Note: Descriptions are shown in the official language in which they were submitted.
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PURIFIED HEPATITIS C VIRUS ENVELOPE PROTEINS FOR DIAGNOSTIC
AND THERAPEUTIC USE
Field of the invention
The present invention relates to the general fields of recombinant ~rotein
expression,
purification of recombinant proteins, synthetic peptides, diagnosis of HCV
infection,
prophylactic treatment against HCV infection and to the prognosis/monitoring
of the clinical
efficiency of treatment of an individual with chronic hepatitis, or the
prognosis/monitoring of
natural disease.
More particularly, the present invention relates to purification methods for
hepatitis C
virus envelope proteins, the use in diagnosis, prophylaxis or therapy of HCV
envelope
proteins purified according to the methods described in the present invention,
the use of
single or specific oligomeric E1 and/or E2 and/or E1/E2 envelope proteins in
assays for
monitoring disease, and/or diagnosis of disease, and/or treatment of disease.
The invention
also relates to epitopes of the E1 and/or E2 envelope proteins and monoclonal
antibodies
thereto, as well their use in diagnosis, prophylaxis or treatment.
Background of the invention
The E2 protein purified from cell lysates according to the methods described
in the
2 0 present invention reacts with approximately 95% of patient sera. This
reactivity is similar to
the reactivity obtained with E2 secreted from CHO cells (Spaete et al., 1992).
However, the
intracellularly expressed form of E2 may more closely resemble the native
viral envelope
protein because it contains high mannose carbohydrate motifs, whereas the E2
protein
secreted from CHO cells is further modified with galactose and sialic acid
sugar moieties.
2 5 When the aminoterminal half of E2 is expressed in the baculovirus system,
only about 13 to
21 % of sera from several patient groups can be detected (moue et al., 1992).
After expression
of E2 from E. coli, the reactivity of HCV sera was even lower and ranged from
14
(Yokosuka et al., 1992) to 17% (Mita et al., 1992). About 75% of HCV sera (and
95% of
chronic patients) are anti-El positive using the purified, vaccinia-expressed
recombinant E1
30 protein of the present invention, in sharp contrast with the results of
Kohara et al. (1992) and
Hsu et al. (1993). Kohara et al. used a vaccinia-virus expressed E1 protein
and detected anti-
E1 antibodies in 7 to 23% of patients, while Hsu et al. only detected 14/50
(28%) sera using
baculovirus-expressed El.
CONFIRMATION COPY
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These results show that not only a good expression system but also a good
purification protocol are required to reach a high reactivity of the envelope
proteins with
human patient sera. This can be obtained using the proper expression system
and/or
purification protocols of the present invention which guarantee the
conservation of the
natural folding of the protein and the purification protocols of the present
invention which
guarantee the elimination of contaminating proteins and which preserve the
conformation,
and thus the reactivity of the HCV envelope proteins. The amounts of purified
HCV
envelope protein needed for diagnostic screening assays are in the range of
grams per year.
For vaccine purposes, even higher amounts of envelope protein would be needed.
Therefore,
the vaccinia virus system may be used for selecting the best expression
constructs and for
limited upscaling, and large-scale expression and purification of single or
specific oligomeric
envelope proteins containing high-mannose carbohydrates may be achieved when
expressed
from several yeast strains. In the case of hepatitis B for example,
manufacturing of HBsAg
from mammalian cells was much more costly compared with yeast-derived
hepatitis B
vaccines.
Clinical importance of necro-inflammation and fibrosis in HCV infection.
The natural history of liver disease after HCV infection does vary
significantly from patient
to patient. About 20% of the acutely infected persons are able to resolve
infection
2 0 spontaneously, while 80% of infected persons progresses to a chronic
infection. Chronic
infection results in an ongoing inflammation and/or necrosis (=necro-
inflammation) in the
liver which can be diagnosed by histological analysis of a liver biopsy or
which can be
diagnosed using a surrogate marker such as the presence of the liver enzyme
ALT in serum.
'This chronic infection increases the risk for development of fibrosis which
can lead to
2 5 development of cirrhosis and ultimately liver carcinoma. Many data suggest
that the ongoing
necro-inflammation drives progression to fibrosis and cirrhosis. It is
estimated that up to 20%
of HCV chronic carriers may develop cirrhosis over a time period of about 20
years and that
of those with cirrhosis between 1 to 4%/year is at risk to develop liver
carcinoma. (Lauer and
Walker 2001, Shiffman 1999). Both cirrhosis and liver carcinoma are end-stage
liver diseases
3 0 for which the treatment options are limited to liver transplantation.
Consequently, the most
important aim of therapy for HCV is to reduce the risk of development of end-
stage liver
disease by reducing liver necro-inflammation and/or reducing fibrosis
progression.
For the documentation and/or diagnosis of liver damage several scoring systems
have been
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developed for histological interpretation of a liver biopsy. These scoring
systems may
combine inflammation, necrosis and fibrosis in a single score such as the
Histology
Activity Index (HAI). Other scoring systems have separated the scores for
necro-
inflammation (=grading) from the one for fibrosis/cirrhosis (=staging). These
systems
include the system proposed by Ishak or the Metavir scoring system. A review
of these
scoring systems was published by Leflcowitch in 1997.
It has been shown in several studies that treatment with interferon, and more
recently
treatment with interferon combined with ribavirin and most recently treatment
with pegylated
interferon with or without ribavirin does change the natural history of HCV
and halts further
progression of liver fibrosis especially in those patients with a sustained
viral response
(Schvarcz et al. 1999, Shiffinan 1999, Reichard et al. 1999, Poynard et al.
2002). The
reduction of the risk for hepatocarcinogenesis in persons with sustained
virological and even
with sustained biochemical response has also been documented (Takimoto et al.
2002).
For persons without sustained virological response to interferon based therapy
a
maintenance interferon therapy may be helpful to prevent histological
progression, but this
only in a subset of patients (Alric et al. 2001 ).
Thus, many patients who do not respond to interferon based therapies or who
are excluded
from these therapies for several reasons (this may mount up to 70% of patients
referred to
clinic, Falck-Ytter et al. 2002), remain without a therapeutic option today to
reduce liver
2 0 necro-inflammation and/or reduce the progression of fibrosis in order to
avoid end-stage
liver disease.
Aims of the invention
It is an aim of the present invention to provide a new purification method for
recombinantly expressed E1 and/or E2 and/or E1/E2 proteins such that said
recombinant
proteins are directly usable for diagnostic and vaccine purposes as single or
specific
oligomeric recombinant proteins free from contaminants instead of aggregates.
It is another aim of the present invention to provide compositions comprising
purified
(single or specific oligomeric) recombinant El and/or E2 and/or E1/E2
glycoproteins
comprising conformational epitopes from the E1 and/or E2 domains of HCV.
It is yet another aim of the present invention to provide novel recombinant
vector
constructs for recombinantly expressing E1 and/or E2 and/or E1/E2 proteins, as
well as host
cells transformed with said vector constructs.
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It is also an aim of the present invention to provide a method for producing
and
purifying recombinant HCV E 1 and/or E2 and/or E 1 /E2 proteins.
It is also an aim of the present invention to provide diagnostic and
immunogenic uses
of the recombinant HCV E1 and/or E2 and/or El/E2 proteins of the present
invention, as
well as to provide kits for diagnostic use, vaccines or therapeutics
comprising any of the
recombinant HCV E1 and/or E2 and/or El/E2 proteins of the present invention.
It is further an aim of the present invention to provide for a new use of E1,
E2, and/or
E1/E2 proteins, or suitable parts thereof, for monitoring/prognosing the
response to treatment
of patients (e.g. with interferon) suffering from HCV infection.
It is also an aim of the present invention to provide for the use of the
recombinant E1,
E2, and/or E1/E2 proteins of the present invention in HCV screening and
confirmatory
antibody tests.
It is also an aim of the present invention to provide E1 and/or E2 peptides
which can
be used for diagnosis of HCV infection and for raising antibodies. Such
peptides may also be
used to isolate human monoclonal antibodies.
It is also an aim of the present invention to provide monoclonal antibodies,
more
particularly human monoclonal antibodies or mouse monoclonal antibodies which
are
humanized, which react specifically with E1 and/or E2 epitopes, either
comprised in peptides
or conformational epitopes comprised in recombinant proteins.
2 0 It is also an aim of the present invention to provide possible uses of
anti-E1 or anti-E2
monoclonal antibodies for HCV antigen detection or for therapy of chronic HCV
infection.
It is also an aim of the present invention to provide kits for
monitoring/prognosing
the response to treatment (e.g. with interferon) of patients suffering from
HCV infection or
monitoring/prognosing the outcome of the disease.
2 5 All the aims of the present invention are considered to have been met by
the
embodiments as set out below.
Definitions
The following definitions serve to illustrate the different terms and
expressions used
3 0 in the present invention.
The term 'hepatitis C virus single envelope protein' refers to a polypeptide
or an
analogue thereof (e.g. mimotopes) comprising an amino acid sequence (and/or
amino acid
analogues) defining at least one HCV epitope of either the E1 or the E2
region. These single
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envelope proteins in the broad sense of the word may be both monomeric or homo-
oligomeric forms of recombinantly expressed envelope proteins. Typically, the
sequences
defining the epitope correspond to the amino acid sequence of either the E1 or
the E2 region
of HCV (either identically or via substitution of analogues of the native
amino acid residue
5 that do not destroy the epitope). In general, the epitope-defining sequence
will be 3 or more
amino acids in length, more typically, 5 or more amino acids in length, more
typically 8 or
more amino acids in length, and even more typically 10 or more amino acids in
length. With
respect to conformational epitopes, the length of the epitope-defining
sequence can be subject
to wide variations, since it is believed that these epitopes are formed by the
three-dimensional
shape of the antigen (e.g. folding). Thus, the amino acids defining the
epitope can be
relatively few in number, but widely dispersed along the length of the
molecule being
brought into the correct epitope conformation via folding. The portions of the
antigen
between the residues defining the epitope may not be critical to the
conformational structure
of the epitope. For example, deletion or substitution of these intervening
sequences may not
affect the conformational epitope provided sequences critical to epitope
conformation are
maintained (e.g. cysteines involved in disulfide bonding, glycosylation sites,
etc.). A
conformational epitope may also be formed by 2 or more essential regions of
subunits of a
homooligomer or heterooligomer.
The HCV antigens of the present invention comprise conformational epitopes
from
2 0 the El and/or E2 (envelope) domains of HCV. The E1 domain, which is
believed to
correspond to the viral envelope protein, is currently estimated to span amino
acids 192-383
of the HCV polyprotein (Hijikata et al., 1991). Upon expression in a mammalian
system
(glycosylated), it is believed to have an approximate molecular weight of 35
kDa as
determined via SDS-PAGE. The E2 protein, previously called NS1, is believed to
span
amino acids 384-809 or 384-746 (Grakoui et al., 1993) of the HCV polyprotein
and to also
be an envelope protein. Upon expression in a vaccinia system (glycosylated),
it is believed to
have an apparent gel molecular weight of about 72 kDa. It is understood that
these protein
endpoints are approximations (e.g. the carboxy terminal end of E2 could lie
somewhere in
the 730-820 amino acid region, e.g. ending at amino acid 730, 735, 740, 742,
744, 745,
3 0 preferably 746, 747, 748, 750, 760, 770, 780, 790, 800, 809, 810, 820).
The E2 protein may
also be expressed together with the E1, P7 (aa 747-809), NS2 (aa 810-1026),
NS4A (aa
1658-1711) or NS4B (aa 1712-1972). Expression together with these other HCV
proteins
may be important for obtaining the correct protein folding.
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It is also understood that the isolates used in the examples section of the
present
invention were not intended to limit the scope of the invention and that any
HCV isolate
from type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any other new genotype of HCV is a
suitable source of
E1 and/or E2 sequence for the practice of the present invention.
The E 1 and E2 antigens used in the present invention may be full-length viral
proteins, substantially full-length versions thereof, or functional fragments
thereof (e.g.
fragments which are not missing sequence essential to the formation or
retention of an
epitope). Furthermore, the HCV antigens of the present invention can also
include other
sequences that do not block or prevent the formation of the conformational
epitope of
interest. The presence or absence of a conformational epitope can be readily
determined
though screening the antigen of interest with an antibody (polyclonal serum or
monoclonal to
the conformational epitope) and comparing its reactivity to that of a
denatured version of the
antigen which retains only linear epitopes (if any). In such screening using
polyclonal
antibodies, it may be advantageous to adsorb the polyclonal serum first with
the denatured
antigen and see if it retains antibodies to the antigen of interest.
The HCV antigens of the present invention can be made by any recombinant
method
that provides the epitope of intrest. For example, recombinant intracellular
expression in
mammalian or insect cells is a preferred method to provide glycosylated E1
and/or E2
antigens in 'native' conformation as is the case for the natural HCV antigens.
Yeast cells and
2 0 mutant yeast strains (e.g. mnn 9 mutant (Kniskern et al., 1994) or
glycosylation mutants
derived by means of vanadate resistence selection (Ballou et al., 1991 )) may
be ideally suited
for production of secreted high-mannose-type sugars; whereas proteins secreted
from
mammalian cells may contain modifications including galactose or sialic acids
which may be
undesirable for certain diagnostic or vaccine applications. However, it may
also be possible
2 5 and sufficient for certain applications, as it is known for proteins, to
express the antigen in
other recombinant hosts (such as E. coli) and renature the protein after
recovery.
The term 'fusion polypeptide' intends a polypeptide in which the HCV antigens)
are
part of a single continuous chain of amino acids, which chain does not occur
in nature. The
HCV antigens may be connected directly to each other by peptide bonds or be
separated by
3 0 intervening amino acid sequences. The fusion polypeptides may also contain
amino acid
sequences exogenous to HCV.
The term 'solid phase' intends a solid body to which the individual HCV
antigens or
the fusion polypeptide comprised of HCV antigens are bound covalently or by
noncovalent
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means such as hydrophobic adsorption.
The term 'biological sample' intends a fluid or tissue of a mammalian
individual (e.g.
an anthropoid, a human) that commonly contains antibodies produced by the
individual,
more particularly antibodies against HCV. The fluid or tissue may also contain
HCV antigen.
Such components are known in the art and include, without limitation, blood,
plasma, serum,
urine, spinal fluid, lymph fluid, secretions of the respiratory, intestinal or
genitourinary tracts,
tears, saliva, milk, white blood cells and myelomas. Body components include
biological
liquids. The term 'biological liquid' refers to a fluid obtained from an
organism. Some
biological fluids are used as a source of other products, such as clotting
factors (e.g. Factor
VIII;C), serum albumin, growth hormone and the like. In such cases, it is
important that the
source of biological fluid be free of contamination by virus such as HCV.
The term 'immunologically reactive' means that the antigen in question will
react
specifically with anti-HCV antibodies present in a body component from an HCV
infected
individual.
The term 'immune complex' intends the combination formed when an antibody
binds
to an epitope on an antigen.
'E1' as used herein refers to a protein or polypeptide expressed within the
first 400
amino acids of an HCV polyprotein, sometimes referred to as the E, ENV or S
protein. In its
natural form it is a 35 kDa glycoprotein which is found in strong association
with
2 0 membranes. In most natural HCV strains, the E1 protein is encoded in the
viral polyprotein
following the C (core) protein. The E 1 protein extends from approximately
amino acid (aa)
192 to about as 383 of the full-length polyprotein.
The term 'E 1' as used herein also includes analogs and truncated forms that
are
immunologically cross-reactive with natural El, and includes El proteins of
genotypes l, 2,
2 5 3, 4, 5, 6, 7, 8, 9, 10, or any other newly identified HCV type or
subtype.
'E2' as used herein refers to a protein or polypeptide expressed within the
first 900
amino acids of an HCV polyprotein, sometimes referred to as the NS 1 protein.
In its natural
form it is a 72 kDa glycoprotein that is found in strong association with
membranes. In most
natural HCV strains, the E2 protein is encoded in the viral polyprotein
following the El
30 protein. The E2 protein extends from approximately amino acid position 384
to amino acid
position 746, another form of E2 extends to amino acid position 809. The term
'E2' as used
herein also includes analogs and truncated forms that are immunologically
cross-reactive
with natural E2. For example, insertions of multiple codons between codon 383
and 384, as
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well as deletions of amino acids 384-387 have been reported by Kato et al.
(1992).
'E1/E2' as used herein refers to an oligomeric form of envelope proteins
containing at
least one El component and at least one E2 component.
The term 'specific oligomeric' El and/or E2 and/or E1/E2 envelope proteins
refers to
all possible oligomeric forms of recombinantly expressed El and/or E2 envelope
proteins
which are not aggregates. El and/or E2 specific oligomeric envelope proteins
are also
referred to as homo-oligomeric El or E2 envelope proteins (see below).
The term 'single or specific oligomeric' E1 and/or E2 and/or E1/E2 envelope
proteins
refers to single monomeric E1 or E2 proteins (single in the strict sense of
the word) as well as
specific oligomeric E1 and/or E2 and/or El/E2 recombinantly expressed
proteins. These
single or specific oligomeric envelope proteins according to the present
invention can be
further defined by the following formula (E1)X(E2)y wherein x can be a number
between 0
and 100, and y can be a number between o and 100, provided that x and y are
not both 0.
With x=1 and y=0 said envelope proteins include monomeric E1.
The term 'homo-oligomer' as used herein refers to a complex of El and/or E2
containing more than one E 1 or E2 monomer, e.g. E 1 /E 1 dimers, E 1 /E 1 /E
1 trimers or
E 1 /E 1 /E 1 /E 1 tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2
tetramers, E 1
pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-
oligomers
of El or E2 are all 'homo-oligomers' within the scope of this definition. The
oligomers may
contain one, two, or several different monomers of E1 or E2 obtained from
different types or
subtypes of hepatitis C virus including for example those described in an
international
application published under WO 94/25601 and European application No.
94870166.9 both
by the present applicants. Such mixed oligomers are still homo-oligomers
within the scope of
this invention, and may allow more universal diagnosis, prophylaxis or
treatment of HCV.
2 5 The term 'purified' as applied to proteins herein refers to a composition
wherein the
desired protein comprises at least 35% of the total protein component in the
composition.
The desired protein preferably comprises at least 40%, more preferably at
least about 50%,
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%, and
most preferably
3 0 at least about 95% of the total protein component. The composition may
contain other
compounds such as carbohydrates, salts, lipids, solvents, and the like,
withouth 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.
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The term 'essentially purified proteins' refers to proteins purified such that
they can be
used for in vitro diagnostic methods and as a therapeutic compound. These
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, 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 like SDS-PAGE and silver
staining.
The term 'recombinantly expressed' used within the context of the present
invention
refers to the fact that the proteins of the present invention are produced by
recombinant
expression methods be it in prokaryotes, or lower or higher eukaryotes as
discussed in detail
below.
The term 'lower eukaryote' refers to host cells such as yeast, fungi and the
like. Lower
eukaryotes are generally (but not necessarily) unicellular. Preferred lower
eukaryotes are
yeasts, particularly species within Saccharomyces, Schizosaccharomyces,
Kluyveromyces,
Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia,
Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like.
Saccharomyces
cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast
hosts, and are
convenient fungal hosts.
The term 'prokaryotes' refers to hosts such as E. coli, Lactobacillus,
Lactococcus,
2 0 Salmonella, Streptococcus, Bacillus subtilis or Streptomyces. Also these
hosts are
contemplated within the present invention.
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
2 5 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 frugiperda). The host cells may be provided
in suspension
or flask cultures, tissue cultures, organ cultures and the like. Alternatively
the host cells may
also be transgenic animals.
3 0 The term 'polypeptide' refers to a polymer of amino acids and does not
refer to a
specific length of the product; thus, peptides, oligopeptides, and proteins
are included within
the definition of polypeptide. This term also does not refer to or exclude
post-expression
modifications of the polypeptide, for example, glycosylations, acetylations,
phosphorylations
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and the like. Included within the definition are, for example, polypeptides
containing one or
more analogues of an amino acid (including, for example, unnatural amino
acids, PNA, etc.),
polypeptides with substituted linkages, as well as other modifications known
in the art, both
naturally occurring and non-naturally occurring.
5 The term 'recombinant polynucleotide or nucleic acid' intends a
polynucleotide or
nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which, by
virtue of its
origin or manipulation : (1) is not associated with all or a portion of a
polynucleotide with
which it is associated in nature, (2) is linked to a polynucleotide other than
that to which it is
linked in nature, or (3) does not occur in nature.
10 The term 'recombinant host cells', 'host cells', 'cells', 'cell lines',
'cell cultures', and
other such terms denoting microorganisms or higher eukaryotic cell lines
cultured as
unicellular entities refer to cells which can be or have been, used as
recipients for a
recombinant vector or other transfer polynucleotide, and include the progeny
of the original
cell which has been transfected. It is understood that the progeny of a single
parental cell may
not necessarily be completely identical in morphology or in genomic or total
DNA
complement as the original parent, due to natural, accidental, or deliberate
mutation.
The term 'replicon' is any genetic element, e.g., a plasmid, a chromosome, a
virus, a
cosmid, etc., that behaves as an autonomous unit of polynucleotide replication
within a cell;
i.e., capable of replication under its own control.
2 0 The term 'vector' is a replicon further comprising sequences providing
replication
and/or expression of a desired open reading frame.
The term 'control sequence' refers to polynucleotide sequences which are
necessary to
effect the expression of coding sequences to which they are ligated. The
nature of such
control sequences differs depending upon the host organism; in prokaryotes,
such control
2 5 sequences generally include promoter, ribosomal binding site, and
terminators; in eukaryotes,
generally, such control sequences include promoters, terminators and, in some
instances,
enhancers. The term 'control sequences' is intended to include, at a minimum,
all components
whose presence is necessary for expression, and may also include additional
components
whose presence is advantageous, for example, leader sequences which govern
secretion.
3 0 The term 'promoter' is a nucleotide sequence which is comprised of
consensus
sequences which allow the binding of RNA polymerase to the DNA template in a
manner
such that mRNA production initiates at the normal transcription initiation
site for the
adjacent structural gene.
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The expression 'operably linked' refers to a juxtaposition wherein the
components so
described are in a relationship permitting them to function in their intended
manner. A
control sequence 'operably linked' to a coding sequence is ligated in such a
way that
expression of the coding sequence is achieved under conditions compatible with
the control
sequences.
An 'open reading frame'. (ORF) is a region of a polynucleotide sequence which
encodes a polypeptide and does not contain stop codons; this region may
represent a portion
of a coding sequence or a total coding sequence.
A 'coding sequence' is a polynucleotide sequence which is transcribed into
mRNA
and/or translated into a polypeptide when placed under the control of
appropriate regulatory
sequences. The boundaries of the coding sequence are determined by a
translation start codon
at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding
sequence can
include but is not limited to mRNA, DNA (including cDNA), and recombinant
polynucleotide sequences.
As used herein, 'epitope' or 'antigenic determinant' means an amino acid
sequence
that is immunoreactive. Generally an epitope consists of at least 3 to 4 amino
acids, and more
usually, consists of at least 5 or 6 amino acids, sometimes the epitope
consists of about 7 to
8, or even about 10 amino acids. As used herein, an epitope of a designated
polypeptide
denotes epitopes with the same amino acid sequence as the epitope in the
designated
2 0 polypeptide, and immunologic equivalents thereof. Such equivalents also
include strain,
subtype (=genotype), or type(group)-specific variants, e.g. of the currently
known sequences
or strains belonging to genotypes la, lb, lc, ld, le, lf, 2a, 2b, 2c, 2d, 2e,
2f, 2g, 2h, 2i, 3a,
3b, 3c, 3d, 3e, 3f, 3g, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 41, Sa,
Sb, 6a, 6b, 6c, 7a, 7b, 7c,
8a, 8b, 9a, 9b, 10a, or any other newly defined HCV (sub)type. It is to be
understood that the
2 5 amino acids constituting the epitope need not be part of a linear
sequence, but may be
interspersed by any number of amino acids, thus forming a conformational
epitope.
The term 'immunogenic' refers to the ability of a substance to cause a humoral
and/or
cellular response, whether alone or when linked to a carrier, in the presence
or absence of an
adjuvant. 'Neutralization' refers to an immune response that blocks the
infectivity, either
3 0 partially or fully, of an infectious agent. A 'vaccine' is an immunogenic
composition capable
of eliciting protection against HCV, whether partial or complete. A vaccine
may also be
useful for treatment of an individual, in which case it is called a
therapeutic vaccine.
The term'therapeutic' refers to a composition capable of treating HCV
infection.
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The term 'effective amount' refers to an amount of epitope-bearing polypeptide
sufficient to induce an immunogenic response in the individual to which it is
administered, or
to otherwise detectably immunoreact in its intended system (e.g.,
immunoassay). Preferably,
the effective amount is sufficient to effect treatment, as defined above. The
exact amount
necessary will vary according to the application. For vaccine applications or
for the
generation of polyclonal antiserum / antibodies, for example, the effective
amount may vary
depending on the species, age, and general condition of the individual, the
severity of the
condition being treated, the particular polypeptide selected and its mode of
administration,
etc. It is also believed that effective amounts will be found within a
relatively large, non-
critical range. An appropriate effective amount can be readily determined
using only routine
experimentation. Preferred ranges of E1 and/or E2 and/or E1/E2 single or
specific oligomeric
envelope proteins for prophylaxis of HCV disease are 0.01 to 100 pg/dose,
preferably 0.1 to
SO pg/dose. Several doses may be needed per individual in order to achieve a
sufficient
immune response and subsequent protection against HCV disease.
Detailed description of the invention
More particularly, the present invention contemplates a method for isolating
or
purifying recombinant HCV single or specific oligomeric envelope protein
selected from the
group consisting of E1 and/or E2 and/or E1/E2, characterized in that upon
lysing the
2 0 transformed host cells to isolate the recombinantly expressed protein a
disulphide bond
cleavage or reduction step is carried out with a disculphide bond cleaving
agent.
The essence of these 'single or specific oligomeric' envelope proteins of the
invention
is that they are free from contaminating proteins and that they are not
disulphide bond linked
with contaminants.
2 5 The proteins according to the present invention are recombinantly
expressed in lower
or higher eukaryotic cells or in prokaryotes. The recombinant proteins of the
present
invention are preferably glycosylated and may contain high-mannose-type,
hybrid, or
complex glycosylations. Preferentially said proteins are expressed from
mammalian cell lines
as discussed in detail in the Examples section, or in yeast such as in mutant
yeast strains also
3 0 as detailed in the Examples section.
The proteins according to the present invention may be secreted or expressed
within
components of the cell, such as the ER or the Golgi Apparatus. Preferably,
however, the
proteins of the present invention bear high-mannose-type glycosylations and
are retained in
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the ER or Golgi Apparatus of mammalian cells or are retained in or secreted
from yeast cells,
preferably secreted from yeast mutant strains such as the mnn9 mutant
(Kniskern et al.,
1994), or from mutants that have been selected by means of vanadate resistence
(Ballou et
al., 1991 ).
Upon expression of HCV envelope proteins, the present inventors could show
that
some of the free thiol groups of cysteines not involved in intra- or inter-
molecular disulphide
bridges, react with cysteines of host or expression-system-derived (e.g.
vaccinia) proteins or
of other HCV envelope proteins (single or oligomeric), and form aspecific
intermolecular
bridges. This results in the formation of'aggregates' of HCV envelope proteins
together with
contaminating proteins. It was also shown in WO 92/08734 that 'aggregates'
were obtained
after purification, but it was not described which protein interactions were
involved. In patent
application WO 92/08734, recombinant El/E2 protein expressed with the vaccinia
virus
system were partially purified as aggregates and only found to be 70% pure,
rendering the
purified aggregates not useful for diagnostic, prophylactic or therapeutic
purposes.
