenting epitopes
similar to the
epitopes of the present invention. Thus, similar epitopes are epitopes
resulting from different
production or purifying procedures than from the present invention, but
recognizable by one
and the same antibody. However, the proteins of the instant invention present
epitopes
extremely efficient. Consequently, the epitopes on the proteins are more
immunogenic.
Therefore, the present invention also pertains to epitopes on proteins, said
epitopes are at
least 10 times, preferentially at least 20 times, preferentially at least 50,
preferentially at least
100 times, preferentially at least 500 times, and most preferentially at least
1000 times more
immunogenic than epitopes on HCV-peptides, which are not produced according to
the
present invention, and which do not have cysteinyl residues with a reversible
redox status. It
wilt be appreciated by the skilled in the art that said immunogenecity can,
for example, be
detected and therefore compared by immunising mammals by means of
administering
comparable quantities of peptides, produced by either method.
More particularly, the term "HCV-antibody" refers to an antibody binding to
the natural,
recombinant or synthetic HCV proteins, in particular binding to the natural,
recombinant or
synthetic E1, Els, E1p and/or NS3 proteins derived from HCV, or any
functionally equivalent
variant or part thereof (anti-HCV-E1-, anti-HCV-E1s-, anti-HCV-E1 p- or HCV-
NS3- antibody,
respectively). HCV-antibody may be present in a sample of body fluid, and may
be an HCV
E1-antibody, HCV-E1 s-antibody, HCV-E1 p-antibody or HCV-NS3-antibody.
The term "monoclonal antibody" used herein refers to an antibody composition
having a
homogeneous antibody population. The term is not limiting regarding the
species or source
of the antibody, nor is it intended to be limited by the manner in which it is
made. Hence, the
term "antibody" contemplates also antibodies derived from camels (Arabian and
Bactrian), or
the genus lama.
Thus, the term "antibody" also refers to antibodies derived from phage display
technology or
drug screening programs.
In addition, the term "antibody" also refers to humanized antibodies in which
at least a portion
of the framework regions of an immunoglobulin are derived from human
immunoglobulin
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sequences and single chain antibodies as described in U.S. patent No 4,946,778
and to
fragments of antibodies such as Fab, F~~ab~2, F", and other fragments which
retain the antigen
binding function and specificity of the parent antibody. The term "antibody"
also refers to
diabodies, triabodies or multimeric (mono-, bi -, tetra- or polyvalent/ mono-,
bi- or polyspecific)
antibodies, as well as enzybodies, ie artificial antibodies with enzyme
activity. Combinations
of antibodies with any other molecule that increases affinity or specificity,
are also
contemplated within the term "antibody". Antibodies also include modified
forms (e.g.
mPEGylated or polysialylated form (Fernandes & Gregoriadis, 1997) as well as
covalently or
non-covalently polymer bound forms.
In addition, the term "antibody" also pertains to antibody-mimicking compounds
of any nature,
such as, for example, derived from lipids, carbohydrates, nucleic acids or
analogues e.g.
PNA, aptamers (see Jayasena, 1999).
HCV antibodies may be induced by vaccination or may be passively transferred
by injection
after the antibodies have been purified from pools of HCV-infected blood or
from blood
obtained from HCV vaccinees.
The present invention relates also to a kit comprising HCV-antibodies for
detecting the HCV
peptides as defined herein.
PURIFICATION PROCEDURE
The invention further pertains to a purification procedure as described
herein, resulting in
HCV proteins of which at least one cysteinyl residue has a reversible redox
status, as well as
the HCV proteins obtainable by said purification procedure. During
purification at least the
cysteine residues are reversibly protected by chemical and/or enzymatic means
(see also
Examples section).
In this regard, the term "reversible redox status" as used herein refers to
sulfur of cysteines
which have the ability to change from the reduced status to the oxidized
status and vice
versa. This change in redox status involves electron transfer. The term
°oxido-reductase
activity" as used herein refers to the redox potential of the redox active
center, and thus to its
ability to transfer electrons from and to substrate molecules. This ability is
dependent of the
redox potential of the substrate molecules and the chemical environment.
Native HCV proteins have a specific conformation and may display biological
activity. The
purification procedure of the present invention results in purified HCV
proteins with a
biological activity and/or conformation which is identical to or almost
identical (native-like) to
the native biological activity and/or conformation of HCV proteins. The
purification procedure
of the present invention is characterised by the following:
-A- The first phase in the purification procedure of the present invention is
intended to
reversibly protect the reactivity of the cysteine residues.
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a
In essence, the first phase consists of the procedure as described extensively
in PCT
EP95/03031 to Maertens et al., but for one fundamental difference, in
particular the cysteine
residues are reversibly protected.
Reversible protection of the cysteine residues can be achieved by one of the
following
conditions (i) a modification group, or by (ii) stabilisation of the thiols
and/or disulfide bridges.
In effect, this protection stabilises the HCV protein, i.e. thiols and/or
disulfide bridges have no
tendency to react.
Hence, the first phase results eventually in a pure product with reversibly
protected cysteines;
to -B- The second phase in the purification procedure of the present invention
is intended to
restore the reactivity of the cysteine residues.
The condition in which the cysteine residues are reversibly protected is
removed, after the
first phase of the purification procedure.
This removal enables the restoration of the reversible redox status of the
cysteine residues.
Thus finally, an HCV peptide, or any functionally equivalent part thereof, is
obtained in which
the cysteine residues have a reversible redox state.
The reversible redox status allows for reactive HCV proteins with biological
activity and/or
with a native-like conformation.
Therefore, the present invention pertains to an HCV protein, or any
functionally equivalent
part thereof, comprising at least two Cys-amino acids, which have a reversible
redox status,
as defined above, obtainable by the following process:
(a) purifying an HCV protein, or any functionally equivalent part thereof, in
which the
cysteine residues are reversibly protected by chemical and/or enzymatic means,
(b) removal of the reversibly protection state of the cysteine residues,
(c) obtaining an HCV protein, or any functionally equivalent part thereof, in
which the
cysteine residues have a reversible redox status.
Thus, the present invention pertains also to the latter process.
Optionally, cofactors and antioxidantia are added to aid in protein
stabilisation.
It is to be understood that the purpose of reversibly protection is to
stabilise the HCV protein.
Especially, after reversibly protection the sulfur-containing functional group
(eg thiols and
disulfides) is retained in a non-reactive condition. The sulfur-containing
functional group is
thus unable to react with other compounds, e.g. no tendency of forming or
exchanging
disulfide bonds, such as, for example
R,-SH + R2-SH -x-> R,-S-S-R2 ;
R,-S-S-RZ + R3-SH -x-> R~-S-S-R3 + RZ-SH ;
R,-S-S-R2 + R3-S-S-R4 -x-> R,-S-S-R3 + R2-S-S-R4.
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The described reactions between thiols a d/or disulphide residues are not
limited to
intermolecular processes, but may also occur intramolecularly.