Therefore, a major aim of the present invention resides in the separation of
single or
specific-oligomeric HCV envelope proteins from contaminating proteins, and to
use the
purified proteins (> 95% .pure) for diagnostic, prophylactic and therapeutic
purposes. To
those purposes, the present inventors have been able to provide evidence that
aggregated
protein complexes ('aggregates') are formed on the basis of disulphide bridges
and non-
2 0 covalent protein-protein interactions. The present invention thus provides
a means for
selectively cleaving the disulphide bonds under specific conditions and for
separating the
cleaved proteins from contaminating proteins which greatly interfere with
diagnostic,
prophylactic and therapeutic applications. The free thiol groups may be
blocked (reversibly
or irreversibly) in order to prevent the reformation of disulphide bridges, or
may be left to
2 5 oxidize and oligomerize with other envelope proteins (see definition homo-
oligomer). It is to
be understood that such protein oligomers are essentially different from the
'aggregates'
described in WO 92/08734 and WO 94/01778, since the level of contaminating
proteins is
undetectable.
Said disuphide bond cleavage may also be achieved by:
30 (1) performic acid oxidation by means of cysteic acid in which case the
cysteine residues are
modified into cysteic acid (Moore et al., 1963).
(2) Sulfitolysis (R-S-S-R -~ 2 R-SO-3) for example by means of sulphite (SOZ-
3) together with
a proper oxidant such as Cuz+ in which case the cysteine is modified into S-
sulpho-cysteine
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(Bailey and Cole, 1959).
(3) Reduction by means of mercaptans, such as dithiotreitol (DDT), 13-mercapto-
ethanol,
cysteine, glutathione Red, ~-mercapto-ethylamine, or thioglycollic acid, of
which DTT and 13-
mercapto-ethanol are commonly used (Cleland, 1964), is the preferred method of
this
invention because the method can be performed in a water envirorunent and
because the
cysteine remains unmodified.
(4) Reduction by means of a phosphine (e.g. Bu3P) (Ruegg and Rudinger, 1977).
All these compounds are thus to be regarded as agents or means for cleaving
disulphide bonds according to the present invention.
Said disulphide bond cleavage (or reducing) step of the present invention is
preferably a partial disulphide bond cleavage (reducing) step (carried out
under partial
cleavage or reducing conditions).
A preferred disulphide bond cleavage or reducing agent according to the
present
invention is dithiothreitol (DTT). Partial reduction is obtained by using a
low concentration
of said reducing agent, i.e. for DTT for example in the concentration range of
about 0.1 to
about 50 mM, preferably about 0.1 to about 20 mM, preferably about 0.5 to
about 10 mM,
preferably more than 1 mM, more than 2 mM or more than 5 mM, more preferably
about 1.5
mM, about 2.0 mM, about 2.5 mM, about 5 mM or about 7.5 mM.
Said disulphide bond cleavage step may also be carried out in the presence of
a
2 0 suitable detergent (as an example of a means for cleaving disulphide bonds
or in combination
with a cleaving agent) able to dissociate the expressed proteins, such as
DecyIPEG,
EMPIGEN-BB, NP-40, sodium cholate, Triton X-100.
Said reduction or cleavage step (preferably a partial reduction or cleavage
step) is
earned out preferably in in the presence of (with) a detergent. A preferred
detergent
2 5 according to the present invention is Empigen-BB. The amount of detergent
used is
preferably in the range of 1 to 10 %, preferably more than 3%, more preferably
about 3.5% of
a detergent such as Empigen-BB.
A particularly preferred method for obtaining disulphide bond cleavage employs
a
combination of a classical disulphide bond cleavage agent as detailed above
and a detergent
3 0 (also as detailed above). As contemplated in the Examples section, the
particular combination
of a low concentration of DTT (1.5 to 7.5 mM) and about 3.5 % of Empigen-BB is
proven to
be a particularly preferred combination of reducing agent and detergent for
the purification of
recombinantly expressed E1 and E2 proteins. Upon gelfiltration chromatography,
said partial
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reduction is shown to result in the production of possibly dimeric E1 protein
and separation
of this E1 protein from contaminating proteins that cause false reactivity
upon use in
immunoassays.
It is, however, to be understood that also any other combination of any
reducing agent
5 known in the art with any detergent or other means known in the art to make
the cysteines
better accessible is also within the scope of the present invention, insofar
as said combination
reaches the same goal of disulphide bridge cleavage as the preferred
combination
exemplified in the present invention.
Apart from reducing the disulphide bonds, a disulphide bond cleaving means
10 according to the present invention may also include any disulphide bridge
exchanging agents
(competitive agent being either organic or proteinaeous, see for instance
Creighton, 1988)
known in the art which allows the following type of reaction to occur:
R1 S-SR2+R3SH-~R1 S-SR3+R2SH
* R1, R2: compounds of protein aggregates
15 * R3 SH: competitive agent (organic, proteinaeous)
The term 'disulphide bridge exchanging agent' is to be interpretated as
including
disulphide bond reforming as well as disulphide bond blocking agents.
The present invention also relates to methods for purifying or isolating HCV
single or
specific oligomeric envelelope proteins as set out above further including the
use of any SH
2 0 group blocking or binding reagent known in the art such as chosen from the
following list:
- Glutathion
- 5,5'-dithiobis-(2-nitrobenzoic acid) or bis-(3-carboxy-4-nitrophenyl)-
disulphide
(DTNB or Ellman's reagent) (Elmann, 1959)
- N-ethylmaleimide (NEM; Benesch et al., 1956)
2 5 - N-(4-dimethylamino-3,5-dinitrophenyl) maleimide or Tuppy's maleimide
which
provides a color to the protein
- P-chloromercuribenzoate (Grassetti et al., 1969)
- 4-vinylpyridine (Friedman and Krull, 1969) can be liberated after reaction
by acid
hydrolysis
30 - acrylonitrile, can be liberated after reaction by acid hydrolysis (Weil
and Seibles,
1961)
- NEM-biotin (e.g. obtained from Sigma B 1267)
- 2,2'-dithiopyridine (Grassetti and Murray, 1967)
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- 4,4'-dithiopyridine (Grassetti and Murray, 1967)
- 6,6'-dithiodinicontinic acid (DTDNA; Brown and Cunnigham, 1970)
- 2,2'-dithiobis-(5'-nitropyridine) (DTNP; US patent 3597160) or other
dithiobis
(heterocyclic derivative) compounds (Grassetti and Murray, 1969)
A survey of the publications cited shows that often different reagents for
sulphydryl
groups will react with varying numbers of thiol groups of the same protein or
enzyme
molecule. One may conclude that this variation in reactivity of the thiol
groups is due to the
steric environment of these groups, such as the shape of the molecule and the
surrounding
groups of atoms and their charges, as well as to the size, shape and charge of
the reagent
molecule or ion. Frequently the presence of adequate concentrations of
denaturants such as
sodium dodecylsulfate, urea or guanidine hydrochoride will cause sufficient
unfolding of the
protein molecule to permit equal access to all of the reagents for thiol
groups. By varying the
concentration of denaturant, the degree of unfolding can be controlled and in
this way thiol
groups with different degrees of reactivity may be revealed. Although up to
date most of the
work reported has been done with p-chloromercuribenzoate, N-ethylmaleimide and
DTNB, it
is likely that the other more recently developed reagents may prove equally
useful. Because
of their varying structures, it seems likely, in fact, that they may respond
differently to
changes in the steric environment of the thiol groups.
Alternatively, conditions such as low pH (preferably lower than pH 6) for
preventing
2 0 free SH groups from oxidizing and thus preventing the formation of large
intermolecular
aggregates upon recombinant expression and purification of E1 and E2
(envelope) proteins
are also within the scope of the present invention.
A preferred SH group blocking reagent according to the present invention is N
ethylmaleimide (NEM). Said SH group blocking reagent may be administrated
during lysis
2 5 of the recombinant host cells and a$er the above-mentioned partial
reduction process or after
any other process for cleaving disulphide bridges. Said SH group blocking
reagent may also
be modified with any group capable of providing a detectable label and/or any
group aiding
in the.immobilization of said recombinant protein to a solid substrate, e.g.
biotinylated NEM.
Methods for cleaving cysteine bridges and blocking free cysteines have also
been
30 described in Darbre (1987), Means and Feeney (1971), and by Wong (1993).
A method to purify single or specific oligomeric recombinant E1 and/or E2
and/or
E1/E2 proteins according to the present invention as defined above is further
characterized as
comprising the following steps:
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- lysing recombinant EI and/or E2 and/or E1/E2 expressing host cells,
preferably in the
presence of an SH group blocking agent, such as N-ethylmaleimide (NEM), and
possibly a suitable detergent, preferably Empigen-BB,
- recovering said HCV envelope protein by affinity purification for instance
by means
lectin-chromatography, such as lentil-lectin chromatography, or immunoaffinity
chromatography using anti-E1 and/or mti-E2 specific monoclonal antibodies,
followed by,
- reduction or cleavage of disulphide bonds with a disulphide bond cleaving
agent,
such as DTT, preferably also in the presence of an SH group blocking agent,
such as
NEM or Biotin-NEM, and,
- recovering the reduced HCV EI and/or E2 and/or EI/E2 envelope proteins for
instance by gelfiltration (size exclusion chromatography or molecular sieving)
and
possibly also by an additional Ni2+-IMAC chromatography and desalting step.
It is to be understood that the above-mentioned recovery steps may also be
carried out
using any other suitable technique known by the person skilled in the art.
Preferred lectin-chromatography systems include Galanthus nivalis agglutinin
(GNA) - chromatography, or Lens culinaris agglutinin (LCA) (lentil) lectin
chromatography
as illustrated in the Examples section. Other useful lectins include those
recognizing high-
mannose type sugars, such as Narcissus pseudonarcissus agglutinin (NPA), Pisum
sativum
2 0 agglutinin (PSA), or Allium ursinum agglutinin (AUA).
Preferably said method is usable to purify single or specific oligomeric HCV
envelope protein produced intracellularly as detailed above.
For secreted E1 or E2 or E1/E2 oligomers, lectins binding complex sugars such
as
Ricinus communis agglutinin I (RCA I), are preferred lectins.
2 5 The present invention more particularly contemplates essentially purified
recombinant HCV single or specific oligomeric envelope proteins, selected from
the group
consisting of E1 and/or E2 and/or E1/E2, characterized as being isolated or
purified by a
method as defined above.
The present invention more particularly relates to the purification or
isolation of
3 0 recombinant envelope proteins which are expressed from recombinant
mammalian cells such
as vaccinia.
The present invention also relates to the purification or isolation of
recombinant
envelope proteins which are expressed from recombinant yeast cells.
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The present invention equally relates to the purification or isolation of
recombinant
envelope proteins which are expressed from recombinant bacterial (prokaryotic)
cells.
The present invention also contemplates a recombinant vector comprising a
vector
sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic
promoter sequence
followed by a nucleotide sequence allowing the expression of the single or
specific
oligomeric E1 and/or E2 and/or E1/E2 of the invention.
Particularly, the present invention contemplates a recombinant vector
comprising a
vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic
promoter
sequence followed by a nucleotide sequence allowing the expression of the
single E1 or E1
of the invention.
Particularly, the present invention contemplates a recombinant vector
comprising a
vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic
promoter
sequence followed by a nucleotide sequence allowing the expression of the
single E1 or E2
of the invention.
The segment of the HCV cDNA encoding the desired E1 and/or E2 sequence
inserted
into the vector sequence may be attached to a signal sequence. Said signal
sequence may be
that from a non-HCV source, e.g. the IgG or tissue plasminogen activator (tpa)
leader
sequence for expression in mammalian cells, or the o,-mating factor sequence
for expression
into yeast cells, but particularly preferred constructs according to the
present invention
2 0 contain signal sequences appearing in the HCV genome before the respective
start points of
the E1 and E2 proteins. The segment of the HCV cDNA encoding the desired E1
and/or E2
sequence inserted into the vector may also include deletions e.g. of the
hydrophobic
domains) as illustrated in the examples section, or of the E2 hypervariable
region I.
More particularly, the recombinant vectors according to the present invention
2 5 encompass a nucleic acid having an HCV cDNA segment encoding the
polyprotein starting
in the region between amino acid positions 1 and 192 and ending in the region
between
positions 250 and 400 of the HCV polyprotein, more preferably ending in the
region between
positions 250 and 341, even more preferably ending in the region between
positions 290 and
341 for expression of the HCV single E1 protein. Most preferably, the present
recombinant
30 vector encompasses a recombinant nucleic acid having a HCV cDNA seqment
encoding part
of the HCV polyprotein starting in the region between positions 117 and 192,
and ending at
any position in the region between positions 263 and 326, for expression of
HCV single E1
protein. Also within the scope of the present invention are forms that have
the first
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hydrophobic domain deleted (positions 264 to 293 plus or minus 8 amino acids),
or forms to
which a 5'-terminal ATG codon and a 3'-terminal stop codon has been added, or
forms which
have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons
have been added.
More particularly, the recombinant vectors according to the present invention
encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein
starting
in the region between amino acid positions 290 and 406 and ending in the
region between
positions 600 and 820 of the HCV polyprotein, more preferably starting in the
region
between positions 322 and 406, even.more preferably starting in the region
between positions
347 and 406, even still more preferably starting in the region between
positions 364 and 406
for expression of the HCV single E2 protein. Most preferably, the present
recombinant vector
encompasses a recombinant nucleic acid having a HCV cDNA seqment encoding the
polyprotein starting in the region between positions 290 and 406, and ending
at any position
of positions 623, 650, 661, 673, 710, 715, 720, 746 or 809, for expression of
HCV single E2
protein. Also within the scope of the present invention are forms to which a
5'-terminal ATG
codon and a 3'-terminal stop codon has been added, or forms which have a
factor Xa
cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.
A variety of vectors may be used to obtain recombinant expression of HCV
single or
specific oligomeric envelope proteins of the present invention. Lower
eukaryotes such as
yeasts and glycosylation mutant strains are typically transformed with
plasmids, or are
2 0 transformed with a recombinant virus. The vectors may replicate within the
host
independently, or may integrate into the host cell genome.
Higher eukaryotes may be transformed with vectors, or may be infected with a
recombinant virus, for example a recombinant vaccinia virus. Techniques and
vectors for the
insertion of foreign DNA into vaccinia virus are well known in the art, and
utilize, for
2 5 example homologous recombination. A wide variety of viral promoter
sequences, possibly
terminator sequences and poly(A)-addition sequences, possibly enhancer
sequences and
possibly amplification sequences, all required for the mammalian expression,
are available in
the art. Vaccinia is particularly preferred since vaccinia halts the
expression of host cell
proteins. Vaccinia is also very much preferred since it allows the expression
of E1 and E2
3 0 proteins of HCV in cells or individuals which are immunized with the live
recombinant
vaccinia virus. For vaccination of humans the avipox and Ankara Modified Virus
(AMV) are
particularly useful vectors.
Also known are insect expression transfer vectors derived from baculovirus
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Autographa californica nuclear polyhedrosis virus (AcNPV), which is a helper-
independent
viral expression vector. Expression vectors derived from this system usually
use the strong
viral polyhedrin gene promoter to drive the expression of heterologous genes.
Different
vectors as well as methods for the introduction of heterologous DNA into the
desired site of
5 baculovirus are available to the man skilled in the art for baculovirus
expression. Also
different signals for posttranslational modification recognized by insect
cells are known in
the art.
Also included within the scope of the present invention is a method for
producing
purified recombinant single or specific oligomeric HCV E1 or E2 or E1/E2
proteins, wherein
10 the cysteine residues involved in aggregates formation are replaced at the
level of the nucleic
acid sequence by other residues such that aggregate formation is prevented.
The recombinant
proteins expressed by recombinant vectors caarying such a mutated E 1 and/or
E2 protein
encoding nucleic acid are also within the scope of the present invention.
The present invention also relates to recombinant E1 and/or E2 and/or E1/E2
proteins
15 characterized in that at least one of their glycosylation sites has been
removed and are
consequently termed glycosylation mutants. As explained in the Examples
section, different
glycosylation mutants may be desired to diagnose (screening, confirmation,
prognosis, etc.)
and prevent HCV disease according to the patient in question. An E2 protein
glycosylation
mutant lacking the GLY4 has for instance been found to improve the reactivity
of certain sera
2 0 in diagnosis. These glycosylation mutants are preferably purified
according to the method
disclosed in the present invention. Also contemplated within the present
invention are
recombinant vectors carrying the nucleic acid insert encoding such a E1 and/or
E2 and/or
E1/E2 glycosylation mutant as well as host cells tranformed with such a
recombinant vector.
2 5 The present invention also relates to recombinant vectors including a
polynucleotide
which also forms part of the present invention. The present invention relates
more
particularly to the recombinant nucleic acids as represented in SEQ ID NO 3,
5, 7, 9, 11, 13,
21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.
The present invention also contemplates host cells transformed with a
recombinant
vector as defined above, wherein said vector comprises a nucleotide sequence
encoding HCV
E1 and/or E2 and/or E1/E2 protein as defined above in addition to a regulatory
sequence
operably linked to said HCV E1 and/or E2 and/or E1/E2 sequence and capable of
regulating
the expression of said HCV El and/or E2 and/or E1/E2 protein.
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Eukaryotic hosts include lower and higher eukaryotic hosts as described in the
definitions section. Lower eukaryotic hosts include yeast cells well known in
the art. Higher
eukaryotic hosts mainly include mammalian cell lines known in the art and
include many
immortalized cell lines available from the ATCC, inluding HeLa cells, Chinese
hamster
ovary (CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK13 and a number of
other
cell lines.
The present invention relates particularly to a recombinant E 1 and/or E2
and/or
E1/E2 protein expressed by a host cell as defined above containing a
recombinany vector as
defined above. These recombinant proteins are particularly purified according
to the method
of the present invention.
A preferred method for isolating or purifying HCV envelope proteins as defined
above is further characterized as comprising at least the following steps:
- growing a host cell as defined above transformed with a recombinant vector
according to the present invention or with a known recombinant vector
expressing E1
and/or E2 and/or E 1 /E2 HCV envelope proteins in a suitable culture medium,
- causing expression of said vector sequence as defined above under suitable
conditions, and,
- lysing said transformed host cells, preferably in the presence of a SH group
blocking
agent, such as N-ethylmaleimide (NEM), and possibly a suitable detergent,
preferably
2 0 Empigen-BB,
- recovering said HCV envelope protein by affinity purification such as by
means of
lectin-chromatography or immunoaffinity chromatography using anti-E1 and/or
anti-
E2 specific monoclonal antibodies, with said lectin being preferably lentil-
lectin or
GNA, followed by,
2 5 - incubation of the eluate of the previous step with a disulphide bond
cleavage means,
such as DTT, preferably followed by incubation with an SH group blocking
agent,
such as NEM or Biotin-NEM, and,
- isolating the HCV single or specific oligomeric El and/or E2 and/or E1/E2
proteins
such as by means of gelfiltration and possibly also by a subsequent Ni2+-IMAC
3 0 chromatography followed by a desalting step.
As a result of the above-mentioned proces, El and/or E2 and/or E1/E2 proteins
may
be produced in a form which elute differently from the large aggregates
containing vector-
derived components and/or cell components in the void volume of the
gelfiltration column or
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22
the IMAC column as illustrated in the Examples section. The disulphide bridge
cleavage step
advantageously also eliminates the false reactivity due to the presence of
host and/or
expression-system-derived proteins. The presence of NEM and a suitable
detergent during
lysis of the cells may already partly or even completely prevent the
aggregation between the
HCV envelope proteins and contaminants.
NiZ+-IMAC chromatography followed by a desalting step is preferably used for
contracts bearing a (His)6 as described by Janknecht et al., 1991, and Hochuli
et al., 1988.
The present invention also relates to a method for producing monoclonal
antibodies
in small animals such as mice or rats, as well as a method for screening and
isolating human
B-cells that recognize anti-HCV antibodies, using the HCV single or specific
oligomeric
envelope proteins of the present invention.
The present invention further relates to a composition comprising at least one
of the
following E1 peptides as listed in Table 3 and described elsewhere herein:
E1-31 (SEQ ID N0:56) spanning amino acids 181 to 200 of the Core/El V1 region,
E1-33 (SEQ ID N0:57) spanning amino acids 193 to 212 of the E1 region,
E1-35 (SEQ ID N0:58) spanning amino acids 205 to 224 of the E1 V2 region
(epitope B),
E1-35A (SEQ ID N0:59) spanning amino acids 208 to 227 of the E1 V2 region
(epitope B),
lbEl (SEQ ID N0:53) spanning amino acids 192 to 228 of E1 regions (V1, C1, and
V2 regions (containing epitope B)),
E1-51 (SEQ ID N0:66) spanning amino acids 301 to 320 of the El region,
E1-53 (SEQ ID N0:67) spanning amino acids 313 to 332 of the E1 C4 region
(epitope A),
E1-55 (SEQ ID N0:68) spanning amino acids 325 to 344 of the E1 region,
The present invention also relates to a composition comprising at least one of
the
following E2 peptides as listed in Table 3:
Env 67 or E2-67 (SEQ ID N0:72) spanning amino acid positions 397 to 416 of the
E2 region (epitope A, recognized by monoclonal antibody 2F10H10, see Figure
19),
3 0 Env 69 or E2-69 (SEQ ID N0:73) spanning amino acid positions 409 to 428 of
the
E2 region (epitope A),
Env 23 or E2-23 (SEQ ID N0:86) spanning positions 583 to 602 of the E2 region
(epitope E),
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Env 25 or E2-25 (SEQ ID N0:87) spanning positions 595 to 614 of the E2 region
(epitope E),
Env 27 or E2-27 (SEQ ID N0:88) spanning positions 607 to 626 of the E2 region
(epitope E),
Env 17B or E2-17B (SEQ ID N0:83) spanning positions 547 to 566 of the E2
region
(epitope D),
Env 13B or E2-13B (SEQ ID N0:82) spanning positions 523 to 542 of the E2
region
(epitope C; recognized by monoclonal antibody 16A6E7, see Figure 19).
The present invention also relates to a composition comprising at least one of
the
following E2 conformational epitopes:
epitope F recognized by monoclonal antibodies 15C8C1, 12D11F1 and 8G10D1H9,
epitope G recognized by monoclonal antibody 9G3E6,
epitope H (or C) recognized by monoclonal antibody l OD3C4 and 4H6B2, or,
epitope I recognized by monoclonal antibody 17F2C2.
The present invention also relates to an E1 or E2 specific antibody raised
upon
immunization with a peptide or protein composition, with said antibody being
specifically
reactive with any of the polypeptides or peptides as defined above, and with
said antibody
being preferably a monoclonal antibody.
The present invention also relates to an El or E2 specific antibody screened
from a
2 0 variable chain library in plasmids or phages or from a population of human
B-cells by means
of a process known in the art, with said antibody being reactive with any of
the polypeptides
or peptides as defined above, and with said antibody being preferably a
monoclonal antibody.
The El or E2 specific monoclonal antibodies of the invention can be produced
by any
hybridoma liable to be formed according to classical methods from splenic
cells of an
2 5 animal, particularly from a mouse or rat, immunized against the HCV
polypeptides or
peptides according to the invention, as defined above on the one hand, and of
cells of a
myeloma cell line on the other hand, and to be selected by the ability of the
hybridoma to
produce the monoclonal antibodies recognizing the polypeptides which has been
initially
used for the immunization of the animals.
3 0 The antibodies involved in the invention can be labelled by an appropriate
label of the
enzymatic, fluorescent, or radioactive type.
The monoclonal antibodies according to this preferred embodiment of the
invention
may be humanized versions of mouse monoclonal antibodies made by means of
recombinant
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24
DNA technology, departing from parts of mouse and/or human genomic DNA
sequences
coding for H and L chains from cDNA or genomic clones coding for H and L
chains.
Alternatively the monoclonal antibodies according to this preferred embodiment
of
the invention may be human monoclonal antibodies. These antibodies according
to the
present embodiment of the invention can also be derived from human peripheral
blood
lymphocytes of patients infected with HCV, or vaccinated against HCV. Such
human
monoclonal antibodies are prepared, for instance, by means of human peripheral
blood
lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID)
mice (for
recent review, see Duchosal et al., 1992).
The invention also relates to the use of the proteins or peptides of the
invention, for
the selection of recombinant antibodies by the process of repertoire cloning
(Persson et al.,
1991).
Antibodies directed to peptides or single or specific oligomeric envelope
proteins
derived from a certain genotype may be used as a medicament, more particularly
for
incorporation into an immunoassay for the detection of HCV genotypes (for
detecting the
presence of HCV El or E2 antigen), for prognosing/monitoring of HCV disease,
or as
therapeutic agents.
Alternatively, the present invention also relates to the use of any of the
above-
specified E1 or E2 specific monoclonal antibodies for the preparation of an
immunoassay kit
2 0 for detecting the presence of E 1 or E2 antigen in a biological sample,
for the preparation of a
kit for prognosing/monitoring of HCV disease or for the preparation of a HCV
medicament.
The present invention also relates to the a method for in vitro diagnosis or
detection
of HCV antigen present in a biological sample, comprising at least the
following steps
(i) contacting said biological sample with any of the E1 and/or E2 specific
2 5 monoclonal antibodies as defined above, preferably in an immobilized form
under appropriate conditions which allow the formation of an immune
complex,
(ii) removing unbound components,
(iii) incubating the immune complexes formed with heterologous antibodies,
3 0 which specifically bind to the antibodies present in the sample to be
analyzed,
with said heterologous antibodies having conjugated to a detectable label
under appropriate conditions,
(iv) detecting the presence of said immune complexes visually or mechanically
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(e.g. by means of densitometry, fluorimetry, colorimetry).
The present invention also relates to a kit for in vitro diagnosis of HCV
antigen
present in a biological sample, comprising:
- at least one monoclonal antibody as defined above, with said antibody being
5 preferentially immobilized on a solid substrate,
- a buffer or components necessary for producing the buffer enabling binding
reaction between these antibodies and the HCV antigens present in the
biological sample,
- a means for detecting the immune complexes formed in the preceding binding
10 reaction,
- possibly also including an automated scanning and interpretation device for
inferring the HCV antigens present in the sample from the observed binding
pattern.
The present invention also relates to a composition comprising E1 and/or E2
and/or
15 E1/E2 recombinant HCV proteins purified according to the method of the
present invention
or a composition comprising at least one peptides as specified above for use
as a
medicament.
The present invention more particularly relates to a composition comprising at
least
one of the above-specified envelope peptides or a recombinant envelope protein
composition
2 0 as defined above, for use as a vaccine for immunizing a mammal, preferably
humans, against
HCV, comprising administering a sufficient amount of the composition possibly
accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune
response.
More particularly, the present invention relates to the use of any of the
compositions
as described here above for the preparation of a vaccine as described above.
2 5 Also, the present invention relates to a vaccine composition for
immunizing a
mammal, preferably humans, against HCV, comprising HCV single or specific
oligomeric
proteins or peptides derived from the E1 and/or the E2 region as described
above.
Immunogenic compositions can be prepared according to methods known in the
art.
The present compositions comprise an immunogenic amount of a recombinant El
and/or E2
and/or E1/E2 single or specific oligomeric proteins as defined above or E1 or
E2 peptides as
defined above, usually combined with a pharmaceutically acceptable carrier,
preferably
further comprising an adjuvant.
The single or specific oligomeric envelope proteins of the present invention,
either El
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26
and/or E2 and/or E1/E2, are expected to provide a particularly useful vaccine
antigen, since
the formation of antibodies to either E1 or E2 may be more desirable than to
the other
envelope protein, and since the E2 protein is cross-reactive between HCV types
and the E1
protein is type-specific. Cocktails including type 1 E2 protein and E1
proteins derived from
several genotypes may be particularly advantageous. Cocktails containing a
molar excess of
E1 versus E2 or E2 versus El may also be particularly useful. Immunogenic
compositions
may be administered to animals to induce production of antibodies, either to
provide a source
of antibodies or to induce protective immunity in the animal.
Pharmaceutically acceptable carriers include any carrier that does not itself
induce the
production of antibodies harmful to the individual receiving the composition.
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 of ordinary
skill in the art.
Preferred adjuvants to enhance effectiveness of the composition include, but
are not
limited to : aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-
isoglutamine (thr-
MDP) as found in U.S. Patent No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-
isoglutamine
(nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-
dipalmitoyl-sn-
glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and RIBI, which contains
three
2 0 components extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate, and cell
wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the 3
components MPL, TDM or CWS may also be used alone or combined 2 by 2.
Additionally,
adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1
(Syntex) may
be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant
2 5 (IFA) may be used for non-human applications and research purposes.
The immunogenic compositions typically will contain pharmaceutically
acceptable
vehicles, such as water, saline, glycerol, ethanol, etc. Additionally,
auxiliary substances, such
as wetting or emulsifying agents, pH buffering substances, preservatives, and
the like, may
be included in such vehicles.
30 Typically, the immunogenic compositions are prepared as injectables, either
as liquid
solutions or suspensions; solid forms suitable for solution in, or suspension
in, liquid vehicles
prior to injection may also be prepared. The preparation also may be
emulsified or
encapsulated in liposomes for enhanced adjuvant effect. The El and E2 proteins
may also be
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27
incorporated into Immune Stimulating Complexes together with saponins, for
example Quil
A (ISCOMS).
Immunogenic compositions used as vaccines comprise a 'sufficient amount' or
'an
immunologically effective amount' of the envelope proteins of the present
invention, as well
as any other of the above mentioned components, as needed. '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 treatment, as defined above. This
amount varies
depending upon the health and physical condition of the individual to be
treated, the
taxonomic group of individual to be treated (e.g. nonhuman primate, primate,
etc.), the
capacity of the individual's immune system to synthesize antibodies, the
degree of protection
desired, the formulation of the vaccine, the treating doctor's assessment of
the medical
situation, the strain of 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 ~g/dose, more particularly
from 0.1 to 100
~g/dose.
The single or specific oligomeric envelope proteins may also serve as vaccine
carriers
to present homologous (e.g. T cell epitopes or B cell epitopes from the core,
NS2, NS3, NS4
or NSS regions) or heterologous (non-HCV) haptens, in the same manner as
Hepatitis B
surface antigen (see European Patent Application 174,444). In this use,
envelope proteins
2 0 provide an immunogenic carrier capable of stimulating an immune response
to haptens or
antigens conjugated to the aggregate. The antigen may be conjugated either by
conventional
chemical methods, or may be cloned into the gene encoding E1 and/or E2 at a
location
corresponding to a hydrophilic region of the protein. Such hydrophylic regions
include the
V 1 region (encompassing amino acid positions 191 to 202), the V2 region
(encompassing
2 5 amino acid positions 213 to 223), the V3 region (encompassing amino acid
positions 230 to
242), the V4 region (encompassing amino acid positions 230 to 242), the VS
region
(encompassing amino acid positions 294 to 303) and the V6 region (encompassing
amino
acid positions 329 to 336). Another useful location for insertion of haptens
is the
hydrophobic region (encompassing approximately amino acid positions 264 to
293). It is
3 0 shown in the present invention that this region can be deleted without
affecting the reactivity
of the deleted E1 protein with antisera. Therefore, haptens may be inserted at
the site of the
deletion.
The immunogenic compositions are conventionally administered parenterally,
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28
typically by injection, 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.
The present invention also relates to a composition comprising peptides or
polypeptides as described above, for in vitro detection of HCV antibodies
present in a
biological sample.
The present invention also relates to the use of a composition as described
above for
the preparation of an immunoassay kit for detecting HCV antibodies present in
a biological
sample.
The present invention also relates to a method for in vitro diagnosis of HCV
antibodies present in a biological sample, comprising at least the following
steps
(i) contacting said biological sample with a composition comprising any of the
envelope peptide or proteins as defined above, preferably in an immobilized
form under appropriate conditions which allow the formation of an immune
complex, wherein said peptide or protein can be a biotinylated peptide or
protein which is covalently bound to a solid substrate by means of
streptavidin or avidin complexes,
(ii) removing unbound components,
2 0 (iii) incubating the irrunune complexes formed with heterologous
antibodies, with
said heterologous antibodies having conjugated to a detectable label under
appropriate conditions,
(iv) detecting the presence of said immune complexes visually or mechanically
(e.g. by means of densitometry, fluorimetry, colorimetry).
2 5 Alternatively, the present invention also relates to competition
immunoassay formats
in which recombinantly produced purified single or specific oligomeric protein
E1 and/or E2
and/or E1/E2 proteins as disclosed above are used in combination with E1
and/or E2 peptides
in order to compete for HCV antibodies present in a biological sample.
The present invention also relates to a kit for determining the presence of
HCV
3 0 antibodies, in a biological sample, comprising
- at least one peptide or protein composition as defined above, possibly in
combination with other ~polypeptides or peptides from HCV or other types of
HCV, with said peptides or proteins being preferentially immobilized on a
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29
solid substrate, more preferably on different microwells of the same ELISA
plate, and even more preferentially on one and the same membrane strip,
- a buffer or components necessary for producing the buffer enabling binding
reaction between these polypeptides or peptides and the antibodies against
HCV present in the biological sample,
- means for detecting the immune complexes formed in the preceding binding
reaction,
- possibly also including an automated scanning and 'interpretation device for
infernng the HCV genotypes present in the sample from the observed binding
pattern.
The immunoassay methods according to the present invention utilize single or
specific oligomeric antigens from the E1 and/or E2 domains that maintain
linear (in case of
peptides) and conformational epitopes (single or specific oligomeric proteins)
recognized by
antibodies in the sera from individuals infected with HCV. It is within the
scope of the
invention to use for instance single or specific oligomeric antigens, dimeric
antigens, as well
as combinations of single or specific oligomeric antigens. The HCV E1 and E2
antigens of
the present invention may be employed in virtually any assay format that
employs a known
antigen to detect antibodies. Of course, a format that denatures the HCV
conformational
epitope should be avoided or adapted. A common feature of all of these assays
is that the
2 0 antigen is contacted with the body component suspected of containing HCV
antibodies under
conditions that permit the antigen to bind to any such antibody present in the
component.
Such conditions will typically be physiologic temperature, pH and ionic
strenght using an
excess of antigen. The incubation of the antigen with the specimen is followed
by detection
of immune complexes comprised of the antigen.
2 5 Design of the immunoassays is subject to a great deal of variation, and
many formats
are known in the art. Protocols may, for example, use solid supports, or
immunoprecipitation.
Most assays involve the use of labeled antibody or polypeptide; the labels may
be, for
example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye
molecules. Assays
which amplify the signals from the immune complex are also known; examples of
which are
3 0 assays which utilize biotin and avidin or streptavidin, and enzyme-labeled
and mediated
immunoassays, such as ELISA assays.
The immunoassay may be, without limitation, in a heterogeneous or in a
homogeneous format, and of a standard or competitive type. In a heterogeneous
format, the
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polypeptide is typically bound to a solid matrix or support to facilitate
separation of the
sample from the polypeptide after incubation. Examples of solid supports that
can be used
are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl
chloride (e.g., in
sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter
plates,
5 polyvinylidine fluoride (known as ImmunolonT"'), diazotized paper, nylon
membranes,
activated beads, and Protein A beads. For example, Dynatech Immunolon'~"' 1 or
Immunlon'~"'' 2 microtiter plates or 0.25 inch polystyrene beads (Precision
Plastic Ball) can be
used in the heterogeneous format. The solid support containing the antigenic
polypeptides is
typically washed after separating it from the test sample, and prior to
detection of bound
10 antibodies. Both standard and competitive formats are know in the art.
In a homogeneous format, the test sample is incubated with the combination of
antigens in solution. For example, it may be under conditions that will
precipitate any
antigen-antibody complexes which are formed. Both standard and competitive
formats for
these assays are known in the art.
15 In a standard format, the amount of HCV antibodies in the antibody-antigen
complexes is directly monitored. This may be accomplished by determining
whether labeled
anti-xenogeneic (e.g. anti-human) antibodies which recognize an epitope on
anti-HCV
antibodies will bind due to complex formation. In a competitive format, the
amount of HCV
antibodies in the sample is deduced by monitoring the competitive effect on
the binding of a
2 0 known amount of labeled antibody (or other competing ligand) in the
complex.
Complexes formed comprising anti-HCV antibody (or in the case of competitive
assays, the amount of competing antibody) are detected by any of a number of
known
techniques, depending on the format. For example, unlabeled HCV antibodies in
the complex
may be detected using a conjugate of anti-xenogeneic Ig complexed with a label
(e.g. an
2 5 enzyme label).
In an immunoprecipitation or agglutination assay format the reaction between
the
HCV antigens and the antibody forms a network that precipitates from the
solution or
suspension and forms a visible layer or film of precipitate. If no anti-HCV
antibody is present
in the test specimen, no visible precipitate is formed.
3 0 There currently exist three specific types of particle agglutination (PA)
assays. These
assays are used for the detection of antibodies to various antigens when
coated to a support.
One type of this assay is the hemagglutination assay using red blood cells
(RBCs) that are
sensitized by passively adsorbing antigen (or antibody) to the RBC. The
addition of specific
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31
antigen antibodies present in the body component, if any, causes the RBCs
coated with the
purified antigen to agglutinate.
To eliminate potential non-specific reactions in the hemagglutination assay,
two
artificial carriers may be used instead of RBC in the PA. The most common of
these are latex
particles. However, gelatin particles may also be used. The assays utilizing
either of these
carriers are based on passive agglutination of the particles coated with
purified antigens.
The HCV single or specififc oligomeric E1 and/or E2 and/or E1/E2 antigens of
the
present invention comprised of conformational epitopes will typically be
packaged in the
form of a kit for use in these immunoassays. The kit will normally contain in
separate
containers the native HCV antigen, control antibody formulations (positive
and/or negative),
labeled antibody when the assay format requires the same and signal generating
reagents (e.g.
enzyme substrate) if the label does not generate a signal directly. The native
HCV antigen
may be already bound to a solid matrix or separate with reagents for binding
it to the matrix.
Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out the assay
usually will be
included in the kit.
Immunoassays that utilize the native HCV antigen are useful in screening blood
for
the preparation of a supply from which potentially infective HCV is lacking.
The method for
the preparation of the blood supply comprises the following steps. Reacting a
body
component, preferably blood or a blood component, from the individual donating
blood with
2 0 HCV E1 and/or E2 proteins of the present invention to allow an
immunological reaction
between HCV antibodies, if any, and the HCV antigen. Detecting whether anti-
HCV
antibody - HCV antigen complexes are formed as a result of the reacting. Blood
contributed
to the blood supply is from donors that do not exhibit antibodies to the
native HCV antigens,
E 1 or E2.
2 5 In cases of a positive reactivity to the HCV antigen, it is preferable to
repeat the
immunoassay to lessen the possibility of false positives. For example, in the
large scale
screening of blood for the production of blood products (e.g. blood
transfusion, plasma,
Factor VIII, limmunoglobulin, etc.) 'screening' tests are typically formatted
to increase
sensitivity (to insure no contaminated blood passes) at the expense of
specificity; i.e. the
3 0 false-positive rate is increased. Thus, it is typical to only defer for
further testing those donors
who are 'repeatedly reactive; i.e. positive in two or more runs of the
immunoassay on the
donated sample. However, for confirmation of HCV-positivity, the
'confirmation' tests are
typically formatted to increase specificity (to insure that no false-positive
samples are
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32
confirmed) at the expense of sensitivity. Therefore the purification method
described in the
present invention for E 1 and E2 will be very advantageous for including
single or specific
oligomeric envelope proteins into HCV diagnostic assays.
The solid phase selected can include polymeric or glass beads, nitrocellulose,
microparticles, microwells of a reaction tray, test tubes and magnetic beads.
The signal
generating compound can include an enzyme, a luminescent compound, a
chromogen, a
radioactive element and a chemiluminescent compound. Examples of enzymes
include
alkaline phosphatase, horseradish peroxidase and beta-galactosidase. Examples
of enhancer
compounds include biotin, anti-biotin and avidin. Examples of enhancer
compounds binding
members include biotin, anti-biotin and avidin. In order to block the effects
of rheumatoid
factor-like substances, the test sample is subjected to conditions sufficient
to block the effect
of rheumatoid factor-like substances. These conditions comprise contacting the
test sample
with a quantity of anti-human IgG to form a mixture, and incubating the
mixture for a time
and under conditions sufficient to form a reaction mixture product
substantially free of
rheumatoid factor-like substance.
The present invention fizrther contemplates the use of E 1 proteins, or parts
thereof,
more particularly HCV single or specific oligomeric E1 proteins as defined
above, for in
vitro monitoring HCV disease or prognosing the response to treatment (for
instance with
Interferon) of patients suffering from HCV infection comprising:
2 0 - incubating a biological sample from a patient with hepatitis C infection
with
an E1 protein or a suitable part thereof under conditions allowing the
formation of an immunological complex,
- removing unbound components,
- calculating the anti-E1 titers present in said sample (for example at the
start of
2 5 and/or during the course of (interferon) therapy),
- monitoring the natural course of HCV disease, or prognosing the response to
treatment of said patient on the basis of the amount anti-E1 titers found in
said sample at the start of treatment and/or during the course of treatment.
Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, or preferably more than
20 times
30 of the initial anti-El titers could be concluded to be long-term, sustained
responders to HCV
therapy, more particularly to interferon therapy. It is illustrated in the
Examples section, that
an anti-El assay may be very useful for prognosing long-term response to IFN
treatment, or
to treatment of Hepatitis C virus disease in general.
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More particularly the following E1 peptides as listed in Table 3 were found to
be
useful for in vitro monitoring HCV disease or prognosing the response to
interferon
treatment of patients suffering from HCV infection:
E1-31 (SEQ ID N0:56) spanning amino acids 181 to 200 of the Core/E1 V1 region,
E1-33 (SEQ ID N0:57) spanning amino acids 193 to 212 of the E1 region,
E1-35 (SEQ ID N0:58) spanning amino acids 205 to 224 of the El V2 region
(epitope B),
E1-35A (SEQ ID N0:59) spanning amino acids 208 to 227 of the E1 V2 region
(epitope B),
lbE1 (SEQ ID N0:53) spanning amino acids 192 to 228 of E1 regions (V1, C1, and
V2 regions (containing epitope B)),
E1-51 (SEQ ID N0:66) spanning amino acids 301 to 320 of the El region,
El-53 (SEQ ID N0:67) spanning amino acids 313 to 332 of the E1 C4 region
(epitope A),
E1-55 (SEQ ID N0:68) spanning amino acids 325 to 344 of the E1 region.
It is to be understood that smaller fragments of the above-mentioned peptides
also fall
within the scope of the present invention. Said smaller fragments can be
easily prepared by
chemical synthesis and can be tested for their ability to be used in an assay
as detailed above
and in the Examples section.
2 0 The present invention also relates to a kit for monitoring HCV disease or
prognosing
the response to treatment (for instance to interferon) of patients suffering
from HCV infection
comprising:
- at least one El protein or E1 peptide, more particularly an El protein or E1
peptide as defined above,
2 5 - a buffer or components necessary for producing the buffer enabling the
binding reaction between these proteins or peptides and the anti-E 1
antibodies
present in a biological sample,
- means for detecting the immune complexes formed in the preceding binding
reaction,
3 0 - possibly also an automated scanning and interpretation device for
inferring a
decrease of anti-E 1 titers during the progression of treatment.
It is to be understood that also E2 protein and peptides according to the
present
invention can be used to a certain degree to monitor/prognose HCV treatment as
indicated
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34
above for the El proteins or peptides because also the anti-E2 levels decrease
in comparison
to antibodies to the other HCV antigens. It is to be understood, however, that
it might be
possible to determine certain epitopes in the E2 region which would also be
suited for use in
an test for monitoring/prognosing HCV disease.
The present invention also relates to a serotyping assay for detecting one or
more
serological types of HCV present in a biological sample, more particularly for
detecting
antibodies of the different types of HCV to be detected combined in one assay
format,
comprising at least the following steps
(i) contacting the biological sample to be analyzed for the presence of HCV
antibodies of one or more serological types, with at least one of the E1
and/or
E2 and/or E1/E2 protein compositions or at least one of the E1 or E2 peptide
compositions as defined above, preferantially in an immobilized form under
appropriate conditions which allow the formation of an immune complex,
(ii) removing unbound components,
(iii) incubating the immune complexes formed with heterologous antibodies,
with
said heterologous antibodies being conjugated to a detectable label under
appropriate conditions,
(iv) detecting the presence of said immune complexes visually or mechanically
(e.g. by means of densitometry, fluorimetry, colorimetry) and inferring the
2 0 presence of one or more HCV serological types present from the observed
binding pattern.
It is to be understood that the compositions of proteins or peptides used in
this
method are recombinantly expressed type-specific envelope proteins or type-
specific
peptides.
2 5 The present invention further relates to a kit for serotyping one or more
serological
types of HCV present in a biological sample, more particularly for detecting
the antibodies to
these serological types of HCV comprising:
- at least one E1 and/or E2 and/or El/E2 protein or E1 or E2 peptide, as
defined
above,
3 0 - a buffer or components necessary for producing the buffer enabling the
binding reaction between these proteins or peptides and the anti-E1 antibodies
present in a biological sample,
- means for detecting the immune complexes formed in the preceding binding
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reaction,
- possibly also an automated scanning and interpretation device for detecting
the presence of one or more serological types present from the observed
binding pattern.
5 The present invention also relates to the use of a peptide or protein
composition as
defined above, for immobilization on a solid substrate and incorporation into
a reversed
phase hybridization assay, preferably for immobilization as parallel lines
onto a solid support
such as a membrane strip, for determining the presence or the genotype of HCV
according to
a method as defined above. Combination with other type-specific antigens from
other HCV
10 polyprotein regions also lies within the scope of the present invention.
The present invention provides a method for purifying recombinant HCV single
or
specific oligomeric envelope proteins selected from E1 and/or E2 and/or E1/E2
proteins
which have been produced by a recombinant process comprising contacting said
proteins
with a disulphide bond cleavage or reducing agent. The contacting of the
method of the
15 invention may be carried out under partial cleavage or reducing conditions.
Preferably, the
disulphide bond cleavage agent is dithiothreitol (DTT), preferably in a
concentration range
of 0.1 to 50 mM, preferably 0.1 to 20 mM, more preferably 0.5 to 10 mM.
Alternatively,
the disulphide bond cleavage agent may be a detergent, such as Empigen-BB
(which is a
mixture containing N-Docecyl-N,N-dimethylglycine as a major component),
preferably at
2 0 a concentration of 1 to 10%, more preferably at a concentration of 3.5%.
Mixtures of
detergents, disulphide bond cleavage agents and/or reducing agents may also be
used. In
one embodiment, disulphide bond reformation is prevented with an SH group
blocking
agent, such as N-ethylmaleimide (NEM) or a derivative thereof. In a preferred
embodiment, the disulphide bond reformation is blocked by use of low pH
conditions.
2 5 The present invention further provides a method as described herein,
further
involving the following steps: lysing recombinant E1 and/or E2 and/or E1/E2
expressing
host cells, optionally in the presence of an SH blocking agent such as N-
ethylmaleimide
(NEM); recovering said HCV envelope proteins by affinity purification such as
by means
of lectin-chromatography, such as lentil-lectin chromatography, or by means of
30 immunoaffinity using anti-E1 and/or anti-E2 specific monoclonal antibodies;
reducing or
cleaving of the disulfide bonds with a disulphide bond cleaving agent, such as
DTT,
preferably also in the presence of an SH blocking agent, such as NEM or Biotin-
NEM;
and, recovering the reduced El and/or E2 and/or E1/E2 envelope proteins by
gelfiltration
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36
and optionally additionally by a subsequent Niz+-IMAC chromatography and
desalting
step.
The present invention provides a composition containing substantially isolated
and/or purified, and/or isolated and/or purified recombinant HCV single or
specific
oligomeric recombinant envelope proteins selected from E1 and/or E2 and/or
E1/E2,
which have preferably been isolated from the methods described herein. In a
preferred
embodiment, the recombinant HCV envelope proteins of the invention have been
expressed in recombinant mammalian cells, such as vaccinia, recombinant yeast
cells.
The present invention provides a recombinant vector containing a vector
sequence,
a prokaryotic, eukaryotic or viral promoter sequence and a nucleotide sequence
allowing
the expression of a single or specific oligomeric E1 and/or E2 and/or E1/E2
protein, in
operable combination. In one embodiment, the nucleotide sequence of the vector
encodes
a single HCV E1 protein starting in the region between amino acid positions 1
and 192 and
ending in the region between amino acid positions 250 and 400, more
particularly ending
in the region between positions 250 and 341, even more preferably ending in
the region
between position 290 and 341. In another embodiment, the nucleotide sequence
of the
vector encodes a single HCV E1 protein starting in the region between amino
acid
positions 117 and 192 and ending in the region between amino acid positions
263 and 400,
more particularly ending in the region between positions 250 and 326. In yet
another
embodiment, the nucleotide sequence of the vector encodes a single HCV El
protein
bearing a deletion of the first hydrophobic domain between positions 264 to
293, plus or
minus 8 amino acids. In a further embodiment, the nucleotide sequence of the
vector
encodes a single HCV E2 protein starting in the region between amino acid
positions 290
and 406 and ending in the region between amino acid positions 600 and 820,
more
2 5 particularly starting in the region between positions 322 and 406, even
more preferably
starting in the region between position 347 and 406 and most preferably
starting in the
region between positions 364 and 406; and preferably ending at any of amino
acid
positions 623, 650, 661, 673, 710, 715, 720, 746 or 809. The vector of the
present
invention, in one embodiment, contains a 5'-terminal ATG codon and a 3'-
terminal stop
3 0 codon operably linked to the nucleotide sequence. The vector further
contains, in one
embodiment, a nucleotide sequence further containing at a factor Xa cleavage
site and/or 3
to 10, preferably 6, histidine codons added 3'-terminally to the coding
region. The vector
of the present invention optionally contains a nucleotide sequence wherein at
least one of
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37
the glycosylation sites present in the El or E2 proteins has been removed at
the nucleic
acid level.
The present invention provides a nucleic acid containing any one of SEQ ID
NOs:
3, 5, 7, 9, 1 l, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and
49, or parts thereof.
The vector of the invention may preferably contain a nucleotide sequence
containing a
nucleic acid containing any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 21, 23, 25,
27, 29, 31,
35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.
The composition of the present invention further contains recombinant HCV
envelope proteins which have been expressed or are the expression product of a
vector
described herein.
The present invention provides a host cell transformed with at least one
recombinant vector as described herein, wherein the vector contains a
nucleotide sequence
encoding HCV El and/or E2 and/or E1/E2 protein as described herein in addition
to a
regulatory sequence operable in the host cell and capable of regulating
expression of the
HCV E1 and/or E2 and/or E1/E2 protein. Moreover; the present invention
provides a
ecombinant E1 and/or E2 and/or E1/E2 protein expressed by a host cell of the
invention.
The present invention further provides a method as described herein and
containing
the following steps: growing a host cell as described herein which has been
transformed
with a recombinant vector as described herein in a suitable culture medium;
causing
2 0 expression of the vector nucleotide sequence of the vector, as described
herein under
suitable conditions; lysing the transformed host cells, preferably in the
presence of an SH
group blocking agent, such as N-ethylmaleimide (NEM); recovering the HCV
envelope
protein by affinity purification by means of for instance lectin-
chromatography or
immunoaffinity chromatography using anti-E1 and/or anti-E2 specific monoclonal
2 5 antibodies, with said lectin being preferably lentil-lectin, followed by,
incubation of the
eluate of the previous step with a disulphide bond cleavage agent, such as
DTT, preferably
also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM;
and,
isolating the HCV single or specific oligomeric El and/or E2 and/or E1/E2
proteins by
means of gelfiltration and possibly also by means of an additional Ni2+-IMAC
3 0 chromatography and desalting step.
The present invention provides a composition containing at least one of the
following E1 and/or E2 peptides:
El-31 (SEQ ID N0:56) spanning amino acids 181 to 200 of the Core/E1
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V 1 region,
E1-33 (SEQ ID N0:57) spanning amino acids 193 to 212 ofthe El region,
E1-35 (SEQ ID N0:58) spanning amino acids 205 to 224 of the E1 V2
region (epitope B),
El-35A (SEQ ID N0:59) spanning amino acids 208 to 227 of the E1 V2
region (epitope B),
lbE1 (SEQ ID N0:53) spanning amino acids 192 to 228 of E1 regions (V1,
C1, and V2 regions (containing epitope B),
El-51 (SEQ ID N0:66) spanning amino acids 301 to 320 of the El region,
El-53 (SEQ ID N0:67) spanning amino acids 313 to 332 of the El C4
region (epitope A),
El-55 (SEQ ID N0:68) spanning amino acids 325 to 344 of the E1 region.
Env 67 or E2-67 (SEQ ID N0:72) spanning amino acid positions 397 to
416 of the E2 region (epitope A),
Env 69 or E2-69 (SEQ ID N0:73) spanning amino acid positions 409 to
428 of the E2 region (epitope A),
Env 23 or E2-23 (SEQ ID N0:86) spanning positions 583 to 602 of the E2
region (epitope E),
Env 25 or E2-25 (SEQ ID N0:87) spanning positions 595 to 614 of the E2
2 0 region (epitope E),
Env 27 or E2-27 (SEQ ID N0:88) spanning positions 607 to 626 of the E2
region (epitope E),
Env 17B or E2-17B (SEQ ID N0:83) spanning positions 547 to 566 of the
E2 region (epitope D),
2 5 Env 13B or E2-13B (SEQ ID N0:82) spanning positions 523 to 542 of the
E2 region (epitope C).
The present invention provides a composition containing at least one of the
following E2 conformational epitopes:
epitope F recognized by monoclonal antibodies 15C8C1, 12D11F1, and
30 8GlOD1H9,
epitope G recognized by monoclonal antibody 9G3E6,
epitope H (or C) recognized by monoclonal antibodies lOD3C4 and 4H6B2,
epitope I recognized by monoclonal antibody 17F2C2.
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The present invention provides an E1 and/or E2 specific monoclonal antibody
raised upon immunization with a composition as described herein. The
antibodies of the
present invention may be used, for example, as a medicament, for incorporation
into an
immunoassay kit for detecting the presence of HCV E1 or E2 antigen, for
prognosis/monitoring of disease or for HCV therapy. The present invention
provides for
the use of an E1 and/or E2 specific monoclonal antibody as described herein
for the
preparation of an immunoassay kit for detecting HCV E1 or E2 antigens, for the
preparation of a kit for prognosing/monitoring of HCV disease or for the
preparation of a
HCV medicament.
The present invention provides a method for in vitro diagnosis of HCV antigen
present in a biological sample, containing at least the following steps:
(i) contacting said biological sample with an El and/or E2 specific monoclonal
antibody as described herein, preferably in an immobilized form under
appropriate
conditions which allow the formation of an immune complex,
(ii) removing unbound components,
(iii) incubating the immune complexes formed with heterologous antibodies,
with
the heterologous antibodies being conjugated to a detectable label under
appropriate
conditions,
(iv) detecting the presence of the immune complexes visually or mechanically.
2 0 The present invention provides a kit for determining the presence of HCV
antigens
present in a biological sample, which includes at least the following: at
least one E I and/or
E2 specific monoclonal antibody as described herein, preferably in an
immobilized form
on a solid substrate, a buffer or components necessary for producing the
buffer enabling
binding reaction between these antibdodies and the HCV antigens present in a
biological
2 5 sample, and optionally a means for detecting the immune complexes formed
in the
preceding binding reaction.
The composition of the present invention may be provided in the form of a
medicament.
The present invention provides a composition, as described herein for use as a
3 0 vaccine for immunizing a mammal, preferably humans, against HCV,
comprising
administrating an effective amount of said composition being optionally
accompanied by
pharmaceutically acceptable adjuvants, to produce an immune response.
The present invention provides a method of using the composition, as described
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herein, for the preparation of a vaccine for immunizing a mammal, preferably
humans,
against HCV, comprising administrating an effective amount of said
composition,
optionally accompanied by pharmaceutically acceptable adjuvants, to produce an
immune
response.
5 The present invention provides a vaccine composition for immunzing a mammal,
preferably humans, against HCV, which contains an effective amount of a
composition
containing an E 1 and/or E2 containing composition as described herein,
optionally also
accompanied by pharmaceutically acceptable adjuvants.