The term "reversibly protecting" as used herein contemplates covalently
binding of
modification agents to the cysteine residue, as well as manipulating the
environment of the
HCV protein such, that the redox state of the thiol-groups remains unaffected
throughout
subsequent steps of the purification procedure (shielding).
Reversible protection of the cysteine residues can be carried out chemically
or enzymatically.
The term "reversible protection by enzymatical means" as used herein
contemplates
reversible protection mediated by enzymes, such as for example acyl-
transferases, e.g. acyl-
transferases that are involved in catalysing thio-esterification, such as
palmitoyl
acyltransferase (see below and Das et al., 1997).
The term "reversible protection by chemical means" as used herein contemplates
reversible protection:
(1 ) by modification agents that reversibly modify cysteinyls such as for
example by
sulphonation and thio-esterification;
Sulphonation is a reaction where thiol or cysteines involved in disulfide
bridges are modified
to S-sulfonate: RSH ~ RS-S03 (Andre Darbre ) or RS-SR-~ 2 RS-S03
(sulfitolysis; Kumar
et al, 1986). Reagents for sulfonation are e.g. Na2S03, or sodium
tetrathionate. The latter
reagents for sulfonation are used in a concentration of 10 - 200 mM, and more
preferentially
in a concentration of 50 - 200 mM. Optionally sulfonation can be performed in
the presence of
a catalysator such as, for example Cu2+ (100 NM - 1 mM) or cysteine (1 - 10
mM).
The reaction can be pertormed under protein denaturing as well as native
conditions (Kumar
et al., 1985; Kumar et al., 1986).
Thioester bond formation, or thio-esterification is characterised by:
RSH + R'COX -~ RS-COR'
in which X is preferentially a halogenide in the compound R'CO-X.
(2) by modification agents that reversibly modify the cysteinyls of the
present invention such
as, for example, by heavy metals, in particular ZnZ+', Cd2+ (Malts et al, 1991
), mono-, dithio
and disulfide- compounds (e.g. aryl- and alkylmethanethiosulfonate,
dithiopyridine,
dithiomorpholine, dihydrolipoamide, Ellmann reagent, aldrothioln"' (Aldrich)
(Rein et al, 1996),
dithiocarbamates), or thiolation agents (e.g. gluthathion, N-Acetyl cysteine,
cysteineamine).
Dithiocarbamate comprise a broad class of molecules possessing an R,R2NC(S)SR3
functional group, which gives them the ability to react with sulphydryl
groups. Thiol containing
compounds are preferentially used in a concentration of 0.1 - 50 mM, more
preferentially in a
concentration of 1 - 50 mM, and even more preferentially in a concentration of
10-50 mM;
(3) by the presence of modification agents that preserve the thiol status
(stabilise), in
particular antioxidantia, such as for example DTT, dihydroascorbate, vitamin s
and derivates,
mannitol, amino acids, peptides and derivates (e.g. histidine, ergothioneine,
carnosine,
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methionine), gallates, hydroxyanisole, hydoxytoluene, hydroquinon,
hydroxymethylphenol and
their derivates in concentration range of 10 NM-10 mM, more preferentially in
a concentration
of 1-10 mM;
(4) by thiol stabilising conditions such as, for example, (i) cofactors as
metal ions (Zn2+, Mg2+),
ATP, (ii) pH control(e.g. for proteins in most cases pH -5 or pH is
preferentially thiol pKa -2;
e.g. for peptides purified by Reverse Phase Chromatography at pH -2).
Combinations of reversible protection as described in (1 ), (2), (3) and (4)
may result in
similarly pure and refolded HCV proteins. In effect, combination compounds can
be used,
such as, for example 2103 (Zn carnosine), preferentially in a concentration of
1 - 10 mM.
It should be clear that reversible protection also refers to, besides the
modification groups or
shielding described above, any cysteinyl protection method which may be
reversed
enzymatically or chemically, without disrupting the peptide backbone. In this
respect, the
present invention specifically refers to peptides prepared by classical
chemical synthesis (see
above), in which, for example, thioester bounds are cleaved by thioesterase,
basic buffer
conditions (Beekman et al., 1997) or by hydroxylamine treatment (Vingerhoeds
et al, 1996).
Thiol containing HCV proteins can be purified, for example, on affinity
chromatography resins
which contain (1 ) a cleavable connector arm containing a disulfide bond (e.g.
immobilised 5,5'
dithiobis(2-nitrobenzoic acid) (Jayabaskaran et al., 1987) and covalent
chromatography on
activated thiol-Sepharose 4B (Pharmacia)) or (2) a aminohexanoyl-4-
aminophenylarsine as
immobilised ligand. The latter affinity matrix has been used for the
purifcation of proteins,
which are subject to redox regulation and dithiol proteins that are targets
for oxidative stress
(Kalef et al., 1993).
Reversible protection may also be used to increase the solubilisation and
extraction of
peptides (Pomroy & Deber, 1998)
The reversible protection and thiol stabilizing compounds may be presented
under a
monomeric, polymeric or liposomic form.
The removal of the reversibly protection state of the cysteine residues can
chemically or
enzymatically accomplished by e.g.:
-a reductant, in particular DTT, DTE, 2-mercaptoethanol, dithionite, SnCl2,
sodium
borohydride, hydroxylamine, TCEP, in particular in a concentration of 1 - 200
mM, more
preferentially in a concentration of 50 - 200 mM;
-removal of the thiol stabilising conditions or agents by e.g. pH increase;
-enzymes, in particular thioesterases, glutaredoxine, thioredoxine, in
particular in a
concentration of 0.01 - 5 NM, even more particular in a concentration of 0.1 -
5 NM.;
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-combinations of the above described chemical and/or enzymatical conditions.
The removal of the reversibly protection state of the cysteine residues can be
carried out in
vitro or in vivo, e.g. in a cell or in an individual.
It will be appreciated that after the second phase of the purification
procedure, the cysteine
residues may or may not be irreversibly blocked, or replaced by any reversible
modification
agent, as listed above.
A reluctant according to the present invention is any agent which achieves
reduction of the
sulfur incysteine residues, e.g. "S-S" disulfide bridges, desulphonation of
the cysteine residue
(RS-S03 -> RSH). An antioxidant is any reagent which preserves the thiol
status or
minimises "S-S" formation and/or exchanges. Reduction of the "S-S" disulfide
bridges is a
chemical reaction whereby the disulfides are reduced to thiol (-SH). The
disulfide bridge
breaking agents and methods disclosed in WO 96/04385 are hereby incorporated
by
reference in the present description. "S-S" Reduction can be obtained by (1 )
enzymatic
cascade pathways or by (2) reducing compounds. Enzymes like thioredoxin,
glutaredoxin are
known to be involved in the in vivo reduction of disulfides and have also been
shown to be
effective in reducing "S-S" bridges in vitro. Disulfide bonds are rapidly
cleaved by reduced
thioredoxin at pH 7.0, with an apparent second order rate that is around 104
times larger than
the corresponding rate constant for the reaction with DTT. The reduction
kinetic can be
dramatically increased by preincubation the protein solution with 1 mM DTT or
dihydrolipoamide (Holmgren, 1979).
Thiol compounds able to reduce protein disulfide bridges are for instance
Dithiothreitol (DTT),
Dithioerythritol (DTE), 3-mercaptoethanol, thiocarbamates, bis(2-
mercaptoethyl) sulfone and
N,N'-bis(mercaptoacetyl)hydrazine, and sodium-dithionite.
Reducing agents without thiol groups like ascorbate or stannous chloride
(SnCIZ), which have
been shown to be very useful in the reduction of disulfide bridges in
monoclonal antibodies
(Thakur et al., 1991 ), may also be used for the reduction of HCV proteins. In
addition,
changes in pH values may influence the redox status of HCV proteins. Sodium
borohydride
treatment has been shown to be effective for the reduction of disulfide
bridges in peptides
(Gailit, 1993). Tris (2-carboxyethyl)phosphine (TCEP) is able to reduce
disulfides at low pH
(Burns et al., 1991 ). Selenol catalyses the reduction of disulfide to thiols
when DTT or sodium
borohydride is used as reluctant. Selenocysteamine, a commercially available
diselenide,
was used as precursor of the catalyst (Singh and Kats, 1995).
It is stressed again that the whole content, including all definitions of the
documents cited
above, are incorporated by reference in the present application. Hence, the
above mentioned
methods and compounds to modify the redox status of HCV proteins are all
contemplated in
the present invention.
BIO-ACTIVE SITE
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O
The present invention further pertains to HCV proteins containing a
biologically active CXXC-
motif. The terms "biologically active" and "oxido-reductase activity" as used
herein
contemplate a CXXC-site in a HCV peptide, or a functionally equivalent part
thereof, with a
reversible redox status, that has the ability to mediate various intra- and
extracellular
biochemical and biological functions, such as, for example, thioUdisulfide
exchange reactions,
binding of transition metals, lipid incorporation, and regulatory activities
(e.g. control of gene
transcription, regulation of signal transduction, including functioning as a
cytokine, and the
like, and control of the (de)thiolation status of proteins).
Structural or conformational changes effected by the cysteinyl redox status
may be followed
with biophysical methods, such as for example by spectrophotometry
(absorbance, Circular
Dichroism, Infrared, fluorescence, NMR) or with immunochemical methods (e.g.
ELISA, EIA,
and the like), which are based on the appearance or disappearance of epitopes.
Sequences
involved in the epitopes can be identified by Mass spectrometry (MS) and
sequencing after
cross-linking and affinity purification of the complex. The conformational or
new detectable
linear epitopes may result from folding processes on tertiary or quaternary
structure level.
Metal ion incorporation in the active site can be measured by radioactive
decay measures or
Atomic absorbance spectrometry.
The binding of the HCV proteins of the present invention to other molecules,
such as for
example receptors, carbohydrates, lipids, nucleic acids (see also above) can
be studied by
e.g. FACS, Biacore, immunological assays (Western blotting, EIA, ELISA, and
the like),
crosslinking and chromatographical methods (e.g. affinity- chromatography, gel
filtration).
Thioredoxin enzymatic activity of HCV proteins can be identified by studying
the potential to
reduce disulphide bridges according to the method as described by Holmgren et
al. (1979).
The effect of cofactors, such as DTT or dihydrolipoamide, can be verified in
this method as
well. Non-proteinaceous compounds (e.g. Ellmann reagent, aldrothiol) as well
as proteins
(e.g. aggregated insulin) can be taken as substrates.
The formation of mixed disulphides (see below), is an activity which is
related to protein
folding, and restoration of the active site. The formation of mixed
disulphides can be
demonstrated by reversible protection or irreversible blocking of the thiol
groups before and
after reductant treatment with different agents (e.g. DTT), such as described
in "purification
procedure", followed by mass spectrometry analysis.
The pKa of the thiol groups in the -CXXC-containing protein is defined by
treament with
alkylation agents in function of the pH (titration). Differential protection
and/or blocking of the
residues and MS give information of the reaction initiating cysteinyl residue
in the CXXC-site.
Amino terminal amino acid sequencing can give information about the
processing, cleavage
products and domain structure of the HCV protein.
The tissue and intracellular distribution of these cleavage products are
localized by immuno-
histochemical methods.
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VACCINE
The present invention also relates to a composition comprising a protein as
defined above.
More particularly the present invention relates to a vaccine composition. The
term "vaccine
composition" relates to an immunogenic composition capable of eliciting
protection against
HCV, whether partial or complete. It therefore includes HCV peptides,
proteins,
polynucleotides, HCV-derived molecules or HCV-derived particles, as defined
above.
Protection against HCV refers in particular to humans, but refers also to non-
human primates,
trimera mouse (Zauberman et al., 1999), or other mammals.
The proteins of the present invention can be used as such, in a biotinylated
form (as
explained in WO 93/18054) and/or complexed to Neutralite Avidin (Molecular
Probes Inc.,
Eugene, OR, USA). It should also be noted that "a vaccine composition"
comprises, in
addition to an active substance, a suitable excipient, diluent, carrier and/or
adjuvant which, by
themselves, do not induce the production of antibodies harmful to the
individual receiving the
composition nor do they elicit protection. Suitable carriers are typically
large slowly
metabolized macromolecules such as proteins, polysaccharides, polylactic
acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers and inactive virus
particles. Such
carriers are well known to those skilled in the art. Preferred adjuvants to
enhance
effectiveness of the composition include, but are not limited to: colloidal
iron hydroxide (Leibl
et al., 1999), aluminium hydroxide, aluminium in combination with 3-0-
deacylated
monophosphoryl lipid A as described in WO 93/19780, aluminium phosphate as
described in
WO 93/24148, N-acetyl-muramyl-L-threonyl-D-isoglutamine as described in U.S.
Patent N°
4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-
alanyl-D-
isoglutamyl-L-alanine2-(1'2'dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)
ethylamine and
RIBI (ImmunoChem Research Inc., Hamilton, MT, USA) which contains
monophosphoryl lipid
A, detoxified endotoxin, trehalose-6,6-dimycolate, and cell wall skeleton (MPL
+ TDM + CWS)
in a 2% squalene/Tween 80 emulsion. Any of the three components MPL, TDM or
CWS may
also be used alone or combined 2 by 2. Additionally, adjuvants such as
Stimulon (Cambridge
Bioscience, Worcester, MA, USA) or SAF-1 (Syntex) may be used, as well as
adjuvants such
as combinations between ~S21 and 3-de-O-acetylated monophosphoryl lipid A
(V11094/00153), or MF-59 (Chiron), or poly[di(carboxylatophenoxy) phosphazene]
based
adjuvants (Virus Research Institute), or blockcopolymer based adjuvants such
as Optivax
(Vaxcel, Cythx) or inulin-based adjuvants, such as Algammulin and Gammalnulin
(Anutech),
Incomplete Freund's Adjuvant (IFA) or Gerbu preparations (Gerbu Biotechnik).