The composition of the present invention may be provided in a form for in
vitro
10 detection of HCV antibodies present in a biological sample. The present
invention also
provides a method of preparing an immunoassay kit for detecting HCV antibodies
present
in a biological sample and a method of detecting HCV antibodies present in a
biological
sample using the kit of the invention to diagnose HCV antibodies present in a
biological
sample. Such a method of the present invention includes at least the following
steps:
15 (i) contacting said biological sample with a composition as described
herein,
preferably in an immobilized form under appropriate conditions which allow the
formation
of an immune complex with HCV antibodies present in the biological sample,
(ii) removing unbound components,
(iii) incubating the immune complexes formed with heterologous antibodies,
with
2 0 the heterologous antibodies being conjugated to a detectable label under
appropriate
conditions,
(iv) detecting the presence of the immune complexes visually or mechanically.
The present invention provides a kit for determining the presence of HCV
antibodies present in a biological sample, containing: at least one peptide or
protein
2 5 composition as described herein, preferably in an immobilized form on a
solid substrate; a
buffer or components necessary for producing the buffer enabling binding
reaction
between these proteins or peptides and the antibodies against HCV present in
the
biological sample; and, optionally, a means for detecting the immune complexes
formed in
the preceding binding reaction.
3 0 The present invention provides a method of in vitro monitoring HCV disease
or
diagnosing the response of a patient suffering from HCV infection to
treatment, preferably
with interferon, the method including: incubating a biological sample from the
patient with
HCV infection with an El protein or a suitable part thereof under conditions
allowing the
39
The present invention provides
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41
formation of an immunological complex; removing unbound components;
calculating the
anti-El titers present in the sample at the start of and during the course of
treatment;
monitoring the natural course of HCV disease, or diagnosing the response to
treatment of
the patient on the basis of the amount anti-E1 titers found in the sample at
the start of
treatment and/or during the course of treatment.
The present invention provides a kit for monitoring HCV disease or prognosing
the
response to treatment, particularly with interferon, of patients suffering
from HCV
infection, wherein the kit contains: at least one El protein or E1 peptide,
more particularly
an El protein or El peptide as described herein; a buffer or components
necessary for
producing the buffer enabling the binding reaction between these proteins or
peptides and
the anti-E1 antibodies present in a biological sample; and optionally, means
for detetecting
the immune complexes formed in the preceding binding reaction, optionally,
also an
automated scanning and interpretation device for inferring a decrease of anti-
E1 titers
during the progression of treatment.
The present invention provides a serotyping assay for detecting one or more
serological types of HCV present in a biological sample, more particularly for
detecting
antibodies of the different types of HCV to be detected combined in one assay
format,
including at least the following steps: (i) contacting the biological sample
to be analyzed
for the presence of HCV antibodies of one or more serological types, with at
least one of
the E1 and/or E2 and/or El/E2 protein compositions as described herein or at
least one of
the E1 or E2 peptide compositions described herein, preferentially in an
immobilized form
under appropriate conditions which allow the formation of an immune complex;
(ii)
removing unbound components; (iii) incubating the immune complexes formed with
heterologous antibodies, with the heterologous antibodies being conjugated to
a detectable
2 5 label under appropriate conditions; and optionally, (iv) detecting the
presence of said
immune complexes visually or mechanically (e.g. by means of densitometry,
fluorimetry,
colorimetry) and infernng the presence of one or more HCV serological types
present from
the observed binding pattern.
The present invention provides a kit for serotyping one or more serological
types of
3 0 HCV present in a biological sample, more particularly for detecting the
antibodies to these
serological types of HCV containing: at least one E1 and/or E2 and/or E1/E2
protein as
described herein or an E 1 or E2 peptide as described herein; a buffer or
components
necessary for producing the buffer enabling the binding reaction between these
proteins or
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42
peptides and the anti-E1 antibodies present in a biological sample;
optionally, means for
detecting the immune complexes formed in the preceding binding reaction,
optionally, also
an automated scanning and interpretation device for detecting the presence of
one or more
serological types present from the observed binding pattern.
The present invention provides a peptide or protein composition as described
herein, for immobilization on a solid substrate and incorporation into a
reversed phase
hybridization assay, preferably for immobilization as parallel lines onto a
solid support
such as a membrane strip, for determining the presence or the genotype of HCV
according
to a method as described herein.
The present invention provides a therapeutic vaccine composition, such as a
therapeutic HCV vaccine composition, containing or comprising a
therapeutically effective
amount o~ a composition containing at least one purified recombinant HCV
single or
specific oligomeric recombinant envelope proteins selected from the group
consisting of an
E1 protein, an E2 protein, a part of said E1 and E2 proteins, an E1/E2 protein
complex
formed from purified HCV single or specific oligomeric recombinant El or E2
proteins or
parts thereof; and optionally a pharmaceutically acceptable adjuvant. Another
therapeutic
vaccine composition, such as a therapeutic HCV vaccine composition, of the
invention
may be comprising a therapeutically effective amount of a combination of at
least two
purified HCV single or specific oligomeric recombinant envelope proteins
selected from
the group consisting of E1 proteins derived from different HCV genotypes or
subtypes, E2
proteins derived from different HCV genotypes or subtypes, parts of said E1
and E2
proteins, and E1/E2 protein complexes formed from purified HCV single or
specific
oligomeric recombinant El or E2 proteins, or parts thereof, derived from
different HCV
genotypes or subtypes; and optionally a pharmaceutically acceptable adjuvant.
2 5 The HCV envelope proteins of the vaccine, or more particularly the HCV
vaccine,
of the present invention are optionally produced by recombinant mammalian
cells, by
recombinant yeast cells, or by or via a recombinant virus. The invention
provides a
therapeutic vaccine composition, such as a therapeutic HCV vaccine
composition,
containing or comprising a therapeutically effective amount of a composition
comprising
at least one of the following E1 and E2 peptides:
E1-31 (SEQ ID N0:56) spanning amino acids 181 to 200 of the Core/E1 V1 region,
E1-33 (SEQ ID N0:57) spanning amino acids 193 to 212 of the E1 region,
E1-35 (SEQ ID N0:58) spanning amino acids 205 to 224 of the E1 V2 region
(epitope
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43
B),
El-35A (SEQ ID N0:59) spanning amino acids 208 to 227 of the E1 V2 region
(epitope B),
lbE1 (SEQ ID N0:53) spanning amino acids 192 to 228 of E1 regions V1, C1, and
V2 regions (containing epitope B),
E1-S1 (SEQ ID N0:66) spanning amino acids 301 to 320 of the E1 region,
E1-53 (SEQ ID N0:67) spanning amino acids 313 to 332 of the E1 C4 region
(epitope
A),
El-55 (SEQ ID N0:68) spanning amino acids 325 to 344 of the E1 region,
Env 67 or E2-67 (SEQ ID N0:72) spanning amino acid positions 397 to 418 of the
E2
region (epitope A),
Env 69 or E2-69 (SEQ ID N0:73) spanning amino acid positions 409 to 428 of the
E2
region (epitope A),
Env 23 or E2-23 (SEQ ID N0:86) spanning positions 583 to 602 of the E2 region
(epitope E),
Env 25 or E2-25 (SEQ ID N0:87) spanning positions 595 to 614 of the E2 region
(epitope E),
Env 27 or E2-27 (SEQ ID N0:88) spanning positions 607 to 626 of the E2 region
(epitope E),
2 0 Env 17B or E2-17B (SEQ ID N0:83) spanning positions 547 to 586 of the E2
region
(epitope D),
Env 13B or E2-13B (SEQ ID N0:82) spanning positions 523 to 542 of the E2
region
(epitope C),
IGP 1626 spanning positions 192-211 of the E1 region (SEQ ID NO:I 12),
2 5 IGP 1627 spanning positions 204-223 of the E1 region (SEQ ID N0:113),
IGP 1628 spanning positions 216-235 of the EI region (SEQ ID N0:114),
IGP 1629 spanning positions 228-247 of the E1 region (SEQ ID NO:115),
IGP 1630 spanning positions 240-259 of the E1 region (SEQ ID NO:l 16),
IGP 1631 spanning positions 252-271 of the E1 region (SEQ ID N0:117),
30 IGP 1632 spanning positions 264-283 of the E1 region (SEQ ID N0:118),
IGP 1633 spanning positions 276-295 of the E1 region (SEQ ID N0:119),
IGP 1634 spanning positions 288-307 of the E1 region (SEQ ID N0:120),
IGP 1635 spanning positions 300-319 of the E1 region (SEQ ID N0:121) and
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44
IGP 1636 spanning positions 312-331 of the E1 region (SEQ ID N0:122);
and wherein said peptides may be of recombinant or synthetic origin, and
optionally
combined with a pharmaceutically acceptable adjuvant.
Any of the above mentioned therapeutic vaccine compositions may also be
considered or regarded as therapeutic HCV vaccine compositions, or as
therapeutic
compositions or therapeutic HCV compositions, or as compositions or HCV
compositions.
The present invention provides a method of treating a mammal, such as a human,
infected with HCV comprising administering an effective amount of a
composition as
described herein, such as the above described vaccines or therapeutic
compositions, and
optionally, a pharmaceutically acceptable adjuvant. In one embodiment, the
composition of
the invention is administered in combination with or at a time in conjunction
with antiviral
therapy, either soon prior to or subsequent to or with administration of the
composition of
the invention. It will be clear that any of the compositions of the invention,
e.g. a
therapeutic HCV vaccine composition, can be used for treating a mammal
chronically
infected with HCV (a "chronic HCV-infected mammal").
The present invention provides a composition, such as a therapeutic HCV
composition or a HCV composition, containing or comprising at least one
purified
recombinant HCV recombinant envelope proteins selected from the group
consisting of an
E1 protein and an E2 protein, and optionally an adjuvant. In a preferred
embodiment, the
2 0 composition contains at least one of the following E1 and E2 peptides:
E1-31 (SEQ ID N0:56) spanning amino acids 181 to 200 of the Core/E1 V1 region,
E1-33 (SEQ ID N0:57) spanning amino acids 193 to 212 of the El region,
E1-35 (SEQ ID N0:58) spanning amino acids 205 to 224 of the E1 V2 region
(epitope
B),
E1-35A (SEQ ID N0:59) spanning amino acids 208 to 227 of the E1 V2 region
(epitope B),
lbE1 (SEQ ID N0:53) spanning amino acids 192 to 228 of E1 regions V1, C1, and
V2 regions (containing epitope B),
E1-51 (SEQ ID N0:66) spanning amino acids 301 to 320 of the E1 region,
E1-53 (SEQ ID N0:67) spanning amino acids 313 to 332 of the E1 C4 region
(epitope
A),
E1-SS (SEQ ID N0:68) spanning amino acids 325 to 344 of the El region,
Env 67 or E2-67 (SEQ ID N0:72) spanning amino acid positions 397 to 418 of the
E2
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region (epitope A),
Env 69 or E2-69 (SEQ ID N0:73) spanning amino acid positions 409 to 428 of the
E2
region (epitope A),
Env 23 or E2-23 (SEQ ID N0:86) spanning positions 583 to 602 of the E2 region
5 (epitope E),
Env 25 or E2-25 (SEQ ID N0:87) spanning positions 595 to 614 of the E2 region
(epitope E),
Env 27 or E2-27 (SEQ ID N0:88) spanning positions 607 to 626 of the E2 region
(epitope E),
10 Env 17B or E2-17B (SEQ ID N0:83) spanning positions 547 to 586 of the E2
region
(epitope D),
Env 13B or E2-13B (SEQ ID N0:82) spanning positions 523 to 542 of the E2
region
(epitope C),
IGP 1626 spanning positions 192-211 of the E1 region (SEQ ID N0:112),
15 IGP 1627 spanning positions 204-223 of the E1 region (SEQ ID N0:113),
IGP 1628 spanning positions 216-235 of the El region (SEQ ID N0:114),
IGP 1629 spanning positions 228-247 of the E1 region (SEQ ID NO:115),
IGP 1630 spanning positions 240-259 of the E1 region (SEQ ID NO:116),
IGP 1631 spanning positions 252-271 of the E1 region (SEQ ID N0:117),
2 0 IGP 1632 spanning positions 264-283 of the E 1 region (SEQ ID N0:118),
IGP 1633 spanning positions 276-295 of the E1 region (SEQ ID N0:119),
IGP 1634 spanning positions 288-307 of the El region (SEQ ID N0:120),
IGP 1635 spanning positions 300-319 of the E1 region (SEQ ID N0:121) and
IGP 1636 spanning positions 312-331 of the E1 region (SEQ ID N0:122),
2 5 and wherein said peptides may be of recombinant or synthetic origin, and
optionally
combined with a pharmaceutically acceptable adjuvant.
Another composition, such as a therapeutic HCV composition or HCV
composition, of the invention may be comprising a therapeutically effective
amount of a
combination of at least two purified HCV single or specific oligomeric
recombinant
30 envelope proteins selected from the group consisting of El proteins derived
from different
HCV genotypes or subtypes, E2 proteins derived from different HCV genotypes or
subtypes, parts of said El and E2 proteins, and E1/E2 protein complexes formed
from
purified HCV single or specific oligomeric recombinant E1 or E2 proteins, or
parts thereof,
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derived from different HCV genotypes or subtypes; and optionally a
pharmaceutically
acceptable adjuvant.
The present invention provides a therapeutic composition or therapeutic
vaccine
composition or composition for inducing HCV-specific antibodies or for
stimulating T-cell
activity or for stimulating cytokine secretion or cytokine production. The
present invention
provides a therapeutic HCV composition or therapeutic HCV vaccine composition
or HCV
composition for inducing HCV-specific antibodies or for stimulating T-cell
activity or for
stimulating cytokine secretion or cytokine production.
The therapeutic composition according to the present invention, such as a
therapeutic HCV vaccine composition or therapeutic HCV composition, may also
be
therapeutically effective in a HCV carrier infected with a HCV genotype
different from the
HCV genotype or HCV genotypes from which said El, E2, or EI/E2 protein
complexes
are derived.
The recombinant HCV envelope proteins may be produced by recombinant
mammalian cells, recombinant HCV envelope proteins are produced by recombinant
yeast
cells, or recombinant HCV envelope proteins are produced by or via a
recombinant virus.
The present invention provides a method of treating a mammal, such as a human,
infected
with HCV including administering an effective amount of a composition
described herein,
such as a therapeutic HCV vaccine composition, and, optionally, a
pharmaceutically
2 0 acceptable adjuvant. It will be clear that any of the compositions of the
invention, such as a
therapeutic HCV vaccine composition, can be used for treating a chronic HCV-
infected
mammal. The present invention provides a therapeutic composition for inducing
HCV-
specific antibodies, for stimulating T-cell activity or for stimulating
cytokine secretion or
cytokine production, said composition containing a therapeutic effective
amount of a
2 5 composition containing at least one purified recombinant HCV single or
specific
oligomeric recombinant envelope protein selected from the group consisting of
an E1
protein and an E2 protein; and optionally a pharmaceutically acceptable
adjuvant.
In particular, any of the compositions according to this invention (including
the
vaccine compositions and therapeutic compositions) may comprise recombinant
HCV
3 0 envelope proteins wherein the cysteines of said recombinant HCV envelope
proteins are
blocked, or may comprise El and/or E2 peptides wherein the cysteines of said
E1 or E2
peptides are blocked.
In another embodiment of the invention, the compositions (including the
vaccine
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47
compositions and therapeutic compositions) according to the invention may
comprise
recombinant HCV envelope proteins which are added to said compositions as
viral-like
particles (VLPs).
In a further embodiment, a composition of the invention such as a therapeutic
HCV
vaccine composition may comprise as recombinant HCV E1 envelope protein an Els
protein. More particularly, said E1 s protein is defined by SEQ ID N0:123.
Another aspect of the invention relates to an immunogenic composition, in
particular a HCV immunogenic composition, comprising a recombinant virus
allowing
expression of at least one HCV recombinant envelope protein chosen from an E1
protein
and/or an E2 protein, and parts of said E1 and E2 proteins; and, optionally, a
pharmaceutically acceptable adjuvant.
In yet a further aspect, the invention is envisaging a vaccine composition
such as a
HCV vaccine composition comprising a recombinant virus allowing expression of
at least
one HCV recombinant envelope protein chosen from an E1 protein and/or an E2
protein,
and parts of said E1 and E2 proteins; and, optionally, a pharmaceutically
acceptable
adjuvant.
In one embodiment, the above recombinant virus compositions may be effective
against a HCV genotype or subtype different from the HCV genotype or subytpe
from
which said E1 protein and/or E2 protein, or said parts thereof, are derived.
2 0 In another embodiment, the above recombinant virus compositions may be
used for
inducing HCV-specific antibodies or for stimulating T-cell activity or for
stimulating
cytokine secretion or cytokine production.
In another embodiment to the recombinant virus compositions, said recombinant
virus is a
vaccinia virus, a recombinant avipox virus or a recombinant Ankara Modified
Virus.
2 5 Another aspect of the invention relates to a method of treating a mammal
infected
with HCV comprising administering an effective amount of a recombinant vaccine
composition as described above.
The mammal in any of the above aspects of the invention may in particular be a
human.
3 0 One further aspect of the present invention relates to a method to reduce
liver
disease in a chronic HCV-infected mammal or human comprising administering a
therapeutic vaccine to said mammal or human.
In another aspect, a method to reduce liver fibrosis progression in a chronic
HCV-
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infected mammal or human comprising administering a therapeutic vaccine to
said
mammal or human is covered.
A further aspect relates to a method to reduce liver fibrosis in a chronic HCV-
infected mammal or human comprising administering a therapeutic vaccine to
said
mammal or human.
A further aspect relates to a method to reduce liver steatosis in a chronic
HCV-
infected mammal or human comprising administering a therapeutic vaccine to
said
mammal or human.
Yet another aspect of the invention embodies a method to reduce liver disease
by at
least 2 points according to the overall Ishak score or Ishak activity score in
a chronic HCV-
infected mammal or human comprising administering a therapeutic vaccine to
said
mammal or human.
Another further aspect relates to a method to reduce liver disease or liver
fibrosis
by at least 1 point according to the Ishak fibrosis score in a chronic HCV-
infected mammal
or human comprising administering a therapeutic vaccine to said mammal or
human.
A further aspect of the invention provides a method to reduce serum ALT levels
in
a chronic HCV-infected mammal or human comprising administering a therapeutic
vaccine to said mammal or human.
Yet another aspect of the invention relates to a method to reduce anti-E1
and/or
2 0 anti-E2 immunoreactivity in the liver of a chronic HCV-infected mammal or
human
comprising administering a therapeutic vaccine to said mammal or human.
Furthermore included in the invention is a method for treating a chronic HCV-
infected mammal or human wherein said method is comprising administration of
multiple
doses of any of the compositions of the invention, such as a therapeutic HCV
vaccine
2 5 composition, to said mammal or human and wherein said multiple doses are
administrated
to said mammal or human separated by a specified time interval. Thus, said
plurality of
administrations of a composition of the invention to treat a chronic HCV-
infected earner
may be separated, e.g., by a time-interval of 4 weeks or less. Thus, said time
intervals
could be 1 or 1.5 or 2 or 2.5 or 3 or 3.5 or 4 weeks, or could be 1, 2, 3, 4,
5, 6, 7, 8, 9, 10,
30 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28
days. In particular,
said method of treating a chronic HCV-infected mammal or human comprises a
plurality
of administrations of a composition of the invention, such as a therapeutic
HCV vaccine
composition, to said mammal or human wherein said administrations are
separated by a
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time interval of 3 weeks. In a further embodiment, said plurality of
administrations
consists of a first series of at least 5 administrations followed by an
administration-free
period of at least 12 weeks followed by a second series of at least 3
administrations. In
particular, a series of administrations may comprise at least 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, or
more administrations. The administration-free period may be at least 6, 7, 8,
9, 10, 1 l, 12,
13, 14, 15, 16, 17, 18 or more weeks.
It will furthermore be clear that any of the compositions of the invention,
such as a
therapeutic HCV vaccine composition, can be used for obtaining any of the
effects aimed
at in the various methods as described above. Said therapeutic vaccine as
applied in the
methods described above may be any composition of the current invention, such
as a
therapeutic HCV vaccine composition.
The use of the HCV E1/E2 proteins or parts thereof or El/E2 peptides as
outlined
throughout the above description for the manufacture of a composition, HCV
composition,
vaccine, HCV vaccine, therapeutic vaccine or therapeutic HCV vaccine for use
in any of
the methods outlined above or used for obtaining any of the effects aimed at
in the various
methods outlined above is also envisaged by the current invention.
In any of these methods said therapeutic vaccine comprises at least one HCV
antigen and, optionally, a pharmaceutically acceptable adjuvant such as alum.
In one
embodiment thereto said HCV antigen is an E1 or E2 antigen, or an immunogenic
part of
2 0 an E 1 or E2 antigen. When referring to the HCV antigen as being an E 1 s
antigen, the E 1 s
antigen may be defined by SEQ ID N0:123.
With the term "liver disease" is meant in this context any abnormal liver
condition
caused by infection with the hepatitis C virus including inflammation,
fibrosis, cirrhosis,
necrosis, necro-inflammation and hepatocellular carcinoma.
2 5 With "steatosis" is meant a histological feature of lipid accumulation in
the
hepatocytes that is indicative of liver involvement in a wide variety of
systemic disorders,
toxic or drug-induced liver injury, as well as of various specific liver
diseases, including
hepatitis C infection, Wilson's disease, and galactosemia.
With "reducing liver disease" is meant any stabilization or reduction of the
liver
3 0 disease status. Liver disease can be determined, e.g., by the Knodell
scoring system or the
Knodell scoring system adapted by Ishak. A reduction of this score by two
points is
accepted as therapeutically beneficial effect in several studies (see, e.g.,
studies published
after 1996 as indicated in Table 2 of Shiffman 1999).
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With "reducing liver fibrosis progression" is meant any slowing down, halting
or
reverting of the normally expected progression of liver fibrosis. Liver
fibrosis progression
can be determined, e.g., by the Metavir scoring system. Normal expected
progression of
liver fibrosis according to this system was published to be an increase of the
Metavir score
of an untreated chronic HCV patient of approximately 0.133 per year (Poynard
et al.
1997). Fibrosis is considered to include any form of fibrosis, e.g. as scored
by the Metavir
or Ishak system, including perisinusoidal fibrosis.
With the term "HCV antigen" is meant any HCV protein or fragment thereof
comprising at least one T cell epitope or B cell epitope.
A further aspect of the current invention provides a method to predict changes
in
liver disease in a chronic HCV-infected mammal or human, said method
comprising:
(i) determining the level of serum anti-E1 antibody level prior to therapeutic
vaccination with a HCV vaccine composition comprising an E1 antigen;
(ii) determining the level of serum anti-E1 antibody level after therapeutic
vaccination with a HCV vaccine composition comprising an E1 antigen;
(iii) inferring the difference in level of serum anti-El antibody level
determined in
(i) and (ii) and, therefrom, predicting the change in liver disease.
It will be clear to the skilled artisan that a significantly high and positive
difference in
level of serum anti-E1 antibody, calculated as the value measured in (ii)
minus the value
measured in (i), would tip the balance in favor of a positive prediction,
i.e., a prediction
that the degree of liver disease will decrease. It will also be clear that a
sustained
significantly high and positive difference would add to the positive character
of the
prediction of decreased liver disease. When said difference is, however, not
significantly
high, is zero, is negative, or is significantly high and positive at one point
in time but is not
sustained, this would then tip the balance in favor of a negative prediction,
i.e., a
prediction that the degree of liver disease will remain unchanged or will
increase. A
sustained high level of serum anti-E1 antibody could be reached trough
additional
immunizations either by administering a new series of immunizations after an
administration free period or by repeating immunizations with a larger time
interval, e.g. 6
weeks, after an initial priming series consisting of administrations with a
short time
interval, e.g. 3 weeks.
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Figure and Table legends
Figure 1 : Restriction map of plasmid pgpt ATA 18
Figure 2 : Restriction map of plasmid pgs ATA 18
Figure 3 : Restriction map of plasmid pMS 66
Figure 4 : Restriction map of plasmid pv HCV-1 lA
Figure 5 : Anti-E1 levels in non-responders to IFN treatment
Figure 6 : Anti-E1 levels in responders to IFN treatment
Figure 7 : Anti-El levels in patients with complete response to IFN treatment
Figure 8 : Anti-E 1 levels in incomplete responders to IFN treatment
Figure 9 : Anti-E2 levels in non-responders to IFN treatment
Figure 10 : Anti-E2 levels in responders to IFN treatment
Figure 11 Anti-E2 levels in incomplete responders to IFN
: treatment
Figure 12 Anti-E2 levels in complete responders to IFN treatment
:
Figure Human anti-E 1 reactivity competed with peptides
13 :
Figure 14 Competition of reactivity of anti-E1 monoclonal
: antibodies with peptides
Figure 15 Anti-El (epitope 1) levels in non-responders to
: IFN treatment
Figure 16 Anti-El (epitope 1 ) levels in responders to IFN
: treatment
Figure 17 Anti-E1 (epitope 2) levels in non-responders to
: IFN treatment
2 0 Figure Anti-E1 (epitope 2) levels in responders to IFN
18 : treatment
Figure 19 Competition of reactivity of anti-E2 monoclonal
: antibodies with peptides
Figure 20: Human anti-E2 reactivity competed with peptides
Figure 21: Nucleic acid sequences of the present invention. The nucleic acid
sequences
encoding an E1 or E2 protein according to the present invention may be
translated (SEQ ID NO 3 to 13, 21-31, 35 and 41-49 are translated in a
reading frame starting from residue number 1, SEQ ID NO 37-39 are
translated in a reading frame starting from residue number 2), into the amino
acid sequences of the respective E1 or E2 proteins as shown in the sequence
listing.
3 0 Figure 22: ELISA results obtained from lentil lectin chromatography eluate
fractions of
4 different E1 purifications of cell lysates infected with wHCV39 (type lb),
wHCV40 (type lb), wHCV62 (type 3a), and wHCV63 (type Sa).
Figure 23: Elution profiles obtained from the lentil lectin chromatography of
the 4
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different E1 constructs on the basis of the values as shown in Figure 22.
Figure 24: ELISA results obtained from fractions obtained after gelfiltration
chromatography of 4 different El purifications of cell lysates infected with
wHCV39 (type lb), wHCV40 (type lb), wHCV62 (type 3a), and wHCV63
(type Sa).
Figure 25: Profiles obtained from purifications of El proteins of type lb (1),
type 3a (2),
and type Sa (3) (from RKl3 cells infected with wHCV39, wHCV62, and
wHCV63, respectively; purified on lentil lectin and reduced as in example
5.2 - 5.3) and a standard (4). The peaks indicated with ' 1', '2', and '3',
represent
pure E1 protein peaks (see Figure 24, E1 reactivity mainly in fractions 26 to
30).
Figure 26: Silver staining of an SDS-PAGE as described in example 4 of a raw
lysate of
E1 wHCV40 (type lb) (lane 1), pool 1 of the gelfiltration of wHCV40
representing fractions 10 to 17 as shown in Figure 25 (lane 2), pool 2 of the
gelfiltration of wHCV40 representing fractions 18 to 25 as shown in Figure
(lane 3), and E1 pool (fractions 26 to 30) (lane 4).
Figure 27: Streptavidine-alkaline phosphatase blot of the fractions of the
gelfiltration of
E1 constructs 39 (type lb) and 62 (type 3a). The proteins were labelled with
NEM-biotin. Lane 1: start gelfiltration construct 39, lane 2: fraction 26
2 0 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28
construct 39,
lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7
fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start
gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11:
fraction
27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29
2 5 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31
construct
62.
Figure 28: Siver staining of an SDS-PAGE gel of the gelfiltration fractions of
vvHCV-
39 (Els, type lb) and wHCV-62 (Els, type 3a) run under identical conditions
as Figure 26. Lane 1: start gelfiltration construct 39, lane 2: fraction 26
construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct
39,
lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7
fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start
gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11:
fraction
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27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29
construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31
construct
62.
Figure 29: Western Blot analysis with anti-E1 mouse monoclonal antibody SElAlO
giving a complete overview of the purification procedure.