It is to be
understood that Complete Freund's Adjuvant (CFA) may be used for non-human
applications
and research purposes as well. "A vaccine composition" will further contain
excipients and
diluents, which are inherently non-toxic and non-therapeutic, such as water,
saline, glycerol,
ethanol, wetting or emulsifying agents, pH buffering substances,
preservatives, and the like.
The reversible modification of cysteinyl residues of the HCV peptides of the
present invention,
allows that these HCV peptides can be coupled covalently to a chemically
activated carrier
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K IG
molecule, such as, for example, polymers or liposomes, or that the HCV peptide
itself
functions as carrier for binding other HCV-related or HCV non-related
immunogenic proteins
(mixed vaccines). HCV peptides linked to liposomes by a thioester have the
advantage that
the bonds are broken in vivo by host thioesterases, resulting in a slow
antigen release and
presentation. Incorporation or binding of the HCV peptide to the polymer or
liposome can
also be based on non-covalent interactions, exploiting affinity between the
ligands.
Typically, a vaccine composition is prepared as an injectable, either as a
liquid solution or
suspension. Solid forms, suitable for solution on, or suspension in, liquid
vehicles prior to
injection may also be prepared. The preparation may also be emulsified or
encapsulated in
liposomes for enhancing adjuvant effect. The polypeptides may also be
incorporated into
Immune Stimulating Complexes together with saponins, for example Quil A
(ISCOMS).
Vaccine compositions comprise an immunologically effective amount of the
polypeptides of
the present invention, as well as any other of the above-mentioned components.
"Immunologically effective amount" means that the administration of that
amount to an
individual, either in a single dose or as part of a series, is effective for
prevention or treatment.
This amount varies depending upon the health and physical condition of the
individual to be
treated, the taxonomic group of the individual to be treated (e.g. human, non-
human primate,
primate, etc.), the capacity of the individual's immune system to mount an
effective immune
response, the degree of protection desired, the formulation of the vaccine,
the treating
doctor's assessment, the strain of the infecting HCV and other relevant
factors. It is expected
that the amount will fall in a relatively broad range that can be determined
through routine
trials. Usually, the amount will vary from 0.01 to 1000 Ng/dose, more
particularly from 0.1 to
100 Ng/dose. The vaccine compositions are conventionally administered
parenterally, typically
by injections for example, subcutaneously or intramuscularly. Additional
formulations suitable
for other methods of administration include oral formulations and
suppositories. Dosage
treatment may be a single dose schedule or a multiple dose schedule. The
vaccine may be
administered in conjunction with other immunoregulatory agents.
DNA vaccine
The intracellular environment of a host can provide the basis for the
reversible redox-status of
the HCV proteins of the present invention. In this regard, it should be clear
that an HCV DNA
vaccine composition comprises a plasmid vector comprising a polynucleotide
sequence
encoding an HCV protein as described above, operably linked to transcription
regulatory
elements. As used herein, a "plasmid vector" refers to a nucleic acid molecule
capable of
transporting another nucleic acid to which it has been linked. Preferred
vectors are those
capable of autonomous replication and/or expression of nucleic acids to which
they have
been linked. In general, but not limited to those, plasmid vectors are
circular double stranded
DNA loops which, in their vector form, are not bound to the chromosome. As
used herein, a
"polynucleotide sequence" refers to polynucleotides such as deoxyribonucleic
acid (DNA),
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and, where appropriate, ribonucleic acid (RNA). The term should also be
understood to
include, as equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and
single (sense or antisense) and double-stranded polynucleotides. As used
herein, the term
"transcription regulatory elements" refers to a nucleotide sequence which
contains essential
regulatory elements, such that upon introduction into a living vertebrate cell
it is able to direct
the cellular machinery to produce translation products encoded by the
polynucleotide. The
term "operably linked" refers to a juxtaposition wherein the components are
configured so as
to perform their usual function. Thus, transcription regulatory elements
operably linked to a
nucleotide sequence are capable of effecting the expression of said nucleotide
sequence.
Those skilled in the art can appreciate that different transcriptional
promoters, terminators,
carrier vectors or specific gene sequences may be used succesfully.
The instant invention pertains thus also to the use of an HCV protein as
defined herein for
prophylactically inducing immunity against HCV (prophylactic vaccine). It
should be noted that
a vaccine may also be useful for treatment of an individual as pointed-out
above, in which
case it is called a "therapeutic vaccine".
It is clear from the above that the present invention also relates to the
usage of a protein as
defined above or a composition as defined above for the manufacture of an HCV
vaccine
composition. In particular, the present invention relates to the usage of a
protein as defined
herein for inducing immunity against HCV in chronic HCV carriers. More in
particular, the
present invention relates to the usage of a protein as defined herein for
inducing immunity
against HCV in chronic HCV carriers prior to, simultaneously to or after any
other therapy,
such as, for example, the well-known interferon therapy either or not in
combination with the
administration of small drugs treating HCV, such as, for example, ribavirin.
Such composition
may also be employed before or after liver transplantation, or after presumed
infection, such
as, for example; needle-stick injury. In addition, the present invention
relates to a kit
containing the HCV proteins of the present invention to detect HCV antibodies
present in a
biological sample.
The term "biological sample" as used herein, refers to a sample of tissue or
fluid isolated from
an individual, including but not limited to, for example, serum, plasma, lymph
fluid, the
external sections of the skin, respiratory intestinal, and genitourinary
tracts, oocytes, tears,
saliva, milk, blood cells, tumors, organs, gastric secretions, mucus, spinal
cord fluid, external
secretions such as, for example, excrement, urine, sperm, and the like.
Since the HCV proteins of the present invention are highly immunogenic, and
stimulate both
the humoral and cellular immune response, the present invention relates also
to a kit for
detecting HCV related T cell response, comprising the HCV protein of the
instant invention.
HCV T cell response can for example be measured as described in PCT/EP
94/03555 to
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2N
Leroux-Roels et al. It should be stressed that the whole content, including
all the definitions,
of this document is incorporated by reference in the present application.
The present invention also relates to a composition as defined above which
also comprises
HCV core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NSSA and/or NSSB protein, or parts
thereof.
E1, E2, and/or E1 E2 particles may, for example, be combined with T cell
stimulating antigens,
such as, for example, core, P7, NS3, NS4A, NS4B, NS5A and/or NSSB.
Moreover, the present invention also features the use of a protein as
described above, or a
composition as described above to detect antibodies against HCV proteins. As
used herein,
the term "to detect" refers to any assay known in the art suitable for
detection. In particular,
the term refers to any immunoassay as described in WO 96/13590.
DRUG SCREENING
The invention provides methods for identifying compounds or agents which can
be used to
treat disorders characterized by (or associated with) HCV infection. These
methods are also
referred to herein as "drug screening assays" or "bioassays" and typically
include the step of
screening a candidate/test compound or agent for the ability to interact with
(e.g., bind to) an
HCV protein to modulate the interaction of an HCV protein and a target
molecule, and/or to
modulate HCV nucleic acid expression and/or HCV protein activity.