Lane 1: crude
lysate, Lane 2: flow through of lentil chromagtography,
Lane 3: wash with
Empigen BB after lentil chromatography, Lane 4: Eluate
of lentil
chromatography, Lane 5: Flow through during concentration
of the lentil
eluate, Lane 6: Pool of E1 after Size Exclusion Chromatography
(gelfiltration).
Figure 30: ODzgo profile (continuous line) of the lentil lectin
chromatography of E2
protein from RK13 cells infected with wHCV44. The
dotted line represents
the E2 reactivity as detected by ELISA (as in example
6).
Figure 31A: ODzBO profile (continuous line) of the lentil-lectin
gelfiltration
chromatography E2 protein pool from RKl3 cells infected
with wHCV44 in
which the E2 pool is applied immediately on the gelfiltration
column (non-
reduced conditions). The dotted line represents the
E2 reactivity as detected
by ELISA (as in example 6).
Figure 31B: ODZBO profile (continuous line) of the lentil-lectin
gelfiltration
2 0 chromatography E2 protein pool from RK13 cells infected
with wHCV44 in
which the E2 pool was reduced and blocked according
to Example 5.3
(reduced conditions). The dotted line represents
the E2 reactivity as detected
by ELISA (as in example 6).
Figure 32: Ni2+-IMAC chromatography and ELISA reactivity of
the E2 protein as
2 5 expressed from wHCV44 after gelfiltration under reducing
conditions as
shown in Figure 31B.
Figure 33: Silver staining of an SDS-PAGE of 0.5 ~g of purified
E2 protein recovered
by a 200 mM imidazole elution step (lane 2) and a
30mM imidazole wash
(lane 1 ) of the Niz+-IMAC chromatography as shown
in Figure 32.
3 0 Figure OD profiles of a desalting step of the purified E2
34: protein recovered by 200
mM imidazole as shown in Figure 33, intended to remove
imidazole.
Figure 35A: Antibody levels to the different HCV antigens (Core
1, Core 2, E2HCVR,
NS3) for NR and LTR followed during treatment and
over a period of 6 to 12
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months after treatment determined by means of the LIAscan method. The
average values are indicated by the curves with the open squares.
Figure 35B: Antibody levels to the different HCV antigens (NS4, NSS, E1 and
E2) for NR
and LTR followed during treatment and over a period of 6 to 12 months after
treatment determined by means of the LIAscan method. The average values
are indicated by the curve with the open squares.
Figure 36: Average E1 antibody (ElAb) and E2 antibody (E2Ab) levels in the LTR
and
NR
groups.
Figure 37: Averages E1 antibody (EIAb) levels for non-responders (NR) and long
term
responders (LTR) for type lb and type 3a.
Figure 38: Relative map positions of the anti-E2 monoclonal antibodies.
Figure 39: Partial deglycosylation of HCV E1 envelope protein. The lysate of
vvHCV l0A-infected RK13 cells were incubated with different concentrations
of glycosidases according to the manufacturer's instructions. Right panel:
Glycopeptidase F (PNGase F). Left panel: Endoglycosidase H (Endo H).
Figure 40: Partial deglycosylation of HCV E2 envelope proteins. The lysate of
wHCV64-infected (E2) and wHCV41-infected (E2s)RK13 cells were
2 0 incubated with different concentrations of Glycopeptidase F (PNGase F)
according to the manufacturer's instructions.
Figure 41: In vitro mutagenesis of HCV E1 glycoproteins. Map of the mutated
sequences and the creation of new restriction sites.
Figure 42A: In vitro mutagenesis of HCV E1 glycoprotein (part 1). First step
of PCR
2 5 amplification.
Figure 42B: In vitro mutagensis of HCV E1 glycoprotein (part 2). Overlap
extension and
nested PCR.
Figure 43: In vitro mutagesesis of HCV E1 glycoproteins. Map of the PCR
mutated
fragments (GLY-# and OVR-#) synthesized during the first step of
3 0 amplification.
Figure 44A: Analysis of El glycoprotein mutants by Western blot expressed in
HeLa (left)
and RK13 (right) cells. Lane 1: wild type W (vaccinia virus), Lane 2:
original E1 protein (wHCV-l0A), Lane 3: E1 mutant Gly-1 (wHCV-81),
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Lane 4: E1 mutant Gly-2 (wHCV-82), Lane 5: E1 mutant Gly-3 (wHCV-
83), Lane 6: El mutant Gly-4 (wHCV-84), Lane 7: El mutant Gly-5
(vvHCV-85), Lane 8: E1 mutant Gly-6 (wHCV-86).
Figure 44B: Analysis of E1 glycosylation mutant vaccinia viruses by PCR
5 amplification/restriction. Lane 1: E1 (wHCV-l0A), BspE I, Lane 2:
E1.GLY-1 (vvHCV-81), BspE I, Lane 4: E1 (wHCV-l0A), Sac l, Lane 5:
E1.GLY-2 (wHCV-82), Sac I, Lane 7: E1 (wHCV-l0A), Sac I, Lane 8:
E1.GLY-3 (wHCV-83), Sac I, Lane 10: E1 (wHCV-l0A), Stu I, Lane 11:
EI.GLY-4 (wHCV-84), Stu I, Lane 13: E1 (wHCV-l0A), Sma I, Lane 14:
10 E1.GLY-5 (vvHCV-85), Sma 1, Lane 16: E1 (wHCV-l0A), Stu 1, Lane 17:
E1.GLY-6 (wHCV-86), Stu I, Lane 3 - 6 - 9 - 12 - 15 : Low Molecular
Weight Marker, pBluescript SK+, Msp I.
Figure 45: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed
in S.
cerevisiae. Innoculates were grown in leucine selective medium for 72 hrs.
15 and diluted 1/15 in complete medium. After 10 days of culture at
28°C,
medium samples were taken. The equivalent of 200 ~1 of culture supernatant
concentrated by speedvac was loaded on the gel. Two independent
transformants were analysed.
Figure 46: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed
in a
2 0 glycosylation deficient S. cerevisiae mutant. Innoculae were grown in
leucine
selective medium for 72 hrs. and diluted 1/15 in complete medium. After 10
days of culture at 28°C, medium samples were taken. The equivalent of
350
pl of culture supernatant, concentrated by ion exchange chromatography, was
loaded on the gel.
2 5 Figure 47: Profile of chimpanzees and immunization schedule.
Figure Cellular response after 3 immunizations.
48:
Figure Evolution of cellular response upon repeated
49: E 1 immunizations.
Figure Cellular response upon NS3 immunizations.
50:
Figure 51: Stimulation index through week 28. The stimulation index (SI;
cellular
30 immune response) was obtained by culturing PBMC (105 cells), drawn from
the individuals before immunization (week 0), two weeks after the third
immunization (week 8), before the booster immunization (week 26) and two
weeks after the booster immunization (week 28), in the presence or absence
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of 3 ~,g of recombinant Els or 2 pg tetanos toxoid
and determining the
amount of tritiated thymidine incorporated in these
cells during a pulse of
18 hours after 5 days of culture. The stimulation
index is the ratio of
thymidine incorporated in the cells cultured with
envelope antigen versus
the ones cultured without antigen. Samples of week
0 and 8 were
determined in a first assay (A), while the samples
of week 26 and 28 were
determined in a second assay (B) in which the samples
of week 0 were
reanalyzed. Results are expressed as the geometric
mean stimulation index
of all 20 (A, experiment) or 19 (B, experiment) volunteers.
Figure Cytokine production of PBMCs. PBMC (105 cells), drawn
52: from the
individuals before the booster immunization (week
26) and two weeks after
the booster immunization (week 28), were cultured
in the presence of 3 ~g
of recombinant Els (El) or 2 pg of tetanos toxoid
(TT) or no antigen (Bl).
Cytokines were measured in the supernatant taken after
24 hours
(interleukin-5) or after 120 hours (interferon-gamma)
by means of ELISA.
The stimulation index is the ratio of cytokine measured
in the supernatants
of cells cultured with envelope antigen versus the
ones cultured without
antigen. Results are expressed as the geometric mean
of pg cytokine/ml
secreted of all 19 volunteers. Samples with a cytokine
amount below
2 0 detection limit were assigned the value of the detection
limit. Similarly
samples with extremely high concentrations of cytokine
out of the linear
range of the assay were assigned the value of the
limit of the linear range of
the assay.
Figure 53: Thymidine incorporation results. The stimulation index
(cellular immune
2 5 response) was obtained by culturing PBMC (3 x105 cells),
in the presence
or absence of peptides and determining the amount
of tritiated thymidine
incorporated in these cells during a pulse after 5-6
days of culture. The
stimulation index is the ratio of thymidine incorporated
in the cells cultured
with peptide versus the ones cultured without peptide.
Results are expressed
3 0 as individual values for vaccinated persons (top panel)
or non vaccinated or
controls (lower panel).
Figure 54: Three-dimensional graph showing for the individual patients (each
represented by a dot) the % change in ALT-levels (absolute change from
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baseline) on the X-axis, the change in serum anti-El antibody levels (in
mU/mL) on the Y-axis, and the change in Ishak fibrosis score on the Z-axis.
Figure 55: Three-dimensional graph showing for the individual patients (each
represented by a dot) the % change in ALT-levels (absolute change from
baseline) on the X-axis, the change in serum anti-E1 antibody levels (in
mU/mL) on the Y-axis, and the change in Metavir fibrosis score on the Z-
axis.
Figure 56: For each individual patient (represented by a dot or a "*"), the
Ishak
fibrosis score (on the X-axis) and the ALT values (on the Y-axis) are given.
In panel A (top), the baseline (pre-vaccination) situation is given whereas
panel B (bottom) is illustrating the situation at the time of taking of liver
biopsies. The seven patients with the highest increase in serum anti-E1
antibody level are represented by a "*".
Figure 57: The influence of the age (on the X-axis) of each individual patient
(represented by a dot or a "*") on the Ishak fibrosis score (on the Y-axis) is
given. In panel A (top), the baseline (pre-vaccination) situation is given
whereas panel B (bottom) is illustrating the situation at the time of taking
of
liver biopsies. The seven patients with the highest increase in serum anti-E1
antibody level are represented by a "*".
Table 1 : Features of the respective clones and primers used for amplification
for
constructing the different forms of the E1 protein as despected in Example 1.
Table 2 : Surrunary of Anti-El tests
Table 3 : Synthetic peptides for competition studies
2 5 Table 4: Changes of envelope antibody levels over time.
Table 5: Difference between LTR and NR
Table 6: Competition experiments between murine E2 monoclonal antibodies
Table 7: Primers for construction of El glycosylation mutants
Table 8: Analysis of E1 glycosylation mutants by ELISA
Table 9: Profile of adjuvanted El Balb/c mice.
Table 10: Humoral responses: No. of immunizations required for different EI-
antibodies levels.
Table 11: Chimpanzee antibody titers.
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Table 12: Human antibody titers.
Table 13: Human antibody titers (8-28 weeks).
Table 14: Stimulation index (SI) of cultured PBMC, drawn from the individuals
four
weeks (W16) after the fourth immunization and two weeks (W26) after the
fifth immunization in the presence or absence of 3 pg of Els. A stimulation
index of >3 is considered a positive signal.
Table 15: Ishak grading of necro-inflammatory intensities for periportal
hepatitis,
confluent necrosis, focal inflammation, portal inflammation and the overall
total inflammation grading. Scores are indicated as the change from
baseline (mean and 95 % confidence interval) and the mean baseline- to
end-values.
Table 16: Overview of frequencies (given as number of patients) of changes of
a
given baseline Metavir score (given in top row; Baseline 0 to 4) to a given
Metavir score at the end of the second course of therapeutic El s vaccination
(given in left row; EOT 0 to 4). For instance, the "5" marked with a "*"
(i.e., "5*") means that 5 patients had a baseline Metavir score of 1 and a
Metavir score of 0 at the end of the second course treatment (EOT = end of
treatment).
Table 17: Correlation between serum anti-El antibody levels induced by
therapeutic
2 0 E1 s vaccination and change in overall Ishak scores. Given are the number
of patients corresponding to the possible criteria as outlined in the Table.
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Example l: Cloning and expression of the hepatitis C virus E1 protein
1. Construction of vaccinia virus recombination vectors
The pgptATAl8 vaccinia recombination plasmid is a modified version of pATAl8
(Stunnenberg et al, 1988) with an additional insertion containing the E. coli
xanthine guanine
phosphoribosyl transferase gene under the control of the vaccinia virus I3
intermediate
promoter (Figure 1). The plasmid pgsATAl8 was constructed by inserting an
oligonucleotide
linker with SEQ ID NO 1/94, containing stop codons in the three reading
frames, into the Pst
I and HindIII-cut pATAl8 vector. This created an extra Pac I restriction site
(Figure 2). The
original HindIII site was not restored.
Oligonucleotide linker with SEQ ID NO 1/94:
5' G GCATGC AAGCTT AATTAATT 3' (SEQ ID N0:1)
3' ACGTC CGTACG TTCGAA TTAATTAA TCGA 5' (SEQ ID N0:94)
PstI SphI HindIII Pac I (HindIII)
In order to facilitate rapid and efficient purification by means of NiZ+
chelation of
engineered histidine stretches fused to the recombinant proteins, the vaccinia
recombination
vector pMS66 was designed to express secreted proteins with an additional
carboxy-terminal
histidine tag. An oligonucleotide linker with SEQ ID NO 2/95, containing
unique sites for 3
2 0 restriction enzymes generating blunt ends (Sma I, Stu I and Pml IlBbr PI)
was synthesized in
such a way that the carboxy-terminal end of any cDNA could be inserted in
frame with a
sequence encoding the protease factor Xa cleavage site followed by a
nucleotide sequence
encoding 6 histidines and 2 stop codons (a new Pac I restriction site was also
created
downstream the 3'end). This oligonucleotide with SEQ ID NO 2/95 was introduced
between
2 5 the Xma I and Pst I sites of pgptATAl 8 (Figure 3).
Oligonucleotide linker with SEQ ID NO 2/95:
5'-CCGGG GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCACCA/
3'-C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGT/
XmaI
30 /TCACTAATAGTTAATTAA CTGCA-3' (SEQ ID
N0:2)
/AGTGATTATCAATTAATT G-5' (SEQ ID
N0:95)
PstI
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Plasmid pgptATA-18 contained within Escherichia coli MC1061 (lambda) has been
deposited under the terms of the Budapest Treaty at BCCM/LMBP (Belgian
Coordinated
Collections of microorganisms/Laboratorium voor Moleculaire Biologie -
Plasmidencollectie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Ghent,
Belgium),
5 and bears accession number LMBP4486. Said deposit was made on January 9,
2002.
Example 2. Construction of HCV recombinant plasmids
2.1. Constructs encoding different forms of the El protein
10 Polymerase Chain Reaction (PCR) products were derived from the serum
samples by
RNA preparation and subsequent reverse-transcription and PCR as described
previously
(Stuyver et al., 1993b). Table 1 shows the features of the respective clones
and the primers
used for amplification. The PCR fragments were cloned into the Sma I-cut pSP72
(Promega)
plasmids. The following clones were selected for insertion into vaccinia
reombination
15 vectors: HCC19A (SEQ ID N0:3), HCC110A (SEQ ID NO:S), HCC111A (SEQ ID
N0:7),
HCC112A (SEQ ID N0:9), HCCI l 3A (SEQ ID NO:11 ), and HCC117A (SEQ ID N0:13)
as
depicted in Figure 21. cDNA fragments containing the E1-coding regions were
cleaved by
EcoRI and HindIII restriction from the respective pSP72 plasmids and inserted
into the
EcoRI/HindIII-cut pgptATA-18 vaccinia recombination vector (described in
example 1),
20 downstream of the 11K vaccinia virus late promoter. The respective plasmids
were
designated pvHCV-9A, pvHCV-10A, pvHCV-11A, pvHCV-12A, pvHCV-13A and pvHCV-
17A, of which pvHCV-11 A is shown in Figure 4.
2.2. Hydrophobic region El deletion mutants
2 5 Clone HCC137, containing a deletion of codons Asp264 to Va1287
(nucleotides 790
to 861, region encoding hydrophobic domain I) was generated as follows: 2 PCR
fragments
were generated from clone HCC110A with primer sets HCPr52 (SEQ ID
N0:16)/HCPr107
(SEQ ID N0:19) and HCPr108 (SEQ ID N0:20)IHCPR54 (SEQ ID N0:18). These primers
are shown in Figure 21. The two PCR fragments were purified from agarose gel
after
3 0 electrophoresis and 1 ng of each fragment was used together as template
for PCR by means
of primers HCPr52 (SEQ ID N0:16) and HCPr54 (SEQ ID N0:18). The resulting
fragment
was cloned into the Sma I-cut pSP72 vector and clones containing the deletion
were readily
identified because of the deletion of 24 codons (72 base pairs). Plasmid
pSP72HCC137
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containing clone HCC137 (SEQ ID 15) was selected. A recombinant vaccinia
plasmid
containing the full-length E1 cDNA lacking hydrophobic domain I was
constructed by
inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I
and BamH
I from the vector pSP72-HCCl37) into the Xma I-Bam H I sites of the vaccinia
plasmid
pvHCV-10A. The resulting plasmid was named pvHCV-37. After confirmatory
sequencing,
the amino-terminal region containing the internal deletion was isolated from
this vector
pvHCV-37 (cleavage by EcoR I and BstE II) and reinserted into the Eco RI and
Bst EII-cut
pvHCV-11A plasmid. This construct was expected to express an E1 protein with
both
hydrophobic domains deleted and was named pvHCV-38. The E1-coding region of
clone
HCC138 is represented by SEQ ID N0:23.
As the hydrophilic region at the E1 carboxyterminus (theoretically extending
to
around amino acids 337-340) was not completely included in construct pvHCV-38,
a larger
E1 region lacking hydrophobic domain I was isolated from the pvHCV-37 plasmid
by EcoR
I/Bam HI cleavage and cloned into an EcoRI/BamHI-cut pgsATA-18 vector. The
resulting
plasmid was named pvHCV-39 and contained clone HCC139 (SEQ ID N0:25). The same
fragment was cleaved from the pvHCV-37 vector by BamH I (of which the sticky
ends were
filled with Klenow DNA Polymerase I (Boehringer)) and subsequently by EcoR I
(5'
cohesive end). This sequence was inserted into the EcoRI and Bbr PI-cut vector
pMS-66.
This resulted in clone HCC140 (SEQ ID N0:27) in plasmid pvHCV-40, containing a
6
2 0 histidine tail at its carboxy-terminal end.
2.3. E 1 of other genotypes
Clone HCCl62 (SEQ ID N0:29) was derived from a type 3a-infected patient with
chronic hepatitis C (serum BR36, clone BR36-9-13, SEQ ID N0:19 in WO 94/25601,
and
see also Stuyver et al. 1993a) and HCCl63 (SEQ ID N0:31) was derived from a
type Sa
infected child with post-transfusion hepatitis (serum BE95, clone PC-4-1, SEQ
ID N0:45 in
WO 94/25601 ).
2.4. E2 constructs
The HCV E2 PCR fragment 22 was obtained from serum BEII (genotype lb) by
means of primers HCPr109 (SEQ ID N0:33) and HCPr72 (SEQ ID N0:34) using
techniques
of RNA preparation, reverse-transcription and PCR, as described in Stuyver et
al., 1993b,
and the fragment was cloned into the Sma I-cut pSP72 vector. Clone HCC122A
(SEQ ID
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N0:35) was cut with Ncol/AIwNI or by BamHI/AIwNI and the sticky ends of the
fragments
were blunted (NcoI and BamHI sites with Klenow DNA Polymerise I (Boehringer),
and
AIwNI with T4 DNA polymerise (Boehringer)). The BamHI/AIwNI cDNA fragment was
then inserted into the vaccinia pgsATA-18 vector that had been linearized by
EcoR I and
Hind III cleavage and of which the cohesive ends had been filled with Klenow
DNA
Polymerise (Boehringer). The resulting plasmid was named pvHCV-41 and encoded
the E2
region from amino acids Met347 to G1n673, including 37 amino acids (from
Met347 to
G1y383) of the E1 protein that can serve as signal sequence. The same HCV cDNA
was
inserted into the EcoR I and Bbr PI-cut vector pMS66, that had subsequently
been blunt
ended with Klenow DNA Polymerise. The resulting plasmid was named pvHCV-42 and
also
encoded amino acids 347 to 683. The NcoI/AIwNI fragment was inserted in a
similar way
into the same sites of pgsATA-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-
44).
pvHCV-43 and pvHCV-44 encoded amino acids 364 to 673 of the HCV polyprotein,
of
which amino acids 364 to 383 were derived from the natural carboxyterminal
region of the
E1 protein encoding the signal sequence for E2, and amino acids 384 to 673 of
the mature E2
protein.
2.5. Generation of recombinant HCV-vaccinia viruses
Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma 143B thymidine
2 0 kinase deficient (TK-) (ATCC CRL 8303), HeLa (ATCC CCL 2), and Hep G2
(ATCC HB
8065) cell lines were obtained from the American Type Culture Collection
(ATCC,
Rockville, Md, USA). The cells were grown in Dulbecco's modified Eagle medium
(DMEM)
supplemented with 10 % foetal calf serum, and with Earle's salts (EMEM) for
RK13 and 143
B (TK-), and with glucose (4 g/1) for Hep G2. The vaccinia virus WR strain
(Western
2 5 Reserve, ATTC VR119) was routinely propagated in either 143B or RKl3
cells, as described
previously (Panicali & Paoletti, 1982; Piccini et al., 1987; Mackett et al.,
1982, 1984, and
1986). A confluent monolayer of 143B cells was infected with wild type
vaccinia virus at a
multiplicity of infection (m.o.i.) of 0.1 (= 0.1 plaque forming unit (PFU) per
cell). Two hours
later, the vaccinia recombination plasmid was transfected into the infected
cells in the form
3 0 of a calcium phosphate coprecipitate containing 500 ng of the plasmid DNA
to allow
homologous recombination (Graham & van der Eb, 1973; Mackett et al., 1985).
Recombinant viruses expressing the E.coli xanthine-guanine phosphoribosyl
transferase (gpt)
protein were selected on rabbit kidney RK13 cells incubated in selection
medium (EMEM
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containing 25 pg/ml mycophenolic acid (MPA), 250 ~g/ml xanthine, and 15 p,g/ml
hypoxanthine; Falkner and Moss, 1988; Janknecht et al, 1991). Single
recombinant viruses
were purified on fresh monolayers of RK13 cells under a 0.9% agarose overlay
in selection
medium. 'Thyrnidine kinase deficient (TK~) recombinant viruses were selected
and then
plaque purified on fresh monolayers of human 143B cells (TK-) in the presence
of 25 pg/ml
5-bromo-2'-deoxyuridine. Stocks of purified recombinant HCV-vaccinia viruses
were
prepared by infecting either human 143 B or rabbit RK13 cells at an m.o.i. of
0.05 (Mackett
et al, 1988). The insertion of the HCV cDNA fragment in the recombinant
vaccinia viruses
was confirmed on an aliquot (50 ~l) of the cell lysate after the MPA selection
by means of
PCR with the primers used to clone the respective HCV fragments (see Table 1
). The
recombinant vaccinia-HCV viruses were named according to the vaccinia
recombination
plasmid number, e.g. the recombinant vaccinia virus vvHCV-l0A was derived from
recombining the wild type WR strain with the pvHCV-l0A plasmid.
Example 3: infection of cells with recombinant vaccinia viruses
A confluent monolayer of RK13 cells was infected at a m.o.i. of 3 with the
recombinant HCV-vaccinia viruses as described in example 2 . For infection,
the cell
monolayer was washed twice with phosphate-buffered saline pH 7.4 (PBS) and the
recombinant vaccinia virus stock was diluted in MEM medium. Two hundred ~l of
the virus
2 0 solution was added per 1 O6 cells such that the m.o.i. was 3, and
incubated for 45 min at 24°C.
The virus solution was aspirated and 2 ml of complete growth medium (see
example 2) was
added per 106 cells. The cells were incubated for 24 hr at 37°C during
which expression of
the HCV proteins took place.
2 5 Example 4: Analysis of recombinant proteins by means of western blotting
The infected cells were washed two times with PBS, directly lysed with lysis
buffer
(50 mM Tris.HCl pH 7.5, 150 mM NaCI, 1 % Triton X-100, 5 mM MgClz, 1 pg/ml
aprotinin
(Sigma, Bornem, Belgium)) or detached from the flasks by incubation in 50 mM
Tris.HCL
pH 7.5/ 10 mM EDTA/ 150 mM NaCI for 5 min, and collected by centrifugation (5
min at
3 0 1 OOOg). The cell pellet was then resuspended in 200 ~l lysis buffer (50
mM Tris.HCL pH
8.0, 2 mM EDTA, 150 mM NaCI, 5 mM MgCIZ aprotinin, 1 % Triton X-100) per 106
cells.
The cell lysates were cleared for 5 min at 14,000 rpm in an Eppendorf
centrifuge to
remove the insoluble debris. Proteins of 20 pl lysate were separated by means
of sodium
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dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins
were then
electro-transferred from the gel to a nitrocellulose sheet (Amersham) using a
Hoefer HSI
transfer unit cooled to 4°C for 2 hr at 100 V constant voltage, in
transfer buffer (25 mM
Tris.HCl pH 8.0, 192 mM glycine, 20% (v/v) methanol). Nitrocellulose filters
were blocked
with Blotto (5 % (w/v) fat-free instant milk powder in PBS; Johnson et al.,
1981) and
incubated with primary antibodies diluted in Blotto/0.1 % Tween 20. Usually, a
human
negative control serum or serum of a patient infected with HCV were 200 times
diluted and
preincubated for 1 hour at room temperature with 200 times diluted wild type
vaccinia virus-
infected cell lysate in order to decrease the non-specific binding. After
washing with
Blotto/0.1 % Tween 20, the nitrocellulose filters were incubated with alkaline
phosphatase
substrate solution diluted in Blotto/0.1 % Tween 20. After washing with 0.1%
Tween 20 in
PBS, the filters were incubated with alkaline phosphatase substrate solution
(100 mM
Tris.HCl pH 9.5, 100 mM NaCI, 5 mM MgCl2, 0,38 ~g/ml nitroblue tetrazolium,
0.165
~g/ml 5-bromo-4-chloro-3-indolylphosphate). All steps, except the
electrotransfer, were
performed at room temperature.
Example 5: Purification of recombinant E1 or E2 protein
5.1. Lysis
2 0 Infected RK13 cells (carrying El or E2 constructs) were washed 2 times
with
phosphate-buffered saline (PBS) and detached from the culture recipients by
incubation in
PBS containing 10 mM EDTA. The detached cells were washed twice with PBS and 1
ml of
lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCI, 1% Triton X-100, 5 mM MgCl2,
1
~g/ml aprotinin (Sigma, Bornem, Belgium) containing 2 mM biotinylated N-
ethylmaleimide
2 5 (biotin-NEM) (Sigma) was added per 105 cells at 4°C. This lysate
was homogenized with a
type B douncer and left at room temperature for 0.5 hours. Another 5 volumes
of lysis buffer
containing 10 mM N-ethylmaleimide (NEM, Aldrich, Bornem, Belgium) was added to
the
primary lysate and the mixture was left at room temperature for 15 min.
Insoluble cell debris
was cleared from the solution by centrifugation in a Beckman JA-14 rotor at
14,000 rpm
3 0 (30100 g at r",aX) for I hour at 4°C.
5.2. Lectin Chromatography
The cleared cell lysate was loaded at a rate of lml/min on a 0.8 by 10 cm
Lentil-lectin
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Sepharose 4B column (Pharmacia) that had been equilibrated with 5 column
volumes of lysis
buffer at a rate of lml/min. The lentil-lectin column was washed with 5 to 10
column
volumes of buffer 1 (O.1M potassium phosphate pH 7.3, S00 mM KCI, S% glycerol,
1 mM
6-NHZ-hexanoic acid, 1 mM MgClz, and 1 % DecyIPEG (KWANT, Bedum, The
5 Netherlands). In some experiments, the column was subsequently washed with
10 column
volumes of buffer 1 containing 0.5% Empigen-BB (Calbiochem, San Diego, CA,
USA)
instead of 1% DecyIPEG. The bound material was eluted by applying elution
buffer (10 mM
potassium phosphate pH 7.3, 5% glycerol, 1 mM hexanoic acid, 1mM MgClz, 0.5%
Empigen-BB, and 0.5 M o,-methyl-mannopyranoside). The eluted material was
fractionated
10 and fractions were screened for the presence of E1 or E2 protein by means
of ELISA as
described in example G. Figure 22 shows ELISA results obtained from lentil
lectin eluate
fractions of 4 different El purifications of cell lysates infected with
vvHCV39 (type lb),
wHCV40 (type 1b), wHCV62 (type 3a), and wHCVG3 (type Sa). Figure 23 shows the
profiles obtained from the values shown in Figure 22. These results show that
the lectin
15 affinity column can be employed for envelope proteins of the different
types of HCV.
5.3. Concentration and partial reduction
The E1- or E2-positive fractions were pooled and concentrated on a Centricon
30
kDa (Amicon) by centrifugation for 3 hours at 5,000 rpm in a Beckman JA-20
rotor at 4°C.
20 In some experiments the El- or E2-positive fractions were pooled and
concentrated by
nitrogen evaporation. An equivalent of 3.108 cells was concentrated to
approximately 200 ~1.
For partial reduction, 30% Empigen-BB (Calbiochem, San Diego, CA, USA) was
added to
this 200 ~1 to a final concentration of 3.5 %, and 1M DTT in HZO was
subsequently added to
a final concentration of 1.5 to 7.5 mM and incubated for 30 min at 37
°C. NEM (1M in
2 5 dimethylsulphoxide) was subsequently added to a final concentration of 50
mM and left to
react for another 30 min at 37°C to block the free sulphydryl groups.
5.4. Gel filtration chromatography
3 0 A Superdex-200 HR 10/20 column (Pharmacia) was equilibrated with 3 column
volumes PBS/3% Empigen-BB. The reduced mixture was injected in a 500 ~1 sample
loop of
the Smart System (Pharmacia) and PBS/3% Empigen-BB buffer was added for
gelfiltration.
Fractions of 250 pl were collected from Vo to V,. The fractions were screened
for the
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presence of E1 or E2 protein as described in example 6.
Figure 24 shows ELISA results obtained from fractions obtained after
gelfiltration
chromatography of 4 different E1 purifications of cell lysates infected with
wHCV39 (type
lb), wHCV40 (type lb), wHCV62 (type 3a), and wHCV63 (type Sa). Figure 25 shows
the
profiles obtained from purifications of El proteins of types 1b, 3a, and Sa
(from RK13 cells
infected with wHCV39, wHCV62, and wHCV63, respectively; purified on lentil
lectin and
reduced as in the previous examples). The peaks indicated with ' 1', '2', and
'3', represent pure
E1 protein peaks (E1 reactivity mainly in fractions 26 to 30). These peaks
show very similar
molecular weights of approximately 70 kDa, corresponding to dimeric E1
protein. Other
peaks in the three profiles represent vaccinia virus and/or cellular proteins
which could be
separated from El only because of the reduction step as outlined in example
5.3. and because
of the subsequent gelfiltration step in the presence of the proper detergent.
As shown in
Figure 26 pool 1 (representing fractions 10 to 17) and pool 2 (representing
fractions 18 to 25)
contain contaminating proteins not present in the E1 pool (fractions 26 to
30). The E1 peak
fractions were ran on SDS/PAGE and blotted as described in example 4. Proteins
labelled
with NEM-biotin were detected by streptavidin-alkaline phosphatase as shown in
Figure 27.
It can be readily observed that, amongst others, the 29 kDa and 45kDa
contaminating
proteins present before the gelfiltration chromatography (lane 1 ) are only
present at very low
levels in the fractions 26 to 30. The band at approximately 65kDa represents
the E1 dimeric
form that could not be entirely disrupted into the monomeric E1 form. Similar
results were
obtained for the type 3a E1 protein (lanes 10 to 15), which shows a faster
mobility on
SDS/PAGE because of the presence of only 5 carbohydrates instead of 6. Figure
28 shows a
silver stain of an SDS/PAGE gel run in identical conditions as in Figure 26. A
complete
overview of the purification procedure is given in Figure 29.
The presence of purified E1 protein was further confirmed by means of western
blotting as described in example 4. The dimeric E1 protein appeared to be non-
aggregated
and free of contaminants. The subtype lb E1 protein purified from wHCV40-
infected cells
according to the above scheme was aminoterlninally sequenced on an 477 Perkin-
Elmer
sequencer and appeared to contain a tyrosine as first residue. This confirmed
that the El
protein had been cleaved by the signal peptidase at the correct position
(between A191 and
Y192) from its signal sequence. This confirms the finding of Hijikata et al.
(1991) that the
aminoterminus of the mature E1 protein starts at amino acid position 192.
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5.5. Purification of the E2 protein
The E2 protein (amino acids 384 to 673) was purified from RK13 cells infected
with
wHCV44 as indicated in Examples 5.1 to 5.4. Figure 30 shows the ODzB°
profile (continuous
line) of the lentil lectin chromatography. The dotted line represents the E2
reactivity as
detected by ELISA (see example 6). Figure 31 shows the same profiles obtained
from
gelfiltration chromatography of the lentil-lectin E2 pool (see Figure 30),
part of which was
reduced and blocked according to the methods as set out in example 5.3., and
part of which
was immediately applied to the column. Both parts of the E2 pool were run on
separate
gelfiltration columns. It could be demonstrated that E2 forms covalently-
linked aggregates
with contaminating proteins if no reduction has been performed. After
reduction and
blocking, the majority of contaminating proteins segregated into the V°
fraction. Other
contaminating proteins copurified with the E2 protein, were not covalently
linked to the E2
protein any more because these contaminants could be removed in a subsequent
step. Figure
32 shows an additional Ni2+-IMAC purification step carried out for the E2
protein
purification. This affinity purification step employs the 6 histidine residues
added to the E2
protein as expressed from wHCV44. Contaminating proteins either run through
the column
or can be removed by a 30 mM imidazole wash. Figure 33 shows a silver-stained
SDS/PAGE of 0.5 ~g of purified E2 protein and a 30 mM imidazole wash. The pure
E2
protein could be easily recovered by a 200 mM imidazole elution step. Figure
34 shows an
2 0 additional desalting step intended to remove imidazole and to be able to
switch to the desired
buffer, e.g. PBS, carbonate buffer, saline.
Starting from about 50,000 cmz of RK13 cells infected with wHCVIIA (or
wHCV40) for the production of E1 or wHCV4l, wHCV42, wHCV43, or wHCV44 for
production of E2 protein, the procedures described in examples 5.1 to 5.5
allow the
2 5 purification of approximately 1.3 mg of E1 protein and 0.6 mg of E2
protein.
It should also be remarked that secreted E2 protein (constituting
approximately 30-
40%, 60-70% being in the intracellular form) is characterized by aggregate
formation
(contrary to expectations). The same problem is thus posed to purify secreted
E2. The
secreted E2 can be purified as disclosed above.
Example 6: ELISA for the detection of anti-E1 or anti-E2 antibodies or for the
detection of E1 or E2 proteins
Maxisorb microwell plates (Nunc, Roskilde, Denmark) were coated with 1 volume
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(e.g. 50 ~l or 100 ~l or 200 ~l) per well of a S ~g/ml solution of
Streptavidin (Boehringer
Mannheim) in PBS for 16 hours at 4°C or for 1 hour at 37°C.
Alternatively, the wells were
coated with 1 volume of 5 ~g/ml of Galanthus nivalis agglutinin (GNA) in 50 mM
sodium
carbonate buffer pH 9.6 for 16 hours at 4°C or for 1 hour at
37°C. In the case of coating with
GNA, the plates were washed 2 times with 400 ~l of Washing Solution of the
Innotest HCV
Ab III kit (Innogenetics, Belgium). Unbound coating surfaces were blocked with
1.5 to 2
volumes of blocking solution (0.1 % casein and 0.1 % NaN3 in PBS) for 1 hour
at 37°C or for
16 hours at 4°C. Blocking solution was aspirated. Purified E1 or E2 was
diluted to 100-1000
ng/ml (concentration measured at A = 280 nm) or column fractions to be
screened for El or
E2 (see example 5), or E1 or E2 in non-purified cell lysates (example 5.1.)
were diluted 20
times in blocking solution, and 1 volume of the E1 or E2 solution was added to
each well and
incubated for 1 hour at 37°C on the Streptavidin- or GNA-coated plates.
The microwells
were washed 3 times with 1 volume of Washing Solution of the Innotest HCV Ab
III kit
(Innogenetics, Belgium). Serum samples were diluted 20 times or monoclonal
anti-E1 or
anti-E2 antibodies were diluted to a concentration of 20 ng/ml in Sample
Diluent of the
Innotest HCV Ab III kit and 1 volume of the solution was left to react with
the E1 or E2
protein for 1 hour at 37°C. The microwells were washed 5 times with 400
pl of Washing
Solution of the Innotest HCV Ab III kit (Innogenetics, Belgium). The bound
antibodies were
detected by incubating each well for 1 hour at 37°C with a goat anti-
human or anti-mouse
2 0 IgG, peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark)
diluted
1/80,000 in 1 volume of Conjugate Diluent of the Innotest HCV Ab III kit
(Innogenetics,
Belgium), and color development was obtained by addition of substrate of the
Innotest HCV
Ab III kit (Innogenetics, Belgium) diluted 100 times in 1 volume of Substrate
Solution of the
Innotest HCV Ab III kit (Innogenetics, Belgium) for 30 min at 24°C
after washing of the
2 5 plates 3 times with 400 pl of Washing Solution of the Innotest HCV Ab III
kit (Innogenetics,
Belgium).
Example 7: Follow up of patient groups with different clinical profiles
3 0 7.1. Monitoring of anti-E 1 and anti-E2 antibodies
The current hepatitis C virus (HCV) diagnostic assays have been developed for
screening and confirmation of the presence of HCV antibodies. Such assays do
not seem to
provide information useful for monitoring of treatment or for prognosis of the
outcome of
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disease. However, as is the case for hepatitis B, detection and quantification
of anti-envelope
antibodies may prove more useful in a clinical setting. To investigate the
possibility of the
use of anti-E1 antibody titer and anti-E2 antibody titer as prognostic markers
for outcome of
hepatitis C disease, a series of IFN-a treated patients with long-term
sustained response
(defined as patients with normal transaminase levels and negative HCV-RNA test
(PCR in
the 5' non-coding region) in the blood for a period of at least 1 year after
treatment) was
compared with patients showing no response or showing biochemical response
with relapse
at the end of treatment.
A group of 8 IFN-a treated patients with long-term sustained response (LTR,
follow
up 1 to 3.5 years, 3 type 3a and S type lb) was compared with 9 patients
showing non-
complete responses to treatment (NR, follow up 1 to 4 years, 6 type lb and 3
type 3a). Type
lb (wHCV-39, see example 2.5.) and 3a E1 (wHCV-62, see example 2.5.) proteins
were
expressed by the vaccinia virus system (see examples 3 and 4) and purified to
homogeneity
(example 5). The samples derived from patients infected with a type lb
hepatitis C virus
were tested for reactivity with purified type lb E1 protein, while samples of
a type 3a
infection were tested for reactivity of anti-type 3a E1 antibodies in an ELISA
as desribed in
example 6. The genotypes of hepatitis C viruses infecting the different
patients were
determined by means of the Inno-LiPA genotyping assay (Innogenetics, Belgium).
Figure 5
shows the anti-E1 signal-to-noise ratios of these patients followed during the
course of
2 0 interferon treatment and during the follow-up period after treatment. LTR
cases consistently
showed rapidly declining anti-E1 levels (with complete negativation in 3
cases), while anti-
E1 levels of NR cases remained approximately constant. Some of the obtained
anti-E1 data
are shown in Table 2 as average S/N ratios ~ SD (mean anti-E1 titer). The anti-
El titer
could be deduced from the signal to noise ratio as show in Figures S, 6, 7,
and 8.
2 5 Already at the end of treatment, marked differences could be observed
between the 2
groups. Anti-El antibody titers had decreased 6.9 times in LTR but only 1.5
times in NR. At
the end of follow up, the anti-E 1 titers had declined by a factor of 22.5 in
the patients with
sustained response and even slightly increased in NR. Therefore, based on
these data,
decrease of anti-E1 antibody levels during monitoring of IFN-~, therapy
correlates with long-
30 term, sustained response to treatment. The anti-E1 assay may be very useful
for prognosis of
long-term response to IFN treatment, or to treatment of the hepatitis C
disease in general.
This finding was not expected. On the contrary, the inventors had expected the
anti-
E1 antibody levels to increase during the course of IFN treatment in patients
with long term
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response. As is the case for hepatitis B, the virus is cleared as a
consequence of the
seroconversion for anti-HBsAg antibodies. Also in many other virus infections,
the virus is
eliminated when anti-envelope antibodies are raised. However, in the
experiments of the
present invention, anti-E1 antibodies clearly decreased in patients with a
long-term response
5 to treatment, while the antibody-level remained approximately at the same
level in non-
responding patients. Although the outcome of these experiments was not
expected, this non-
obvious finding may be very important and useful for clinical diagnosis of HCV
infections.
As shown in Figures 9, 10, 11, and 12, anti-E2 levels behaved very differently
in the same
patients studied and no obvious decline in titers was observed as for anti-El
antibodies.
10 Figure 35 gives a complete overview of the pilot study.
As can be deduced from Table 2, the anti-E1 titers were on average at least 2
times
higher at the start of treatment in long term responders compared with
incomplete responders
to treatment. Therefore, measuring the titer of anti-E1 antibodies at the
start of treatment, or
monitoring the patient during the course of infection and measuring the anti-
E1 titer, may
15 become a useful marker for clinical diagnosis of hepatitis C. Furthermore,
the use of more
defined regions of the El or E2 proteins may become desirable, as shown in
example 7.3.
7.2. Analysis of E1 and E2 antibodies in a larger patient cohort
The pilot study lead the inventors to conclude that, in case infection was
completely
2 0 cleared, antibodies to the HCV envelope proteins changed more rapidly than
antibodies to the
more conventionally studied HCV antigens, with El antibodies changing most
vigorously.
We therefore included more type lb and 3a-infected LTR and further
supplemented the
cohort with a matched series of NR, such that both groups included 14 patients
each. Some
partial responders (PR) and responders with relapse (RR) were also analyzed.
25 Figure 36 depicts average El antibody (EIAb) and E2 antibody (E2Ab) levels
in the
LTR and NR groups and Tables 4 and 5 show the statistical analyses. In this
larger cohort,
higher E1 antibody levels before IFN-~, therapy were associated with LTR (P <
0.03). Since
much higher E1 antibody levels were observed in type 3a-infected patients
compared with
type lb-infected patients (Figure 37), the genotype was taken into account
(Table 4). Within
30 the type lb-infected group, LTR also had higher E1 antibody levels than NR
at the initiation
of treatment [P < 0.05]; the limited number of type 3a-infected NR did not
allow statistical
analysis.
Of antibody levels monitored in LTR during the 1.5-year follow up period, only
E1
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antibodies cleared rapidly compared with levels measured at initiation of
treatment [P =
0.0058, end of therapy; P = 0.0047 and P = 0.0051 at 6 and 12 months after
therapy,
respectively]. This clearance remained significant within type 1- or type 3-
infected LTR
(average P values c 0.05). These data confirmed the initial finding that EIAb
levels decrease
rapidly in the early phase of resolvement. This feature seems to be
independent of viral
genotype. In NR, PR, or RR, no changes in any of the antibodies measured were
observed
throughout the follow up period. In patients who responded favourably to
treatment with
normalization of ALT levels and HCV-RNA negative during treatment, there was a
marked
difference between sustained responders (LTR) and responders with a relapse
(RR). In
contrast to LTR, RR did not show any decreasing E1 antibody levels, indicating
the presence
of occult HCV infection that could neither be demonstrated by PCR or other
classical
techniques for detection of HCV-RNA, nor by raised ALT levels. The minute
quantities of
viral RNA, still present in the RR group during treatment, seemed to be
capable of anti-E 1 B
cell stimulation. Anti-El monitoring may therefore not only be able to
discriminate LTR
from NR, but also from RR.
7.3. Monitoring of antibodies of defined regions of the E1 protein
Although the molecular biological approach of identifying HCV antigens
resulted in
unprecedented breakthrough in the development of viral diagnostics, the method
of immune
screening of ~,gtl l libraries predominantly yielded linear epitopes dispersed
throughout the
core and non-structural regions, and analysis of the envelope regions had to
await cloning
and expression of the E1/E2 region in mammalian cells. This approach sharply
contrasts with
many other viral infections of which epitopes to the envelope regions had
already been
mapped long before the deciphering of the genomic structure. Such epitopes and
2 5 corresponding antibodies often had neutralizing activity useful for
vaccine development
and/or allowed the development of diagnostic assays with clinical or
prognostic significance
(e.g. antibodies to hepatitis B surface antigen).
As no HCV vaccines or tests allowing clinical diagnosis and prognosis of
hepatitis C disease
are available today, the characterization of viral envelope regions exposed to
immune
3 0 surveillance may significantly contribute to new directions in HCV
diagnosis and
prophylaxis.
Several 20-mer peptides (Table 3) that overlapped each other by 8 amino acids,
were
synthesized according to a previously described method (EP-A-0 489 968) based
on the HC-
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Jl sequence (Okamoto et al., 1990). None of these, except peptide env35 (also
referred to as
E1-35), was able to detect antibodies in sera of approximately 200 HCV cases.
Only 2 sera
reacted slightly with the env35 peptide. However, by means of the anti-El
ELISA as
described in example 6, it was possible to discover additional epitopes as
follows: The anti-
s E1 ELISA as described in example 6 was modified by mixing 50 ~g/ml of E1
peptide with
the 1/20 diluted human serum in sample diluent. Figure 13 shows the results of
reactivity of
human sera to the recombinant El (expressed from vvHCV-40) protein, in the
presence of
single or of a mixture of El peptides. While only 2% of the sera could be
detected by means
of El peptides coated on strips in a Line Immunoassay format, over half of the
sera contained
anti-E1 antibodies which could be competed by means of the same peptides, when
tested on
the recombinant El protein. Some of the marine monoclonal antibodies obtained
from
Balb/C mice after injection with purified E1 protein were subsequently
competed for
reactivity to El with the single peptides (Figure 14). Clearly, the region of
env53 contained
the predominant epitope, as the addition of env53 could substantially compete
reactivity of
several sera with E1, and antibodies to the env31 region were also detected.
This finding was
surprising, since the env53 and env31 peptides had not shown any reactivity
when coated
directly to the solid phase.
Therefore peptides were synthesized using technology described by applicant
previously (in WO 93/18054). The following peptides were synthesized:
peptide env35A-biotin
NHZ-SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO:51)
spanning amino acids 208 to 227 of the HCV polyprotein in the El region
peptide biotin-env53 ('epitope A')
biotin-GG-ITGHRMAWDMMMNWSPTTAL-COOH (SEQ ID N0:52)
2 5 spanning amino acids to 313 of 332 of the HCV polyprotein in the E1 region
peptide lbEl ('epitope B')
HZN-YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCGK -biotin
(SEQ ID N0:53)
spanning amino acids 192 to 228 of the HCV polyprotein in the El region
3 0 and compared with the reactivities of peptides E 1 a-BB (biotin-GG-
TPTVATRDGKLPATQLRRHIDLL, SEQ ID N0:54) and Elb-BB (biotin-GG-
TPTLAARDASVPTTTIRRHVDLL, SEQ ID NO:55) which are derived from the same
region of sequences of genotype 1 a and I b respectively and which have been
described at the
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IXth international virology meeting in Glasgow, 1993 ('epitope C'). Reactivity
of a panel of
HCV sera was tested on epitopes A, B and C and epitope B was also compared
with env35A
(of 47 HCV-positive sera, 8 were positive on epitope B and none reacted with
env35A).
Reactivity towards epitopes A, B, and C was tested directly to the
biotinylated peptides (50
~g/ml) bound to streptavidin-coated plates as described in example 6. Clearly,
epitopes A
and B were most reactive while epitopes C and env35A-biotin were much less
reactive. The
same series of patients that had been monitored for their reactivity towards
the complete E1
protein (example 7.1.) was tested for reactivity towards epitopes A, B, and C.
Little reactivity
was seen to epitope C, while as shown in Figures 15, 16, 17, and 18, epitopes
A and B
reacted with the majority of sera. However, antibodies to the most reactive
epitope (epitope
A) did not seem to predict remission of disease, while the anti-lbE1
antibodies (epitope B)
were present almost exclusively in long term responders at the start of IFN
treatment.
Therefore, anti-lbEl (epitope B) antibodies and anti-env53 (epitope A)
antibodies could be
shown to be useful markers for prognosis of hepatitis C disease. The env53
epitope may be
advantageously used for the detection of cross-reactive antibodies (antibodies
that cross-react
between major genotypes) and antibodies to the env53 region may be very useful
for
universal E1 antigen detection in serum or liver tissue. Monoclonal antibodies
that
recognized the env53 region were reacted with a random epitope library. In 4
clones that
reacted upon immunoscreening with the monoclonal antibody SElAlO, the sequence
-GWD-
2 0 was present. Because of its analogy with the universal HCV sequence
present in all HCV
variants in the env53 region, the sequence AWD is thought to contain the
essential sequence
of the env53 cross-reactive murine epitope. The env31 clearly also contains a
variable region
which may contain an epitope in the amino terminal sequence -YQVRNSTGL- (SEQ
ID
N0:93) and may be useful for diagnosis. Env31 or El-31 as shown in Table 3, is
a part of the
peptide lbEl. Peptides E1-33 and E1-51 also reacted to some extent with the
murine
antibodies, and peptide E1-55 (containing the variable region 6 (V6); spanning
amino acid
positions 329-336) also reacted with some of the patient sera.
Anti-E2 antibodies clearly followed a different pattern than the anti-E1
antibodies,
especially in patients with a long-term response to treatment. Therefore, it
is clear that the
3 0 decrease in anti-envelope antibodies could not be measured as efficiently
with an assay
employing a recombinant E1/E2 protein as with a single anti-E1 or anti-E2
protein. The anti-
E2 response would clearly blur the anti-E1 response in an assay measuring both
kinds of
antibodies at the same time. Therefore, the ability to test anti-envelope
antibodies to the
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single E1 and E2 proteins, was shown to be useful.
7.4. Mapping of anti-E2 antibodies
Of the 24 anti-E2 Mabs only three could be competed for reactivity to
recombinant
E2 by peptides, two of which reacted with the HVRI region (peptides E2-67 and
E2-69,
designated as epitope A) and one which recognized an epitope competed by
peptide E2-13B
(epitope C). The majority of murine antibodies recognized conformational anti-
E2 epitopes
(Figure 19). A human response to HVRI (epitope A), and to a lesser extent
HVRII (epitope
B) and a third linear epitope region (competed by peptides E2-23, E2-25 or E2-
27,
designated epitope E) and a fourth linear epitope region (competed by peptide
E2-17B,
epitope D) could also frequently be observed, but the majority of sera reacted
with
conformational epitopes (Figure 20). These conformational epitopes could be
grouped
according to their relative positions as follows: the IgG antibodies in the
supernatant of
hybridomas 15C8C1, 12D11F1, 9G3E6, BGlODIH9, lOD3C4, 4H6B2, 17F2C2, SH6A7,
1 SB7A2 recognizing conformational epitopes were purified by means of protein
A affinity
chromatography and 1 mg/ml of the resulting IgG's were biotinylated in borate
buffer in the
presence of biotin. Biotinylated antibodies were separated from free biotin by
means of
gelfiltration chromatography. Pooled biotinylated antibody fractions were
diluted 100 to
10,000 times. E2 protein bound to the solid phase was detected by the
biotinylated IgG in the
2 0 presence of 100 times the amount of non-biotinylated competing antibody
and subsequently
detected by alkaline phosphatase labeled streptavidin.
Percentages of competition are given in Table 6. Based on these results, 4
conformational anti-E2 epitope regions (epitopes F, G, H and I) could be
delineated (Figure
38). Alternatively, these Mabs may recognize mutant linear epitopes not
represented by the
2 5 peptides used in this study. Mabs 4H6B2 and l OD3C4 competed reactivity of
16A6E7, but
unlike 16A6E7, they did not recognize peptide E2-13B. These Mabs may recognize
variants
of the same linear epitope (epitope C) or recognize a conformational epitope
which is
sterically hindered or changes conformation after binding of 16A6E7 to the E2-
13B region
(epitope H).
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Example 8: El glycosylation mutants
8.1. Introduction
The E1 protein encoded by wHCVlOA, and the E2 protein encoded by wHCV41 to
5 44 expressed from mammalian cells contain 6 and 11 carbohydrate moieties,
respectively.
This could be shown by incubating the lysate of wHCV l0A-infected or wHCV44-
infected
RK13 cells with decreasing concentrations of glycosidases (PNGase F or
Endoglycosidase
H, (Boehringer Mannhein Biochemica) according to the manufacturer's
instructions), such
that the proteins in the lysate (including E1) are partially deglycosylated
(Fig. 39 and 40,
10 respectively).
Mutants devoid of some of their glycosylation sites could allow the selection
of
envelope proteins with improved immunological reactivity. For HIV for example,
gp120
proteins lacking certain selected sugar-addition motifs, have been found to be
particularly
useful for diagnostic or vaccine purpose. The addition of a new
oligosaccharide side chain in
15 the hemagglutinin protein of an escape mutant of the A/Hong Kong/3/68
(H3N2) influenza
virus prevents reactivity with a neutralizing monoclonal antibody (Skehel et
al, 1984). When
novel glycosylation sites were introduced into the influenza hemaglutinin
protein by site-
specific mutagenesis, dramatic antigenic changes were observed, suggesting
that the
carbohydrates serve as a modulator of antigenicity (Gallagher et al., 1988).
In another
2 0 analysis, the 8 carbohydrate-addition motifs of the surface protein gp70
of the Friend Murine
Leukemia Virus were deleted. Although seven of the mutations did not affect
virus
infectivity, mutation of the fourth glycosylation signal with respect to the
amino terminus
resulted in a non-infectious phenotype (Kayman et al., 1991). Furthermore, it
is known in the
art that addition of N-linked carbohydrate chains is important for
stabilization of folding
2 5 intermediates and thus for efficient folding, prevention of malfolding and
degradation in the
endoplasmic reticulum, oligomerization, biological activity, and transport of
glycoproteins
(see reviews by Rose et al., 1988; Doms et al., 1993; Helenius, 1994).
After aligmnent of the different envelope protein sequences of HCV genotypes,
it
may be inferred that not all 6 glycosylation sites on the HCV subtype lb E1
protein are
3 0 required for proper folding and reactivity, since some are absent in
certain (sub)types. The
fourth carbohydrate motif (on Asn251), present in types lb, 6a, 7, 8, and 9,
is absent in all
other types known today. This sugar-addition motif may be mutated to yield a
type 1 b E 1
protein with improved reactivity. Also the type 2b sequences show an extra
glycosylation site
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in the VS region (on Asn299). The isolate S83, belonging to genotype 2c, even
lacks the first
carbohydrate motif in the V 1 region (on Asn), while it is present on all
other isolates (Stuyver
et al., 1994). However, even among the completely conserved sugar-addition
motifs, the
presence of the carbohydrate may not be required for folding, but may have a
role in evasion
of immune surveillance. Therefore, identification of the carbohydrate addition
motifs which
are not required for proper folding (and reactivity) is not obvious, and each
mutant has to be
analyzed and tested for reactivity. Mutagenesis of a glycosylation motif (NXS
or NXT
sequences) can be achieved by either mutating the codons for N, S, or T, in
such a way that
these codons encode amino acids different from N in the case of N, and/or
amino acids
different from S or T in the case of S and in the case of T. Alternatively,
the X position may
be mutated into P, since it is known that NPS or NPT are not frequently
modified with
carbohydrates. After establishing which carbohydrate-addition motifs are
required for folding
and/or reactivity and which are not, combinations of such mutations may be
made.