Candidate/test
compounds or agents which have one or more of these abilities can be used as
drugs to treat
disorders characterized by HCV infection, HCV nucleic acid expression and/or
HCV protein
activity. Candidate/test compounds such as small molecules, e.g., small
organic molecules,
and other drug candidates can be obtained, for example, from combinatorial and
natural
product libraries.
In one embodiment, the invention provides assays for screening candidate/test
compounds
which interact with (e.g., bind to) HCV protein, or any functionally
equivalent part thereof.
Typically, the assays are cell-free assays which include the steps of
combining the HCV
proteins of the present invention, its catalytic, i.e. oxido-reductase
activity, or immunogenic
fragments thereof, and a candidate/test compound, e.g., under conditions which
allow for
interaction of (e.g., binding of) the candidate/test compound to the HCV
protein or portion
thereof to form a complex, and detecting the formation of a complex, in which
the ability of the
candidate compound to interact with (e.g., bind to) the HCV protein or portion
thereof is
indicated by the presence of the candidate compound in the complex. Formation
of
complexes between the HCV protein and the candidate compound can be
quantitated, for
example, using standard immunoassays.
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The HCV proteins, its catalytic or immunogenic fragments or oligopeptides
thereof employed
in such a test may be free in solution, affixed to a solid support, borne on a
cell surface, or
located intracellularly
In another embodiment, the invention provides screening assays to identify
candidate/test
compounds which modulate (e.g., stimulate or inhibit) the interaction (and
most likely HCV
protein activity as well) between an HCV protein and a molecule (target
molecule) with which
the HCV protein normally interacts, or antibodies which specifically recognize
the HCV
protein. Examples of such target molecules include proteins in the same
signaling path as the
l0 HCV protein, e.g., proteins which may function upstream (including both
stimulators and
inhibitors of activity) or downstream of the HCV protein signaling pathway [Zn-
fingers,
protease activity, regulators of cysteine redox status].
Typically, the assays are cell-free assays which include the steps of
combining an HCV
protein of the present invention, its catalytic or immunogenic fragments
thereof, an HCV
protein target molecule (e.g., an HCV protein ligand) or a specific antibody
and a
candidate/test compound, e.g., under conditions wherein but for the presence
of the
candidate compound, the HCV protein or biologically active portion thereof
interacts with
(e.g., binds to) the target molecule or the antibody, and detecting the
formation of a complex
which includes the HCV protein and the target molecule or the antibody, or
detecting the
interaction/reaction of the HCV protein and the target molecule or antibody.
Detection of complex formation can include direct quantitation of the complex
by, for example,
measuring inductive effects of the HCV protein. A statistically significant
change, such as a
decrease, in the interaction of the HCV protein and target molecule (e.g., in
the formation of a
complex between the HCV protein and the target molecule) in the presence of a
candidate
compound (relative to what is detected in the absence of the candidate
compound) is
indicative of a modulation (e.g., stimulation or inhibition) of the
interaction between the HCV
protein and the target molecule. Modulation of the formation of complexes
between the HCV
protein and the target molecule can be quantitated using, for example, an
immunoassay.
Therefore, the present invention contemplates a method for identifying
compounds that
modulate the interaction between binding partners in a complex, in which at
least one of said
binding partners is the HCV protein as defined above, and said method
comprising:
(a) contacting a test compound with the complex, for a time sufficient to
modulate the
interaction in the complex; and thereafter
(b) monitoring said complex for changes in interactions, so that if a change
in the
interaction is detected, a compound that modulates the interaction is
identified .
In particular, the present invention contemplates the latter method in which
at least one of the
binding partners is selected from the group of:
(i) HCV-derived molecules, eg nucleic acids (promoters or enhancers) (HCV RNA
packed in HCV particles) or proteins (structural or non-structural proteins)
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(ii) Intracellular, host-derived molecules (modifiers of redox status of HCV
peptides,
(TRX, GRX, thioesterase, etc), )
(iii) Extracellular host-derived molecules (receptors, glucosamines, heparine)
It should be clear that modulators for interaction between binding partners in
a complex, when
identified by any of the herein described methods is contemplated in the
invention.
To perform the above described drug screening assays, it is feasible to
immobilize either HCV
protein or its target molecule to facilitate separation of complexes from
uncomplexed forms of
one or both of the proteins, as well as to accommodate automation of the
assay. Interaction
(e.g., binding of) of HCV protein to a target molecule, in the presence and
absence of a
candidate compound, can be accomplished in any vessel suitable for containing
the
reactants. Examples of such vessels include microtitre plates, test tubes, and
microcentrifuge
tubes. In one embodiment, a fusion protein can be provided which adds a domain
that allows
the protein to be bound to a matrix. For example, HCV protein-His tagged can
be adsorbed
onto Ni-NTA microtitre plates (Paborsky et al., 1996), or HCV protein-ProtA
fusions adsorbed
to IgG, which are then combined with the cell lysates (e.g. (35)S-labeled) and
the candidate
compound, and the mixture incubated under conditions conducive to complex
formation (e.g.,
at physiological conditions for salt and pH). Following incubation, the plates
are washed to
remove any unbound label, and the matrix immobilized and radiolabel determined
directly, or
in the supernatant after the complexes are dissociated. Alternatively, the
complexes can be
dissociated from the matrix, separated by SDS-PAGE, and the level of HCV
protein-binding
protein found in the bead fraction quantitated from the gel using standard
electrophoretic
techniques.
Other techniques for immobilizing protein on matrices can also be used in the
drug screening
assays of the invention. For example, either HCV protein or its target
molecule can be
immobilized utilizing conjugation of biotin and streptavidin. Biotinylated HCV
protein
molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques well
known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IIL),
and immobilized in
the wells of streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies
reactive with HCV protein but which do not interfere with binding of the
protein to its target
molecule can be derivatized to the wells of the plate, and HCV protein trapped
in the wells by
antibody conjugation. As described above, preparations of a HCV protein-
binding protein and
a candidate compound are incubated in the HCV protein-presenting wells of the
plate, and the
amount of complex trapped in the well can be quantitated. Methods for
detecting such
complexes, in addition to those described above for the GST-immobilized
complexes, include
immunodetection of complexes using antibodies reactive with the HCV protein
target
molecule, or which are reactive with HCV protein and compete with the target
molecule; as
well as enzyme-linked assays which rely on detecting an enzymatic activity
associated with
the target molecule.
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Another technique for drug screening which provides for high throughput
screening of
compounds having suitable binding affinity to the HCV protein is described in
detail in
"Determination of Amino Acid Sequence Antigenicity" by Geysen HN, WO
Application
84/03564, published on 13/09/84, and incorporated herein by reference. In
summary, large
numbers of different small peptide test compounds are synthesized on a solid
substrate, such
as plastic pins or some other surtace. The protein test compounds are reacted
with fragments
of HCV protein and washed. Bound HCV protein is then detected by methods well
known in
the art. Purified HCV protein can also be coated directly onto plates for use
in the
aforementioned drug screening techniques. Alternatively, non-neutralizing
antibodies can be
used to capture the peptide and immobilize it on a solid support.