8.2. Mutagenesis of the E1 protein
All mutations were performed on the El sequence of clone HCC110A (SEQ ID
NO:S). The first round of PCR was performed using sense primer 'GPT' (see
Table 7)
targetting the GPT sequence located upstream of the vaccinia 11 K late
promoter, and an
antisense primer (designated GLY#, with # representing the number of the
glycosylation
2 0 site, see Fig. 41 ) containing the desired base change to obtain the
mutagenesis. The six GLY#
primers (each specific for a given glycosylation site) were designed such
that:
- Modification of the codon encoding for the N-glycosylated Asn (AAC or AAT)
to a Gln
codon (CAA or CAG). Glutamine was chosen because it is very similar to
asparagine (both
amino acids are neutral and contain non-polar residues, glutamine has a longer
side chain
2 5 (one more -CHZ- group).
- The introduction of silent mutations in one or several of the codons
downstream of the
glycosylation site, in order to create a new unique or rare (e.g. a second
SmaI site for
ElGlyS) restriction enzyme site. Without modifying the amino acid sequence,
this mutation
will provide a way to distinguish the mutated sequences from the original E1
sequence
30 (pvHCV-l0A) or from each other (Figure 41) . This additional restriction
site may also be
useful for the construction of new hybrid (double, triple, etc.) glycosylation
mutants.
- 18 nucleotides extend 5' of the first mismatched nucleotide and 12 to 16
nucleotides extend
to the 3' end. Table 7 depicts the sequences of the six GLY# primers
overlapping the
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sequence of N-linked glycosylation sites.
For site-directed mutagenesis, the 'mispriming' or 'overlap extension'
(Horton, 1993)
was used. The concept is illustrated in Figures 42 and 43. First, two separate
fragments were
amplified from the target gene for each mutated site. The PCR product obtained
from the 5'
end (product GLY#) was amplified with the 5' sense GPT primer (see Table 7)
and with the
respective 3' antisense GLY# primers. The second fragment (product OVR#) was
amplified
with the 3' antisense TK,t primer and the respective 5' sense primers (OVR#
primers, see
Table 7, Figure 43).
The OVR# primers target part of the GLY# primer sequence. Therefore, the two
groups of PCR products share an overlap region of identical sequence. When
these
intermediate products are mixed (GLY-1 with OVR-1, GLY-2 with OVR-2, etc.),
melted at
high temperature, and reannealed, the top sense strand of product GLY# can
anneal to the
antisense strand of product OVR# (and vice versa) in such a way that the two
strands act as
primers for one another (see Fig. 42.B.). Extension of the annealed overlap by
Taq
polymerase during two PCR cycles created the full-length mutant molecule E 1
Gly#, which
carries the mutation destroying the glycosylation site number #. Sufficient
quantities of the
E1GLY# products for cloning were generated in a third PCR by means of a common
set of
two internal nested primers. These two new primers are respectively
overlapping the 3' end of
the vaccinia I 1 K promoter (sense GPT-2 primer) and the 5' end of the
vaccinia thymidine
2 0 kinase locus (antisense TK,t-2 primer, see Table 7). All PCR conditions
were performed as
described in Stuyver et al. (1993).
Each of these PCR products was cloned by EcoRI/BamHI cleavage into the
EcoRI/BaniHI-cut vaccinia vector containing the original E1 sequence (pvHCV-
l0A).
The selected clones were analyzed for length of insert by EcoRI/BamH I
cleavage
2 5 and for the presence of each new restriction site. The sequences
overlapping the mutated sites
were confirmed by double-stranded sequencing.
8.3. Analysis of E1 glycosylation mutants
Starting from the 6 plasmids containing the mutant E1 sequences as described
in
3 0 example 8.2, recombinant vaccinia viruses were generated by recombination
with wt
vaccinia virus as described in example 2.5. Briefly, 175 cmz-flasks of
subconfluent RK13
cells were infected with the 6 recombinant vaccinia viruses carrying the
mutant E1
sequences, as well as with the wHCV-IOA (carrying the non-mutated El sequence)
and wt
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vaccinia viruses. Cells were lysed after 24 hours of infection and analyzed on
western blot as
described in example 4 (see Figure 44A). All mutants showed a faster mobility
(corresponding to a smaller molecular weight of approximately 2 to 3 kDa) on
SDS-PAGE
than the original El protein; confirming that one carbohydrate moiety was not
added.
Recombinant viruses were also analyzed by PCR and restriction enzyme analysis
to confirm
the identity of the different mutants. Figure 44B shows that all mutants (as
shown in Figure
41 ) contained the expected additional restriction sites. Another part of the
cell lysate was
used to test the reactivity of the different mutant by ELISA. The lysates were
diluted 20 times
and added to microwell plates coated with the lectin GNA as described in
example 6.
Captured (mutant) E1 glycoproteins were left to react with 20-times diluted
sera of 24 HCV-
infected patients as described in example 6. Signal to noise (S/N) values (OD
of GLY#/OD
of wt) for the six mutants and E1 are shown in Table 8. The table also shows
the ratios
between S/N values of GLY# and El proteins. It should be understood that the
approach to
use cell lysates of the different mutants for comparison of reactivity with
patient sera may
result in observations that are the consequence of different expression levels
rather then
reactivity levels. Such difficulties can be overcome by purification of the
different mutants as
described in example S, and by testing identical quantities of all the
different E1 proteins.
However, the results shown in table 5 already indicate that removal of the 1
st (GLY1 ), 3rd
(GLY3), and 6th (GLY6) glycosylation motifs reduces reactivity of some sera,
while removal
2 0 of the 2nd and 5th site does not. Removal of GLY4 seems to improve the
reactivity of certain
sera. These data indicate that different patients react differently to the
glycosylation mutants
of the present invention. Thus, such mutant E1 proteins may be useful for the
diagnosis
(screening, confirmation, prognosis, etc.) and prevention of HCV disease.
2 5 Example 9: Expression of HCV E2 protein in glycosylation-deficient yeasts
The E2 sequence corresponding to clone HCC141 was provided with the ~,-mating
factor pre/pro signal sequence, inserted in a yeast expression vector and S.
cerevisiae cells
transformed with this construct secreted E2 protein into the growth medium. It
was observed
that most glycosylation sites were modified with high-mannose type
glycosylations upon
3 0 expression of such a construct in S. cerevisiae strains (Figure 45). This
resulted in a too high
level of heterogeneity and in shielding of reactivity, which is not desirable
for either vaccine
or diagnostic purposes. To overcome this problem, S. cerevisiae mutants with
modified
glycosylation pathways were generated by means of selection of vanadate-
resistant clones.
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Such clones were analyzed for modified glycosylation pathways by analysis of
the molecular
weight and heterogeneity of the glycoprotein invertase. This allowed us to
identify different
glycosylation deficient S. cerevisiae mutants. The E2 protein was subsequently
expressed in
some of the selected mutants and left to react with a monoclonal antibody as
described in
example 7, on western blot as described in example 4 (Figure 46).
Example 10. General utility
The present results show that not only a good expression system but also a
good
purification protocol are required to reach a high reactivity of the HCV
envelope proteins
with human patient sera. This can be obtained using the proper HCV envelope
protein
expression system and/or purification protocols of the present invention which
guarantee the
conservation of the natural folding of the protein and the purification
protocols of the present
invention which guarantee the elimination of contaminating proteins and which
preserve the
conformation, and thus the reactivity of the HCV envelope proteins. The
amounts of purified
HCV envelope protein needed for diagnostic screening assays are in the range
of grams per
year. For vaccine purposes, even higher amounts of envelope protein would be
needed.
Therefore, the vaccinia virus system may be used for selecting the best
expression constructs
and for limited upscaling, and large-scale expression and purification of
single or specific
oligomeric envelope proteins containing high-mannose carbohydrates may be
achieved when
2 0 expressed from several yeast strains. In the case of hepatitis B for
example, manufacturing of
HBsAg from mammalian cells was much more costly compared with yeast-derived
hepatitis
B vaccines.
The purification method dislcosed in the present invention may also be used
for 'viral
envelope proteins' in general. Examples are those derived from Flaviviruses,
the newly
2 5 discovered GB-A, GB-B and GB-C Hepatitis viruses, Pestiviruses (such as
Bovine viral
Diarrhoea Virus (BVDV), Hog Cholera Virus (HCV), Border Disease Virus (BDV)),
but also
less related virusses such as Hepatitis B Virus (mainly for the purification
of HBsAg).
The envelope protein purification method of the present invention may be used
for
intra- as well as extracellularly expressed proteins in lower or higher
eukaryotic cells or in
3 0 prokaryotes as set out in the detailed description section.
Example 11. Demonstration of Prophylactic and Therapeutic Utility
Liver disease in chimpanzees chronically infected with HCV can be reduced by
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immunization with El. Multiple immunizations, however, were required in order
to reach
a significant immune response. One of ordinary skill will appreciate that
viral persistence
is produced with immune modulation which is either orchestrated by the virus
itself or by
the host. In order to analyze if such an immune modulation does exist in HCV,
the immune
5 responses against E1 and NS3 in naive and chronically infected chimpanzees
were
compared. Since a lower response in the chronically infected animals was
anticipated,
this group of animals was selected for a more rigorous immunization schedule
including
the following: use of an adjuvant proven in mice to be more potent for
inducing cellular
responses (Table 9) compared to alum, which was the adjuvant used for naive
animals; and
10 the immunization schedule for chronically infected animals consisted of 12
immunizations
compared to 6 for naive animals (Fig. 47).
Although the number of immunized animals does not allow statistical analysis,
the
following clear tendency can be detected in the humoral responses (Table 10):
the number
of immunizations for seroconversion is lower in naive animals; and the
magnitude of the
15 immune response is substantially greater in the naive animals, 2/3 infected
animals do not
reach the level of 10 internal units, even after 12 immunizations.
The analysis of the cellular responses, after three immunizations, reveals an
even
larger difference (Fig. 48a-d), including the following: E1-specific T-cell
proliferation is
almost absent in the chronically infected animals, while a clear stimulation
can be seen in
2 0 the naive setting;1L-2 measurements confirmed that the low stimulation of
the T-cell
compartment in chronic carriers; and, a clear Th2 (IL-4) response in naive
animals is
induced, as expected for an alum-adjuvant containing vaccine.
This confirms that at least E1 immunization provides a prophylactic effect in
naive
animals and suggest that E2 and/or combinations of E1 and E2 proteins and/or
peptides
2 5 may provide useful therapeutic and/or prophylactic benefits in naive
animals.
The'impainnent' to induce both cellular and humoral responses against an HCV
E1
antigen can be only partially overcome by multiple immunizations, as
demonstrated by the
following results: an increase in antibody titer after each injection was
noted but the levels
as in naive animals were not reached in 2/3 animals; and the T-cell
proliferative responses
3 0 remain very low (Fig. 49). The ELISPOT results show, however, a minor
increase in IL-2
(not shown), no change in IFN-g (not shown) and an increase in IL-4 (Fig. 49)
which
indicates that Th2 type responses are more readily induced. IL-4 was noted to
remain at a
low level compared to the level reached after three immunizations in naive
animals.
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A quite similar observation was made for NS3 immunizations where an even
stronger adjuvant (RIBI) was used in the chronic chimpanzee. As compared with
an alum
formulation in naive animals the following has been noted: the induced
antibody titers are
comparable in both groups (not shown); and both cytokine secretion and T-cell
proliferation are almost absent in the chronic animals compared to the
responses in naive
animals (Fig. 49a-b).
Currently there have been some indications that immune responses against HCV
in
chronic carriers are low or at least insufficient to allow clearance of
infection. The above
results support the hypothesis that the immune system of HCV chronic earners
may be
impaired and that they do not respond to HCV antigens as efficiently as in a
naive
situation.
In a study by Wiedmann et al., (Hepatology 2000; 31: 230-234), vaccination for
HBV was less effective in HCV chronic carriers, which indicates that such an
immune
impairment is not limited to HCV antigens. De Maria et al. (Hepatology 2000;
32: 444-
445) confirmed these data and have proposed adapted vaccine dosing regimens
for HCV
patients. The data presented herein indicates that increasing the number of
immunizations
may indeed augment humoral responses but that cellular (especially Thl )
responses are
difficult to induce, even when powerful adjuvants are used. It may be
advantageous to
begin immunization at the time of antiviral therapy, when the immune system is
more
2 0 prone to respond.
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Table 1: Recombinant vaccinia plasmids and viruses
Plasmid HCV cDNA Vector
Name sublcone used for
Name Construction insertion
Length
(nt/aa)
pvHCV-13A E1 s EcoR I - Hind III 472/157 pgptATA-18
pvHCV-12A Els EcoR I - Hind III 472/158 pgptATA-18
~!,
pvHCV-9A E1 EcoR I - Hind III 631/211 pgptATA-18
pvHCV-11 E 1 s EcoR I - Hind III 625/207 pgptATA-18
A
pvHCV-17A Els EcoR I - Hind III 625/208 pgptATA-18
pvHCV-l0A E1 EcoR I - Hind III 783/262 pgptATA-18
pvHCV-18A COREs Acc I (Kl) - EcoR 403/130 pgptATA-18
I (Kl)
pvHCV-34 CORE Acc I (Kl) - Fsp 595/197 pgptATA-18
I
pvHCV-33 CORE-E1 Acc I (Kl) 1150/380pgptATA-18
pvHCV-35 CORE-Elb.hisEcoR I - BamH I (Kl)1032/352pMS-66
pvHCV-36 CORE-Eln.hisEcoR I - Nco I (Kl) 1106/376pMS-66
pvHCV-37 E10 Xma I - BamH I 711/239 pvHCV-l0A
pvHCV-38 ElOs EcoR I - BstE II 553/183 pvHCV-1
lA
pvHCV-39 Elpb EcoR I - BamH I 960/313 pgsATA-18
pvHCV-40 El~b.his EcoR I - BamH I (Kl)960/323 pMS-66
pvHCV-41 E2bs BamH I (Kl)-AIwN 1005/331pgsATA-18
I (T4)
pvHCV-42 E2bs.his BamH I (Kl)-AIwN 1005/341pMS-66
I (T4)
pvHCV-43 E2ns Nco I (Kl) - AIwN 932/314 pgsATA-18
I (T4)
pvHCV-44 E2ns.his Nco I (Kl) - AIwN 932/321 pMS-66
I (T4)
pvHCV-62 Els (type EcoR I - Hind III 625/207 pgsATA-18
3a)
pvHCV-63 Els (type EcoR I - Hind III 625/207 pgsATA-18
5)
pvHCV-64 E2 BamH I - Hind III 1410/463pgsATA-18
pvHCV-65 El-E2 BamH I - Hind III 2072/691pvHCV-l0A
pvHCV-66 CORE-El-E2 BamH I - Hind III 2427/809pvHCV-33
nt: nucleotide; aa: amino acid; Kl: Klenow DNA Pol filling; T4: T4 DNA Pol
filling
Position: amino acid position in the HCV polyprotein sequence
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Table 1 - continued: Recombinant vaccinia plasmids and viruses
Plasmid HCV cDNA Vector
Name subclone used for
Name Construction insertion
Length
(nt/aa)
pvHCV-81 E1 *-GLY EcoRI - BamH I 783/262 pvHCV-l0A
1
pvHCV-82 E1 *-GLY EcoRI - BamH I 783/262 pvHCV-l0A
2
pvHCV-83 El *-GLY EcoRI - BamH I 783/262 pvHCV-l0A
3
pvHCV-84 E1 *-GLY EcoRI - BamH I 783/262 pvHCV-l0A
4
pvHCV-85 E1 *-GLY EcoRI - BamH I 783/262 pvHCV-1
5 OA
pvHCV-86 E1 *-GLY EcoRI - BamH I 783/262 pvHCV-l0A
6
nt: nucleotide; aa: amino acid; KI: Klenow DNA Pol filling; T4: T4 DNA Pol
filling
Position: amino acid position in the HCV polyprotein sequence
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Table 2 : Summary of anti-E1 tests
S/N + SD (mean anti-E1 titer)
Start of treatmentEnd of treatment Follow-up
LTR 6.94 + 2.29 (1:3946)4.48 + 2.69 (1:568)2.99 + 2.69 (1:175)
NR 5.77 + 3.77 (1:1607)5.29 + 3.99 (1:1060)6.08 + 3.73 (1:1978)
LTR : Long-term, sustained response for more than 1 year
NR : No response, response with relapse, or partial response
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Table 3 : Synthetic peptides for competition studies
PROTEIN PEPTIDE AMINO ACID SEQUENCE POSITION SEQ ID NO
E1 E1-31 LLSCLTVPASAYQVRNSTGL 181-200 56
E1-33 QVRNSTGLYHVTNDCPNSSI 193-212 57
E1-35 NDCPNSSIVYEAHDAILHTP 205-224 58
E1-35A SNSSIVYEAADMIMHTPGCV 208-227 59
E1-37 HDAILHTPGCVPCVREGNVS 217-236 60
E1-39 CVREGNVSRCWVAMTPTVAT 229-248 61
E1-41 AMTPTVATRDGKLPATQLRR 241-260 62
El-43 LPATQLRRHIDLLVGSATLC 253-272 63
E1-45 LVGSATLCSALYVGDLCGSV 265-284 64
E1-49 QLFTFSPRRHWTTQGCNCSI 289-308 65
E1-51 TQGCNCSIYPGHITGHRMAW 301-320 66
El-53 ITGHRMAWDMMMNWSPTAAL 313-332 67
E1-55 NWSPTAALVMAQLLRIPQAI 325-344 68
E1-57 LLRIPQAILDMIAGAHWGVL 337-356 69
E1-59 AGAHWGVLAGIAYFSMVGNM 349-368 70
E1-63 VVLLLFAGVDAETIVSGGQA 373-392 71
E2 E2-67 SGLVSLFTPGAKQNIQLINT 397-416 72
E2-69 QNIQLINTNGSWHINSTALN 409-428 73
E2-$3B LNCNESLNTGWWLAGLIYQHK 427-446 74
E2-$1B AGLIYQHKFNSSGCPERLAS 439-458 75
E2-1B GCPERLASCRPLTDFDQGWG 451-470 76
E2-3B TDFDQGWGPISYANGSGPDQ 463-482 77
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Table 3 - continued : Synthetic peptides for competition studies
E2-5B ANGSGPDQRPYCWHYPPKPC 475-494 78
E2-7B WHYPPKPCGIVPAKSVCGPV 487-506 79
E2-9B AKSVCGPVYCFTPSPVVVGT 499-518 80
E2-11B PSPVVVGTTDRSGAPTYSWG 511-530 81
E2-13B GAPTYSWGENDTDVFVLNNT 523-542 82
E2-17B GNWFGCTWMNSTGFTKVCGA 547-566 83
E2-19B GFTKVCGAPPVCIGGAGNNT 559-578 84
E2-21 IGGAGNNTLHCPTDCFRKHP 571-590 85
E2-23 TDCFRKHPDATYSRCGSGPW 583-602 86
E2-25 SRCGSGPWITPRCLVDYPYR 595-614 87
E2-27 CLVDYPYRLWHYPCTINYTI 607-626 88
E2-29 PCTINYTIFKIRMYVGGVEH 619-638 89
E2-31 MYVGGVEHRLEAACNWTPGE 631-650 90
E2-33 ACNWTPGERCDLEDRDRSEL 643-662 91
E2-35 EDRDRSELSPLLLTTTQWQV 655-674 92
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0
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CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
88
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CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
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CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
u v u u r-~w - c~
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th FC U C- mC H i - c''~U
-
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H H H C7 H U C7 U ~ U C7 U
cY7U U H U H U U C7 H H H U U
c~o I FC H Ch U H FC ~ C7 U C7 C7 H U
I U C7 U H C7 r.~U C7 FC C9 ~ C7 CJ E-~H
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H H U H A,'FC H ~C C7 H FC U U C7
~C U U ~ U Ch H U U C7 U C7 ~ U C7 U
C7 C7 H C7 C7 C7 U' U H ~C CJ U U FC U o
H CJ H H FC U C7 C~ C7 C7 C7 FC H C7 H C7
FC U FC ~ U ~C C7 C.7H H H U FC C7 ~C U
U C7 U ~7 C7 U ~C FC FC U U U U U U
C7 H r~ U C~ C7 U H U H C7 ~C r.C ~ x
H C7 H r.~U C7 U C7 U C7 H U U ~ H U
U FC C~ U C7 C~ C7 H r~ H U U U U H H
r~ C7 C7 FC ~C H CJ H H C.JC7 U C~ ~ r.~U 'bp
U U H H U C7 H C7 C7 H U U U H C7 C7 'o
U H ~ r.~C7 C7 C7 U H H U H Ch U U U
FC U U FC H H U FC U C7 U U E-ir~
H U FC H U U FC C7 ~ U H U U C7 ~C H
U C7 U U U H C7 C7 U U U U U' H FC o
H U H U U H H U C7 H C9 H ~C ~ U U Q.,
H H C7 C7 r~ C7 C7 r.>rU U ~ U H C7 H U ~
U C7 U H H H U U U C7 ( U C7 E- W7
I I I I I I I I I I I U I I I I ~ ~.
- _ - - _ _ - - _ _ - I _ - - -
-
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cr7~ u7 to v-IN M ~ ~ lflI Irx
f~'f~'fy 0.ifx fy H
r-IN a a a a ~ ~ ~ ~ ~ ~ W Y,
w x a a ~ '~ ~ '~ ~ o o o o o c~ H ',
C7 H C7 C7
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v~ ~, r.
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
O O O O O O O O O O O O O O O O
z z z z z z z z z z z z z z z z b b
n ~ ~ a n ~ ~ n ~ ~ ~ ~ ca ~ ca n
H H H H H H H H H H H H H H H H
a~ O O
a of of of o~ a o~ o~ of of o~ of a of a of
ee
H ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ L~ ~ ~ ~ O
L7
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N
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91
Table 8. Analysis of E1 glycosylation mutants by ELISA
M M N t~h ~D~O p~ h h h
O~h N O M h ~ h M ~ O O ~O
N V10101~ pp~ M M ~ -~,-~O~
O ooN O ood'~ r.,v1~ v~N
M ooO ~ O O D h ~ h ~ N C~Ov
h ~D~Dh h h 00 ~O~Oh ~t~ ~
-,M ~ -... ~Oh Vi~OSrii h
. . ' r
W
M ~ h ~O~OV7V1
~
M h OvN ~D0100 .--~N W M ~ -~~t N o0~tt~~ h O~
0oh O~~ M o0Q\ N O~O ~ N O ~r1~ N .-.~ O O O .
M ~ ~ h h M 00N M M V~N h ~ ~OV1Ov
~
~OV7N 01~D~ ~Y ~ I~M V1~'~ M O w 01O 00h 00V100
~Dp o0~nh O~V~ .~M ~ O v~~Oo0 ~ Own N oo~1.
0ov1~ O~v1d;0\ M O W ~O~ M 01~ N N N d'N N M
1
.~r ~ M ~ .-~r. N N N U1N N N
~ M N oo~ ~-00
~nN ~ M v~Ovo00oN V1~ ~ M ~ h h ,-,oo~O
' ~ O
~ O N ~nN OvV1 O ~tN Ov.-~ooM ~ n N o0~ W ~ O
M M 01h ,~~Dh ~p-~~D~Od'O~M M ~ 00V101~ ~ON
~
h O ~D~O00OvO h ,~V1~1~!1'~t00N O ~ 00~OO ~jN M
~n~ ~OOvM N o0 0ov1d'00M h o0 OvOvN O Ov.--~O
Oy~ O~- ,~O N OvO OWO M v>N ~n~Ov0~--~~O~Oh
N ~ ~'N N N ~ M N ~ M N M
N ~n ~n-~M
M M N v1t~00 0000Ovo0.~~t~ Ovo0~Do0~ N N
O~~ N ~ON N O~N O ~O~ O ~O ~ 01h h ~1N ~ ~
OvOvh O h O ~ ~ ~ OvQ ~ ~ h N N v0N M .-.oov0N
~OM M M ~ ~ ~ r.,M ~ ~Oh ~ ~ (~ O M O 00M ~ 00
~D~ M ~ ~Ooo~O ~ON ~ O N N ~ h ~ON ~ ~Oh h
0oO ooh O~h o0 h ~OO t~~ ~OO
r~.~~ N ,-,.--~.
N ~ d'~ ~'~'~ M M M ~ M N M
~ ~tV7 ~ N M
.~Q100 ~ M 01 ~ M V7N ~'N r., ~ '~h M O ~OM
-i ~--~M ('~ O ~ V'1N M V1h M ooO\h --~01O M
O oo M oo~n ~ v0~OO ~ M Ovh N o0.~~ON h v1M
O 01~ V7~ ~ ,~M h M ~ O 01M V7~OM 00.~h 00
N ~ 01~ .~01M M ~ ~ ~Od'Q100 .-r~OM ~O00~ O
d'~1,-.h d'r. N v~h v101~ ~ .
c~j.-~ ri
N N ~ M ~ N M M N N ~ON N M
l~000oh o0~ ~ ~ N o0~Do0.-.N o0,-, ~ ~OM ~O
OvO ~ oo~nN oo ~OooM ~ O ~O,- ~ 00h ~ ~n~to0
~ M h M M ~ 00 ~ ~OV100'~~ ~ N h h ~ ~ M
M p p O h
v~01~ Ovh h O v1~OO ~DM v1v1 N p
r., oo~nh OWn o0~O
O M V~~ N N Ov o0~nM l~ooO N
,~,~p M ,~N ,
N ~OM ~ ~ ulh ~OV1~ -ro0O o0 ,
-~N N ~ N N N ~ I~h o0000000 ~'""'O N
~OV't~ M N M d'~'M ~ ~p M ~-'M 00 M
l~O~V'O d'~ Ov O N ~D~ ooN o0 M O M M h ~OM
0oW O ~OM ~Ot~- N '~t~nr -~h N oo '~h _ ~ O
o O o M
M ~ ,~~tO~r.N ~ OvO --~v1N ~ M N o 0
, h o0~DO~~ oov1
O N ~ h O M ~ N I~N ~nh d-O~
M ~pN N ~Oh M
~ M N o0~ .-~v~ ~OO ('~OvO h v~
~ N N M N N N ~ N '--~M N ~ N ~ ~ ~ ~ ~ N
.-, h o0.~h ~D ~ N ~D~ N o0 0od'V~,-~M o0
h 00h M ~OM M V100~ 00O~O ~ ~ 0000M ~OM ~
Ov~ ~ O h h O h N M v'1h ~ O
O
N O o0~n~ M v~h ~ O ~ ~ O ~ ~ h N -~,~h N v0~no0
N ~ ~ N 01O~N N ~O~OV7~ON ~ h N ~ O N ~OI~
h h o0h ~tN N d'~ N v1v Wn ~ O wnO ~ ~O~D
N ~ ~ M ~ .~N r:,~r-.~ ,~.-~,~ N N N M M N cn
N ~no0'~~ h V1 N N 00,~h h N N
p ~ 00
01.~l!7V~00O ~D~ ~f1d'O ~ N ~O~ 00N
h h ,~O ~ N O --~N V10000h 00 p~h h ~-'M 00h N
~'N O N o0N ~ 00 ,~00N ~ O ~ h ~D ~ ~ h O M N ~D
O O ~ t~ooM N ~Do0O~h N h - M N o0~ ~ O h
0od y0v~~tO o0 d'~ ~ ,~O ~O~O O~,~O~ood'V~h
~ N ~ N N N N N N N V1M N N ~ N ~ M N ~ N
N M d'~1~ .-rN M ~i'~ ~ ~ N M ~ u1~O
a a a a w a ~ a a a w a a ~ a a a a a a ~
C7C7c7c7c7c7w c7W 7 c7c7c7w c7t7c7c7c7c7w
w z z z z z z z z z z z z z z z z z z z z z
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Table 8 (continued). Analysis of E1 glycosylation mutants by ELISA
,mn oo~t ~ o~o\
~OM 00V70001 ~ 00N N O
N d'M O ~ N o0~--~N O v0.-N
00~ I~~ ~Od' -~00d'M ~ ~ N
~ M 00V7~ ~ N ~OO 01V1N
OvOvo0O OvD\ OvO o000l~t~
O O O N O O O '--rO O O O
~
00N ~ ~ ~Ot~N l~~ U" .,V1I~l~ M 00
''
~ M ~ l~~ 00 ~DI~~D00l~\O 00l~~O0000M
' ~ a
Q1~ d ~ ~Ol ~ ~DM 01V1V7O~ 00O 01O I~V1
~
~ ~O00~D~Ov7 ~ l~~ M O O N (7~OM O ~OI~~D
V~~ v~N O ~O t~00~ 00OvOvW ~ O O O~~ O
Ovo0t~O oot~ t~Ovo0000ot~ ~'00O W v)O~00
~
O O O N O O O O O ~ O O ~ W O O O .~O O
~O ~ I~,--~~ M VI~ON t~~t ~,~t~t'~ N Ov
I~~D~ M ~ ~nO v0O ~noo N ooN O W Ov
a ~
V1~ [~M O N ~D~ V1014100~ ~ ~ M O~V7
N o001~!1N M ,-,~!1.~~ N l~~ (7~Ot~Cno000Ov
V7M V1M M 00 O M ~ M ~ 00~ ~ M \O~ M
00Qy00000100 ~OO~00l~O I~C/~ ~' .