This invention also contemplates the use of competitive drug screening assays
in which
neutralizing antibodies capable of binding HCV protein specifically compete
with a test
compound for binding HCV protein. In this manner, the antibodies can be used
to detect the
presence of any protein which shares one or more antigenic determinants with
HCV protein.
In yet another embodiment, the invention provides a method for identifying a
compound (e.g.,
a screening assay) capable of use in the treatment of a disorder characterized
by (or
associated with) HCV infection, HCV nucleic acid expression or HCV protein
activity. This
method typically includes the step of assaying the ability of the compound or
agent to
modulate the expression of the HCV nucleic acid or the activity of the HCV
protein thereby
identifying a compound for treating a disorder characterized by HCV infection,
HCV nucleic
acid expression or HCV protein activity.
Modulators of HCV infection, HCV protein activity and/or HCV nucleic acid
expression
identified according to these drug screening assays can be used to treat, for
example, HCV
infection or disorders related to HCV infection.
These methods of treatment include the steps of administering the modulators
of HCV protein
activity and/or HCV nucleic acid expression, e.g., in a pharmaceutical
composition as
described above, to a subject in need of such treatment, e.g., a subject with
an HCV infection.
The tissue or cell specificity of the drug may be enhanced by using the drug
targeting
methods (see Davis, 1997) or intracellular immunisation. Liver targeting tools
are for example
biluribin coupled drugs (Kramer et al. 1992), asialoglycoprotein receptor or
lipoprotein
mediated transfer of drugs (Vingerhoeds et al. 1996). Drugs may even
intracellularly targeted
with cell organel targeting of DNA expressed molecule via cell organel
specific targeting tags
(Persic et al 1997).
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Methods for assaying the ability of the compound or agent to modulate the
infection of HCV,
the expression of the HCV nucleic acid or activity of the HCV protein are
typically cell-based
assays. However, HCV infected animals are also contemplated herein. For
example, HCV
infected or transfected cells which are sensitive to reductants or oxidants,
or which transduce
signals via a pathway involving HCV protein can be induced to overexpress an
HCV protein in
the presence and absence of a candidate compound.
Candidate compounds which produce a statistically significant change in HCV
protein -
dependent responses (either stimulation or inhibition) can be identified.
In one embodiment, infection of target cells by HCV, expression of the HCV
nucleic acid or
the oxido-reductase activity of an HCV protein is modulated in cells and the
effects of
candidate compounds on the readout of interest (such as rate of infection,
cell proliferation or
differentiation, or oxido-reductase activity) are measured. For example, the
transition rate
from the thiolated form to the S-conjugated, I.e. S-S bridge, form can be
assayed. For
example, the expression of genes which are up- or down-regulated in response
to an HCV
protein -dependent signal cascade can be assayed. In preferred embodiments,
the regulatory
regions of such genes, e.g., the 5' flanking promoter and enhancer regions,
are operably
linked to a detectable marker (such as luciferase) which encodes a gene
product that can be
readily detected. Phosphorylation of HCV protein or HCV protein target
molecules can also be
measured, for example, by immunoblotting.
Therefore the present invention pertains to a bioassay for identifying
compounds that
modulate the oxido-reductase activity of HCV proteins as defined above, said
bioassay
comprising:
(a) exposing cells expressing HCV proteins, or any functionally equivalent
part thereof,
as defined above to at least one compound whose ability to modulate
the oxido-reductase activity of said proteins is sought to be determined;
and thereafter
(b) monitoring said proteins for changes in oxido-reductase activity.
The reversibly protected HCV peptide may be used for diagnostic coupling
purposes in, for
example, an oligomerised state as (1 ) chemical polyantigen preparations (the
E1 s coupled
antigens are not necessary HCV related and may thus be used for multi-disease
screening);
(2) targets for immobilisation and immunodetection (e.g. biotinylation,
fluorescence) or (3) for
antibody conjugation, which in turn may result in supramolecular antibodies
(e.g. Antibodies
on virus-like particles). The labelling and antibody conjugation result in an
increase of
sensitivity due to the amplification step by oligomerisation of the protein.
It has to be mentioned that any reactive group on the peptide (sugars, amino,
carboxyl, thiol,
histidine, and the like) may be exploited for the coupling or conjugation. The
reversible
protected group can be used to enhance the specificity of reaction and the
thiol reactivity can
be exploited in a later step / phase of conjugation after deprotection.
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Finally, the present invention relates to an immunoassay for detecting HCV
antibody, which
immunoassay comprises: (1 ) providing the purified HCV protein as defined
herein, or a
functional equivalent thereof, (2) incubating a biological sample with said
HCV protein under
conditions that allow the formation of antibody-antigen complex, (3)
determining whether said
antibody-antigen complex comprising said HCV protein is formed.
The present invention will now be illustrated by reference to the following
examples which set
forth particularly advantageous embodiments. However, it should be noted that
these
embodiments are merely illustrative and can not be construed as to restrict
the invention in
any way.
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EXAMPLES
Example 1: Determination of the thiol-disulphide status in vaccinia expressed
E1s
HCV E1 s protein (amino acids 192-326) was expressed and purified from Vero
cells using
recombinant vaccinia virus pv-HCV11A according to the protocol as described in
Maertens et
al. (PCT/EP 95/03031 ), except that the blocking of the thiol groups was done
with lodo
acetamide and N-ethylmaleimide (NEM) during the lysis and after the reduction
with DTT,
respectively. Thus, blocking free thiols with IAA (lodoacetamide) in the lysis
buffer, and
alkylation with NEM after the reduction step with DTT.
The purified E1 s was concentrated by ultrafiltration (Centricon 10,
Millipore), deglycosylated
with N-glycosidase F (PGNase F; Boehringer Mannheim) as described by the
manufacturer,
after which the E1s was loaded on a 15% PolyAcrylAmide minigel. SDS-PAGE was
performed as described by Laemmli. The protein bands were cut in the ca. 18
kDalton region
after size separation and staining.
Proteins were cleaved by in situ trypsinolysis, and the resulting peptide
digest was analysed
by mass spectroscopy (MS; MALDI-TOFF) to determine the derivatisation state of
the
different cysteine-residues.
The MS results show for the cysteines in the CXXC-motif that:
(1 ) in ca. 10% of the CXXC-motifs, both cysteines were present as IAA
derivatised
products;
(2) in ca. 30% of the CXXC-motifs, one cysteine was blocked with IAA, the
other cysteine
was present as NEM-derivatised product;
(3) in ca. 60% of the CXXC-motifs, both cysteines were retrieved as NEM-
derivatised
product.
These data show surprisingly that
(1 ) either the two cysteines were present in a fully reduced ("thiol")
status; and
(2) either one of the cysteines was involved in a mixed disulfide bridge and
the second
cysteine was present as free thiol (intermediate form); and
(3) that both cysteines were present in the oxidised form (disulfide bridge).
Although the experiment is performed with an HCV peptide, ie derived from an
infectious
pathogen, these three forms correlate well with the different oxidation status
which has been
described for the -CXXC-motif in the TRX superfamily, and correspond with the
activity
pattern described for the thiol oxidoreductases , ie molecules involved in
regulating oxido
reduction environment in the cell (Rietsch & Beckwith, 1998; Loferer &
Hennecke, 1994;
Aslund & Beckwith, 1999 ; Huppa & Ploegh, 1999).
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.~ i
Since the cysteines in the active site of thioredoxin are oxidised and the
disulfide bridge in the
substrate is reduced, the excess of the oxidised form (60%) is in agreement
with thioredoxin
activity. Surprisingly, these results tend to indicate that E1 s is involved
in an (auto)folding
mechanism, that is dependent on on the intracellular oxidative status for the
regeneration of
the active site to the reduced form. Therefore, the -S-S- protein based
aggregate consisting of
E1 s, vaccinia and host proteins can be diminished by interfering at the level
of protein folding
or by addition of compounds in the culture media which intertere/ influence
the intracellular
redox status of cysteines.
Example 2: Purification of Yeast E1 s-His after reversible modification of
cysteines
Saccharomyces cerevesiae (yeast) cells producing his-tagged HCV E1s were
harvested by
microfiltration and centrifugation. The cell pellets are resuspended in 5
volumes lysis buffer
(50mM phosphate, 6M Guanidinium-HCI, pH 7.4 (=buffer A) ) and solid Na2S03 ,
Na2S406 are
added to the solution till a final concentration of 160 mM and 65 mM,
respectively. Cuz+ (100
mM stock solution in NH3) is added as catalysator till a concentration of 100
NM and the
solution is incubated overnight at room temperature. The lysate is stored at -
70°C and
cleared by centrifugation (JA 20 rotor, 27 kg at 4°C) after the freeze-
thaw cycle.
Imidazole and EmpigenT"" (Albright & Wilson, UK) are added to the supernatant,
respectively,
till a final concentration of 20 mM and 1% (w/v) and the sample is applied on
a Ni-IDA
Sepharose FF column (Pharmacia) after dilution with the equilibration buffer
(Buffer A, 20 mM
Imidazole, 1 % Empigen).
The resin was washed with the equilibration buffer till the absorbance at 280
nm reaches
baseline level and the bound proteins are eluted by applying an imidazole step
gradient.
SDS-PAGE and Western blot analysis show that >90 % pure E1s-His protein is
retrieved in
the 200 mM Imidazole elution pool after sulfitolysis under denaturing
conditions and IMAC
(Fig 6B).
Sulphonated HCV E1s is desulphonated by addition of DTT to restore the thiol
status and
allow the formation of intra -and inter molecular disulphide bridge.
Example 3: Purification and immunolog~ical reactivity of E. coli NS3 fusion
protein
E. coli cells producing the mTNF(His)6NS3 B9 fusion protein were harvested and
the cells
were resuspended in buffer A (see Example 2). Sulfonation, sample preparation
and metal
chromatography run on Ni-IDA Sepharose FF (Pharmacia) were done as described
for yeast
(Example 2). The mTNF(His)6 NS3 b9 fusion protein was retrieved in the 200 mM
Imidazole
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elution pool. Coomassie staining of SDS-PAGE gels and Western blot showed that
the HCV
fusion protein is >90% pure after sulfitolysis and IMAC (see Fig. 6A).
The immune reactivity of the fusion protein was checked by ELISA with HCV
positive human
sera. The purified fusion protein was reduced with 200 mM DTT and the protein
was desalted
to 35 mM acetate, 6 M ureum, pH 4 on a Sephadex G25 column (Pharmacia). The
effect of
anti-oxidants and reversible protecting agents (dithiocarbamate, GSH,
cysteine) on the NS3
fusion protein reactivity was verified by adding these agents either before
freezing at -70° or
by adding these compounds during the dilution in the ELISA coating buffer.
NS3 fusion protein coated in the presence of 10 mM or 200 mM DTT were included
as
positive and negative control, respectively. Sera (17790, 17832) are difficult
detectable sera
(HCV NS3 converting sera) and sera (17826, 17838) are easily detectable HCV
positive sera.
HCV sera which are difficult to detect are (1) sera which react not or minimal
with other HCV
antigens (NS3 onlies) or (2) sera which react with NS3- epitopes which are
only presented
and recognized by antibodies after treatment of sulfonated NS3 b9 with 200 mM
DTT. In
contrast, for easy detectable sera a treatment with 10 mM DTT of sulfonated
NS3b9 is
sufficient for restoring the immunological reactivity. The ELISA results are
given in Figures
5A and 5B.
The results show that the disponibility of the epitopes is strongly dependent
on the thiol redox
state, i.e. the difficult HCV sera are only detected either (1) after
reduction of the NS3 with
200 mM DTT in the coating buffer or (2) by incubation of the sample diluent in
the presence of
10 mM or 3mM DTT, provided that the NS3 sample is diluted with thiol
containing
antioxidantia and/or reversible protection agents. The restoration of the
immunological
reactivity was more pronounced with dithiocarbamates than with gluthathion,
cystein or
thiophenecarboxylic acid (TPCB) or thiodiethyleneglycol (TEG). Gluthathion was
in turn
superior to Cystein or other tested mono-SH (TEG, TPCB) products.The best
ELISA signals
were obtained for NS3B9 fusion protein, which was incubated at -70°C
and diluted in the
presence of thiol stabilising and reversible protective agents.
The need of the DTT reduction step to restore the immune reactivity after the
addition of thiol
containing compounds showed the formation of mixed disulfide bridges between
the thiol
agents and the cysteine residues of NS3 b9 fusion protein. The addition of
thiol compounds
have inhibited the reformation of the very stable intramolecular disulphide
bond, that only
could be reduced with 200 mM DTT. This mixed disulfide bridge status resembles
the in vivo
thiolation of proteins, which is known to be a regulator biological activity
and is with minimal
energy input transferred (enzymatically or by a'S-S' reductant) to the reduced
status.
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Example 4: Mapping of monoclonal antibodies against an E1 epitope overlapping
with the
Cvsteine residues from the CXXC site
Ten monoclonal antibodies, directed against E1, were identified which
recognize the N-
terminal region of E1. These monoclonals were characterized regarding their
minimal epitope.
In order to do so two peptides were synthesized and reactivity of each
monoclonal towards
these peptides was analyzed by assessing competition. Recombinant E1 was
adsorbed to
microtiterplates and the monoclonal antibody was allowed to react in the
presence of an
excess of the peptide. Based on these results the ten monoclonal antibodies
can be split in
two groups (Table 1 ). For the first group the minimal epitope is as 209-227,
especially the
lack of reactivity with a peptide not containing the aminoacids 225-227 proves
that these
monoclonals cover an epitope overlapping with the thioredoxine-like site, more
specifically
with the first cysteine of this site. The minimal epitope of the monoclonals
of group 2 does not
reach into the thioredoxine-like site. These results are summarized in Table
1.
Table 1: minimal enitope delineation of monoclonal antibodies directed against
E1
E1 monoclonal antibodies oroup 1: IGH 198. 199 and 200
Sequence as region IGP* result
NDCPNSSIVYEAHDAILHTP 205-224 263 no competition
Bio-GG-SNSSIVYEAADMIMHTPGCV 208-227 436 competition
E1 monoclonal antibodies groupe 2: IGH 201. 202. 203. 204. 205. 206 and 208
Sequence as region IGP* result
NDCPNSSIVYEAHDAILHTP 205-224 263 competition
Bio-GG-SNSSIVYEAHDAILHTPGCV 208-227 436 competition
The minimal epitope for each group is underlined
*IGP refers to the peptide code number
note 1
Also monoclonals are available recognizing an epitope in the C-terminal part
of E1 (IGH 207,
209 and 210, as 307-326; see PCT/EP99/02154). These monoclonals may be used as
controls since they recognize a region which is not at all in the
neighbourhood of the
thioredoxine-like site.
note 2
IGH 198 = 23C12 IGH 203 = 1566 IGH 208 = 5C6
IGH 199 = 1585 IGH 204 = 8A8 IGH 209 = 5E1
IGH 200 = 25CF3 IGH 205 = 3H2 IGH 210 = 7D2
IGH 201 = 11 B7 IGH 206 = 7C4
IGH 202 = 3F3 IGH 207 = 14H11
Thus, monoclonals are available which can be used as tools to determine
changes in the
biological activity and/or conformation of peptides of the present invention.
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Example 5: HCV E1 s purification after reversible modification of Cys-residues
Vaccinia RK13 cells were lysed as described in Maertens et al. (PCT EP95/03031
), but solid
sodium tetrathionate was added to the lysate up to 65 mM instead of the
irreversible thiol
blocking agent N-ethylmaleimide (NEM). The lysate was incubated overnight at
4°C and the
purification steps (Lentil lectin (LCA) chromatography, the concentration of
the LCA eluate
and reduction with DTT) were pertormed as described in Maertens et al. (PCT
EP95/03031 ).
The concentrate was split in 2 and was either sulfonated overnight at
4°C by NaZS406 or
irreversible blocked with N-ethyl-maleimide (NEM) as reference material. The
sulfonated as
well as the NEM-treated E1s were applied on Superdex 6200 (Pharmacia) in the
presence of
Empigen (see Figurel) and the E1s peak was analysed by SDS-PAGE and Western
blot.
The chromatogram overlays as well as the ELISA profiles show that the
irreversible protected
and sulfonated E1s product behave analogously on SEC in the presence of
Empigen. SDS-
PAGE and silverstaining show a similar purity degree of the 2 products.
Example 6: HCV E1 s purification under non -denaturing conditions after Iysis
in the
presence of thiol stabilisin4 agents
Vaccinia infected RK13 cells were lysed as described in Maertens et al. (PCT
EP95/03031 ),
but ascorbate (1 mM) was added to the lysate as thiol stabiliser instead of
NEM. The sample
was applied on the LCA resin and the LCA eluate was acidified till pH 5.5 with
1 M acetic acid.
The acidified eluate was concentrated, after which the pH was adjusted to 7.2
and treated
with DTT as described in Maertens et al. (PCT EP95/03031).
The reduced protein solution was split and treated as follows: either (1 )
acidified to pH 6 (thiol
stabilising conditions) or (2) sulfonated with sodium tetrathionate
(reversibly protected) or (3)
treated with NEM.bio (irreversible blocking). The SEC of the acidified (pH 6)
sample was also
performed at pH 6Ø The other 2 samples were separated on Superdex 6200 in
the presence
of Empigen as decribed in Maertens et al. (PCT EP95/03031 ). The elution
fractions were
analysed by ELISA, by SDS-PAGE and Western blotting.
The material, prepared as described in Maertens et al. (PCT EP95/03031 ) is
included as
reference material for the SEC.
Fraction analysis shows that pure E1s is recovered for the different
conditions (Fig. 3A.1 and
Fig 3A.2). The higher apparent Mr of NEM.bio E1s materials is probably caused
by the
insertion of voluminous blocking group on Els.
Figure 3B shows a Western blot of E1s pools, obtained by different procedures
as described
in Examples 5 and 6.
CA 02387666 2002-03-27
WO 01/30815 PCT/EP00/10499
~S
Examples 5 and 6 illustrate that pure E1s is obtained under non-denaturing
conditions by (1)
using reversible modification agents or (2) running the chromatography under
thiol stabilising
conditions (antioxidant, low pH).
Example 7: Processin4 of Vero E1s and cleavage analogy with growth factors
such as
thioredoxine
Vaccinia infected Vero cells were lysed as described in Maertens et al. (PCT
EP95/03031 ),
but lodoacetamide (IAA) was added as irreversible blocking agent and aprotinin
was added
after an overnight incubation at 4°C.
Chromatography on LCA resin (Pharmacia), reduction with DTT and gel
filtrations were
performed as described, except that IAA was used as irreversible blocking
agent instead of
NEM. The E1s pool was analyzed by silver staining and Western blotting.
Western blot analysis of the semi purified product showed besides the quartet
band in the
region 27-32 kDa also an E1 s band with an Mr of about 18 kDa. The bands were
characterized by NH2-terminal amino acid sequencing.
Main signal sequence of the different bands of the quartet:
YEVR?VSG
(amino- terminus of correctly processed E1s)
Sequence of E1s degradation product:
??VALTPTLAA
This degradation product results from a specific cleavage at the carboxy-
terminus after Arg
237, which is localized upstream of the CVPC-site. The first and second
residue are not
identified, because the cysteine and tryptophan amino acids are destroyed by
the Edman
sequencing method.
Surprisingly, no other degradation products were retrieved although other
basic residues and
even dibasic sequences are present in Els. This specific cleavage pattern
corresponds with a
E1 s domain structure, which has been described for the processing of growth
factors, such as
thioredoxine, which cleavage has resulted in the formation of ECEF (Balcewicz-
Sablinska, et
al., 1991; Newman et al., 1994).
Example 8: Titration of the gKa of the cysteines in the E1s CVPC-site
In order to establish the sequence of reaction steps, ie which cysteine of the
C,VPC2-site
reacts first, the pKa of these cysteines is titrated. The pKa of the cysteines
in the C,VPCz-site
of E1 s is determined by modification of the cysteines in E1 s or synthetic
peptides in function
of the pH.
CA 02387666 2002-03-27
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The modification is pertormed by treatment with IAA at the preset pH,
whereafter the sample
is loaded on RPC after lowering the pH to 2 with Trifluotroacetic acid (TFA).
In order to determine the most reactive cysteine, the excess of IAA reagent is
removed by
RPC. The non-reacted thiol-groups are modified by raising the pH after
addition of
ethyleneimine (El) or Bromo-ethanolamine (BEA).
The treatment with EI or BEA results in the introduction of a lysine mimicking
cysteine adduct,
which creates a supplementary trypsinolysis site. This supplementary site
allows the
identification of most reactive cysteine in the -C,VPC2-site via peptide
fingerprinting and MS
(see also Example 1 ).
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