O O O ~ O O O O O ~ ~ O W ~ N ~ M N
~ M M Q1N N ooM O~M ~Dt~ 0000~ ooN Ov
M M ~ N 00M ~ M O M N 01
I~01I~~ ~OV7 V70100M 00O t~ ch~ 01M ~
00V1N t~OvN --W O ~
p
. M O Ov~ N l ~ ~ N Q\N
00M 01~D~ 00 N 0000~ .-~~ ~ .~I~p~,~~D
~nO 01Q\O Ov N ~ Ovo0O o0 01O WOM OvOv
O ~ O O ~ O ~ ~ O ~ ~ O O O O ~ O O
~ M N N ~DU N O W N 00l~ I~Q1N N M
O
M M ~ 00~ lp ~ \OV7O M .-~ ~p..~V)N O~M
O N M o0I~~ ~ ~O~ 0000~1N M N l~~O~ ~nO
I~~ 00V7I~~O .~V)~pl~O M ~ N ~Ol~~ (~N 00
I~00O O d'a1 ~ O ~D~ON o0 M 01W O l~O
~OI~v~O v~t~ O o00oO a\I~ O two l~00l~
O O O -~O O ~ O O N O O ~ O O ~ O O
l~ ~ 00 00 M \Od' ~D 00~hM l~
t~OvM V'O o0 ~tt~M ~ O M 00~DV1~ON Ov
C~~Ot~N ~ t~ M N N ~ ~ ~O~ N M o0V7O M ~
,~v~M l~~ ~r1 ,~~ ~Ooo~O~Dl~ N o0~ l~O oot~
M d'~ M ~ O cY~ O~N O O ~ 00M ~ 00O~
~ 0100~ O~O~ ~O0000Q1O O~ O Q100O O~O
O O O O O O O O O O ~ O ~ O O N O ~
M M N -rv1 ~ N t~00N ~n ~ ~ ~ M N ~
O~~O~ V~M O l~M d'a1O M ~n~D~ t~~ 01
~O~ O CnooM N ~ N vW~ ~ ~t ~ t~O N t~~ M
00V7O ~ 0000 .-~0000M ~tO~N N l~d'l~~ t~Q1
v1N O ~ ~n~ N OWD N OW~ ~ l~Q\O
V101Ov~ D\00 ~Ol~~D~ t~~D 0000l~0000~
O O O ~ O O O O O ~ O O O O O O O O
~Ot~l~~ N ~ d' 0000 M N ~OV1Q1M
t~~ 0101~tN o0V7Ovo0~to0 ~-~ O M O o0
N M OvN O d'~ ~ 00~tN t~~ N O O N M M w 0
N M O t~~n.~ .-.M N O OvM N N N ~ V1~DO ~
v~OWE ~ O t~ ood'~ N O o0 t~OvOv,-~~nN
Q1t~l~t~00~O (~O~D1t~O O~ ~Ot~~ 0000t~
O O O ~ O O O O O N ~ O O O O O O O
~O~D~ l~l~~O o0 N ~O N d-~1~ N
- l~M ooO Q\ D\~ oo ~Ooo v0O W ~1'~ O
1
M o000~WO N O ~ v~~ t~N Ov~ l~v~O ~ t~
t~00O ~ (~00 .~M 00t~~ ~ M ~ 00l~d'~p~ M
M cf'00.-rt~,-.r ~ d'M t~U1N O~~ ~ t~00
~O00V)~ 00t~ 01Ov00Ov,--~O ~ l~t~M 00~
O O O O O O O O O ~ ~ ~ O O O .-~O O
w w w w w w w w w w w w w
N M d'~ ~ .-~N M ~ V1~O ~ N M ~ VWp
a ~ a a a a a a .~a a a a a a a a a
c7c7c7c7C7c7 c7c7c~c7c7c7 c7c7c~C~c~~
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Table 9. Profile of adjuvated El in Balb/c mice
alum T-cell adjuvantRBI
antibody titre 96000 ~ 101000 62000 ~ 176000 ~ 149000
(mean ~ 60000
SD, n=6)
antibody isotypesIgG 1 IgG 1 /2b IgG 1 /2a
T-cell preliferation11750 (2/3) 48300 (3/3)26000 (3/3)
in
spleen' (n=3)
T-cell proliferationno specific stimulation4000 8000
in
lymph nodez
cytokine profile Il-4 IFN-g/Il-4 IFN-g/Il-4
(spleen)
' after three s.c./i.m. immunizations, 3 randomly selected mice were analyzed
individually,
the result is expressed as the mean specific cpm obtained after 4 days of E1
stimulation (1
pg/ml), the number in brackets refers to the number of mice with specific
stimulation
above background
2 after one single intra footpath immunization (n=2), the result is expressed
as the mean
specific cpm obtained after 5 days of El stimulation (1 pg/ml)
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Table 10. Humoral Responses: No. of immunizations required for different E-1
antibodies levels
Animal status seroconversion'> 1 U/m12 > 10 U/ml
Marcel chronic 3 4 5
Peggy chronic 3 5 >12
Femma chronic 4 5 >12
Yoran naive 3 4 5
Marti naive 2 3 S
defined as ELISA signal higher than cut-off level if no E1-antibodies were
present prior to
immunization, in the other cases the observation of a titer higher than the 3
individual time
points of pre-immunization titers was considered as the point of
seroconversion.
Z the unit is defined as follows: the level of E1 antibodies in human chronic
carriers prior to
interferon therapy and infected with genotype lb is < 0.1 U/ml for 50% of the
patients,
between 0.1 to 1 U/ml for 25% of the patients and > 1 U/ml in the remaining
25% of
patients, n=58
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Example 12: Immunization of a chimpanzee chronically infected with HCV subtype
lb
A chimpanzee (Phil) already infected for over 13 years (5015 days before
immunization)
5 with an HCV subtype Ib strain was vaccinated with El (aa 192-326) which was
derived
from a different strain of genotype 1 b, with a 95.1 % identity on the amino
acid level (see
also Table 2 of WO 99/67285 the whole of which is incorporated herein by
reference), and
which was prepared as described in examples 1-3 of WO 99/97285. The chimpanzee
received in total 6 intramuscular immunizations of each 50 pg E1 in PBS/0.05%
CHAPS
10 mixed with RIBI R-730 (MPLA+TDM+CWS) according to the manufacturer's
protocol
(Ribi Inc. Hamilton, MT). The 6 immunizations were given in two series of
three shots
with a three week interval and with a lag period of 6 weeks between the two
series. Starting
150 days prior to immunization, during the immunization period and until 1
year post
immunization (but see below and WO 99/67285) the chimpanzee was continuously
15 monitored for various parameters indicative for the activity.of the HCV
induced disease.
These parameters included blood chemistry, ALT ,AST, gammaGT, blood
chemistry, viral load in the serum, viral load in the liver and liver
histology. In addition,
the immune answer to the immunization was monitored both on the humoral and
cellular
level. During this period the animal was also monitored for any adverse
effects of the
2 0 immunization, such as change in behaviour, clinical symptoms, body weight,
temperature
and local reactions (redness, swelling, indurations). Such effects were not
detected.
Clearly, ALT (and especially gammaGT, data not shown) levels decreased as soon
as the
antibody level against El reached its maximum (see, Figure 8 of WO 99/67285).
ALT
2 5 rebounded rather rapidly as soon as the antibody levels started to
decline, but gammaGT
remained at a lower level as long as anti-El remained detectable.
E2 antigen in the liver decreased to almost undetectable levels during the
period in which
anti-El was detectable and the E2 antigen rebounded shortly after the
disappearance of
3 0 these antibodies. Together with the Core and E2 antigen becoming
undetectable in the
liver, the inflammation of the liver markedly decreased (see also Table 3 of
WO 99/67285).
This is a major proof that the vaccine induces a reduction of the liver
damage, probably by
clearing, at least partially, the viral antigens from its major target organ,
the liver .
The viraemia level, as measured by Amplicor HCV Monitor (Roche, Basel,
Switzerland),
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remained approximately unchanged in the serum during the whole study period.
More detailed analyses of the humoral response revealed that the maximum end-
point titer
reached 14.5 x 103 (after the sixth immunization) and that this titer dropped
to undetectable
1 year post immunization (Figure 8 of WO 99/67285). Figure 9 of WO 99/67285
shows
that the main epitopes, which can be mimicked by peptides, recognized by the B-
cells are
located at the N-terminal region of E2 (peptides V 1 V2 and V2V3, for details
on the
peptides used see Table 4 of WO 99/67285). Since the reactivity against the
recombinant
El is higher and longer lasting, it can also be deduced from this figure, that
the antibodies
recognizing these peptides represent only part of the total antibody
population against El.
The remaining part is directed against epitopes which cannot be mimicked by
peptides, i.e discontinuous epitopes. Such epitopes are only present on the
complete E1
molecule or even only on the particle-like structure. Such an immune response
against EI is
unique, at least compared to what is normally observed in human chronic HCV
carriers
(WO 96/13590 to Maertens et al.) and in chimpanzees (van Doorn et al., 1996),
who raise
anti-E1 antibodies in their natural course of infection. In those patients,
anti-E1 is in part
also directed to discontinuous epitopes but a large proportion is directed
against the C4
epitope (~50% of the patient sera), a minor proportion against V 1 V2 (ranging
from 2-70%
depending on the genotype), and reactivity against V2V3 was only exceptionally
recorded
2 0 (Maertens et al., 1997).
Analysis of the T-cell reactivity indicated that also this compartment of the
immune system
is stimulated by the vaccine in a specific way, as the stimulation index of
these T-cells rises
from 1 to 2.5, and remains somewhat elevated during the follow up period
(Figure 10 of
2 5 WO 99/67285). It is this T cell reactivity that is only seen in Long term
responders to
interferon therapy (see: PCT/EP 94/03555 to Leroux-Roels et al.;Leroux-Roels
et al.,
1996).
Example 13: Immunization of a chronic HCV carrier with different sub pe
30 A chimpanzee (Ton) already infected for over 10 years (3809 days before
immunization)
with HCV from genotype la was vaccinated with El from genotype lb, with only a
79.3
identity on the amino acid level (see also Table 2 of WO 99/67285), and
prepared as
described in the previous examples. The chimpanzee received a total of 6
intramuscular
immunizations of 50 p.g El in PBS/0.05% CHAPS each mixed with RIBI R-730
according
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to the manufacturer's protocol (Ribi Inc. Hamilton, MT). The 6 immunizations
were given
in two series of three shots with a three week interval and with a lag period
of 4 weeks
between the two series. Starting 250 days prior to immunization, during the
immunization
period and until 9 months (but see below and WO 99/67285) post immunization
the
chimpanzee was continuously monitored for various parameters indicative for
the activity
of the HCV induced disease. These parameters included blood chemistry, ALT,
AST,
gammaGT, viral load in the serum, viral load in the liver and liver histology.
In addition,
the immune answer to the immunization was monitored both on the humoral and
cellular
level. During this period the animal was also monitored for any adverse
effects of the
immunization, such as change in behaviour, clinical symptoms, body weight,
temperature
and local reactions (redness, swelling, indurations). Such effects were not
detected.
Clearly, ALT levels (and gammaGT levels, data not shown) decreased as soon as
the
antibody level against El reached its maximum (Figure 11 of WO 99/67285). ALT
and
gammaGT rebounded as soon as the antibody levels started to decline, but ALT
and
gammaGT remained at a lower level during the complete follow up period. ALT
levels
were even significantly reduced after vaccination (62 ~ 6 U/1) as compared to
the period
before vaccination (85 ~ 11 U/1). Since less markers of tissue damage were
recovered in
the serum, these findings were a first indication that the vaccination induced
an
improvement of the liver disease.
E2 antigen levels became undetectable in the period in which anti-El remained
above a
titer of 1.0 x 103, but became detectable again at the time of lower El
antibody levels.
Together with the disappearance of HCV antigens, the inflammation of the liver
markedly
decreased from moderate chronic active hepatitis to minimal forms of chronic
persistent
2 5 hepatitis (Table 3 of WO 99/67285). This is another major proof that the
vaccine induces a
reduction of the liver damage, probably by clearing, at least partially, the
virus from its
major target organ, the liver .
The viraemia level, as measured by Amplicor HCV Monitor (Roche, Basel,
Switzerland),
3 0 in the serum remained at approximately similar levels during the whole
study period. More
detailed analysis of the humoral response revealed that the maximum end-point
titer
reached was 30 x 103 (after the sixth immunization) and that this titer
dropped to 0.5 x 103
nine months after immunization (Figure 11 of WO 99/67285). Figure 12 of WO
99/67285
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shows that the main epitopes, which can be mimicked by peptides and are
recognized by
the B-cells, are located at the N-terminal region (peptides V 1 V2 and V2V3,
for details on
the peptides used see Table 4 of WO 99/67285). Since the reactivity against
the
recombinant El is higher and longer lasting, it can also be deduced from this
figure, that the
antibodies recognizing these peptides represent only part of the total
antibody population
against El. The remaining part is most likely directed against epitopes which
cannot be
mimicked by peptides, i.e. discontinuous epitopes. Such epitopes are probably
only present
on the complete E1 molecule or even only on the particle-like structure. Such
an immune
response against El is unique, at least compared to what is normally observed
in human
chronic HCV carriers, which have detectable anti-El. In those patients, anti-
E1 is in part
also discontinuous, but a large proportion is directed against he C4 epitope
(50% of the
patient sera), a minor proportion against V I V2 (ranging from 2-70% depending
on the
genotype) and exceptionally reactivity against V2V3 was recorded (Maertens et
al., 1997).
As this chimpanzee is infected with an la isolate the antibody response was
also evaluated
for cross-reactivity towards a El-la antigen. As can be seen in Figure 13 of
WO 99/67285,
such cross-reactive antibodies are indeed generated, although, they form only
part of the
total antibody population. Remarkable is the correlation between the
reappearance of
viral antigen in the liver and the disappearance of detectable anti-la El
antibodies in the
serum.
Analysis of the T-cell reactivity indicated that also this compartment of the
immune system
is stimulated by the vaccine in a specific way, as the stimulation index of
these T-cells rises
from 0.5 to 5, and remains elevated during the follow up period (Figure 14 of
WO
99/67285).
Example 14: Reboosting of HCV chronic carriers with El
As the El antibody titers as observed in examples 12 and 13 were not stable
and declined
over time, even to undetectable levels for the lb infected chimp, it was
investigated if this
antibody response could be increased again by additional boosting. Both
chimpanzees were
3 0 immunized again with three consecutive intramuscular immunization with a
three week
interval (50 ~g El mixed with RIBI adjuvant). As can be judged from Figures 8
and 11 of
WO 99/67285, the anti-El response could indeed be boosted, once again the
viral antigen in
the liver decreased below detection limit. The viral load in the serum
remained constant
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although in Ton (Figure 11 of WO 99/67285). A viremia level of < 105 genome
equivalents
per ml was measured for the first time during the follow up period.
Notable is the finding that, as was already the case for the first series of
immunizations, the
chimpanzee infected with the subtype lb HCV strain (Phil) responds with lower
anti-El
titers, than the chimpanzee infected with subtype la HCV strain (maximum titer
in the first
round 14.5 x 10' versus 30 x 103 for Ton and after additional boosting only
1.2 x 103 for
Phil versus 40 x 103 for Ton). Although for both animals the beneficial effect
seems to be
similar, it could be concluded from this experiment that immunization of a
chronic carrier
with an El protein derived from another subtype or genotype may be especially
beneficial
to reach higher titers, maybe circumventing a preexisting and specific immune
suppression
existing in the host and induced by the infecting subtype or genotype.
Alternatively, the
lower titers observed in the homologous setting (lb vaccine +lb infection) may
indicate
binding of the bulk of the antibodies to virus. Therefore, the induced
antibodies may
possess neutralizing capacity .
Example 15: Demonstration of prophylactic utility of E1-vaccination in
chimpanzee
The HCV Els protein (amino acids 192-326) was expressed in Vero cells using
recombinant vaccinia virus HCV11B. This vaccinia virus is essentially
identical to
2 0 vvHCV 11 A (as described in U.S. Patent No. 6, I 50,134, the entire
contents of which is
hereby incorporated by reference) but has been passaged from RK13 to Vero
cells. The
protein was purified (by means of lentil chromatography, reduction-alkylation
and size
exclusion chromatography) essentially as described in example 9 of
PCT/E99/04342
(WO 99/67285), making use of iodoacetamide as alkylating agent for the
cysteines. After
2 5 purification the 3% empigen-BB was exchanged to 3% betain by size
exclusion
chromatography as described in example 1 of PCT/E99/04342 this process allows
to
recover Els as a particle. Finally the material was desalted to PBS containing
0.5% betain
and an Els concentration of 500 pg/ml. This El was mixed with an equal volume
of
Alhydrogel 1.3% (Superfos, Denmark) and finally further diluted with 8 volumes
of 0.9%
30 NaCI to yield alum-adjuvanted El at a concentration of 50 ~g El/ml and
0.13% of
Alhydrogel.
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The HCV E2deltaHVRI (amino acids 412-715) was expressed in and purified from
Vero
essentially as described for El using recombinant vaccinia virus HCV101 which
has been
recombined from pvHCV-101 described in Example 8 of PCT/E99/04342 and wild
type
vaccinia virus. Also E2deltaHVRI behaves as a particle (measured by dynamic
light
scattering) after exchange of empigen to betain.
Five chimpanzees were selected which tested negative for HCV-RNA and HCV-
antibodies. One of the animals (Huub) was not immunized, 2 animals received 6
immunizations with 50 ~g El adjuvanted with alum (Marti and Yoran) while the
remaining
2 animals received 6 immunizations with 50 ~.g E2deltaHVRI adjuvanted with
alum (Joost
and Karlien). All immunizations were administered intra-muscularly with a 3
week
interval. Humoral and cellular immune responses were assessed in each animal
against the
antigen with which they where immunized and in each animal both type of
responses was
detected as shown in Table 11.
All chimpanzees became HCV-RNA positive (determined with Monitor HCV, Roche,
Basel, Switzerland) on day 7 post challenge and a first ALT and gammaGT peak
was
measured between days 35 and 63. This evidences that all chimps developed
acute
hepatitis. Remarkably, both El immunized animals resolved their infection
while the
2 0 E2deltaHVRI and the control animal did not. This is evidenced by the fact
that the El
immunized animals lost HCV-RNA (determined with Monitor HCV, Roche, Basel,
Switzerland) at day 98 (Yoran) and 133 (Marti) and remained negative so far
until day 273
with monthly testing. All the other animals stayed RNA-positive during the
entire
follow up period of 273 days so far with ALT and gammaGT values not returning
to
2 5 normal as for the El immunized chimpanzees but gradually increasing.
Table 11: antibody titers were determined by ELISA two weeks after the 6"'
immunization.
A serial dilution of the sample was compared to an in house standard (this in
house
standard defined as having 1000 mU/ml of El or anti-E2deltaHVR I antibody is a
mixture
3 0 of three sera from HCV chronic carriers selected based on a high anti-
envelope titer). The
stimulation index, which reflects the cellular immune response, was obtained
by culturing
PBMC, drawn from the animals two weeks after the third immunization, in the
presence or
absence of envelope antigen and determining the amount of tritiated thymidine
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incorporated in these cells during a pulse of 18 hours after 5 days of
culture. The
stimulation index is the ratio of thymidine incorporated in the cells cultured
with envelope
antigen versus the ones cultured without antigen. A stimulation index of >3 is
considered a
positive signal.
Chimpanzee Anti-E1 response Anti-E2deltaHVRI
response
Antibody StimulationAntibody titerStimulation
titer index Index
Yoran 14110 10.9
Marti 5630 14.2
Joost 3210 8.5
Karlien 1770 11.2
Three weeks after the last of 6 immunizations all animals including the
control were
challenged with 100 CID (chimpanzee infectious doses) of a genotype lb
inoculum (J4.91,
kindly provided by Dr. J. Bukh, NIH, Bethesda, Maryland). The amino acid
sequence
divergence between the vaccine proteins and the J4.91 isolate (of which the
sequence
information is available under accession number BAA01583) is 7% (9 out of 135
amino
acids) for Els and 11% (32 out of 304 amino acids) for E2delta HVRI;
Consequently this
challenge is considered heterologous and reflects a real life challenge.
In conclusion we have shown that El-immunization changes the natural history
of HCV
infection by preventing evolution to a chronic infection, which is the major
health problem
related with HCV.
Example 16: Similar EI responses which allowed clearing of infection in
chimpanzee
2 0 can be induced in man
In order to obtain a prophylactic effect of El immunization in man it is
required that similar
immune responses can be induced in man compared to chimpanzee. Therefore we
vaccinated 20 male human volunteers, in which no anti-El responses (humoral or
cellular)
could be detected, with 3 doses of 20 pg Els formulated on 0.13% Alhydrogel in
0.5 ml.
2 5 All immunizations were given intramuscularly with a 3 week interval. As
evidenced in
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Table 12, 17 out of 20 volunteers indeed mounted a significant humoral and
cellular
immune response against El and this without serious adverse events. Only 1
volunteer
(subject 021) should be considered as a non-responder since neither humoral
nor cellular
responses were above the cut-off level after 3 El immunizations. The
observation that the
humoral response is lower compared to chimpanzee relates to the fact that only
3 immunizations with 20 ~g were given and not 6 with SO pg.
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Table 12: antibody titers were determined by ELISA two weeks after the third
immunization. A serial dilution of the sample was compared to an in house
standards (this
in house standard defined as having 1000 mU/ml of El or anti-E2deltaHVR I
antibody is a
mixture of three sera from HCV chronic carriers selected based on a high anti-
envelope
titer). The stimulation index (cellular immune response) was obtained by
culturing PBMC,
drawn from the individuals two weeks after the third immunization, in the
presence or
absence of 1 ~tg of Els and determining the amount of tritiated thymidine
incorporated in
these cells during a pulse of 18 hours after 5 days of culture. The
stimulation index is the
ratio of thymidine incorporated in the cells cultured with envelope antigen
versus the ones
cultured without antigen. A stimulation index of >3 is considered a positive
signal.
SubjectAntibody StimulationSubjectAntibody Stimulation
no titer index no titer index
002 1370 30.9 014 49 4.6
003 717 13.2 O15 228 3.8
004 800 9.1 016 324 4.1
007 680 3.8 017 <20* 6.2
008 1026 3.9 018 <20 6.7
009 325 4.6 019 624 3.1
010 898 7.7 020 84 S.5
011 284 4.1 021 <20 2.1
012 181 3.6 022 226 2.7
013 <20 3.5 023 163 7.6
*this individual is considered anti-E1 positive after immunization since a
significant
increase in ELISA signal was seen between the preimmune sample and the sample
after
three immunization, the titer however is very low and does not allow accurate
determination.
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Example 17: Boosting of E1 responses in vaccinated healthy volunteers
19 out of the 20 human volunteers of example 16 were boosted once more with 20
pg Els
formulated on 0.13% Alhydrogel in 0.5 ml at week 26 (i.e. 20 weeks after the
third
immunization). Again antibody titers and cellular immune responses were
determined 2
weeks after this additional immunization. In all individuals the antibody
titer had decreased
during the 20 week interval but could easily be boosted by this additional
immunization to
a level equal or higher of that observed at week 8. On average the antibody
titer was double
as high after this boost compared to the week 8 titer, and 7 times as high
compared to the
week 26 titer (Table 13).
Remarkably the T-cell responses were for the majority of individuals still
high after the 20
week interval. Taking in account a normalization to the tetanos response,
which is present
in most individuals as a consequence of previous vaccinations, there is no
change in the
geomeatric mean of the stimulation index. After the additional boost, taking
in account a
normalization to the tetanos response, no change is noted (figure 51). This
confirms that a
strong T-help response was induced after 3 E 1 immunizations and indicates
that these
immunizations induced already a very good T-help memory which requires, at
leeast for a
period of 6 months, no further boosting.
25
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Table 13: antibody titers were determined by ELISA two weeks (= week 8) and 20
weeks
(= week 26) after the third immunization and finally also 2 weeks after the
boost (= Week
28). A serial dilution of the sample was compared to an in house standards
(this in house
standard defined as having 1000 mU/ml of E1 antibody, is a mixture of three
sera from
HCV chronic Garners selected based on a high anti-envelope titer). For
accurate
comparison the determination of the titer at week 8 was repeated within the
same assay as
for the week 26 and 28 samples, which explains the differences with table 12
of example
16.
Subject Antibody titer
no
Week 8 Week 26 Week 28
002 1471 443 3119
003 963. 95 2355
004 1006 409 2043
007 630 65 541
008 926 81 819
009 704 77 269
010 1296 657 3773
011 253 65 368
012 254 148 760
013 36 <20 166
014 53 40 123
016 159 45 231
017 109 39 568
018 43 23 50
019 425 157 1894
020 73 33 113
021 25 <20 26
022 280 150 357
024 177 81 184
average 467 138 936
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Legend to figure 51: The stimulation index (cellular immune response) was
obtained by
culturing PBMC (105 cells), drawn from the individuals before immunization
(week 0),
two weeks after the third immunization (week 8), before the booster
immunization (week
26) and two weeks after the booster immunization (week 28), in the presence or
absence of
3 pg of recombinant Els or 2 pg tetanos toxoid and determining the amount of
tritiated
thymidine incorporated in these cells during a pulse of 18 hours after 5 days
of culture. The
stimulation index is the ratio of thymidine incorporated in the cells cultured
with envelope
antigen versus the ones cultured without antigen. Samples of week 0 and 8 were
determined in a first assay (A), while the samples of week 26 and 28 were
determined in a
second assay (B) in which the samples of week 0 were reanalyzed. Results are
expressed as
the geometric mean stimulation index of all 20 (A, experiment) or 19 (B,
experiment)
volunteers.
In addition the Thl cytokine interferon-gamma and Th2 cytokine interleukin-5
were
measured in the supernatants of the PBMC cultures of samples taken at week 26
and 28
and restimulated with El. As can be judged from figure 52 the predominant
cytokine
secreted by the E1 stimulated PBMC is interferon-gamma. It is highly
surprising to see that
a strong Thl biased response is observed with an alum adjuvanted El, since
alum is known
to be a Th2 inducer. Once more the results confirm that a good T-cell memory
response is
2 0 induced, as prior to the final boost (week 26) already a very strong
response is observed.
The interferon-gamma secretion was found to be specific as in an additional
experiment we
saw no difference in interferon-gamma secretion between E 1 stimulated cell
cultures and
non-stimulated cell cultures of these volunteers using samples drawn at week
0.
Legend to figure 52: PBMC (105 cells), drawn from the individuals before the
booster
immunization (week 26) and two weeks after the booster immunization (week 28),
were
cultured in the presence of 3 pg of recombinant Els (E1) or 2 pg of tetanos
toxoid (TT) or
no antigen (Bl). Cytokines were measured in the supernatant taken after 24
hours
(interleukin-5) or after 120 hours (interferon-gamma) by means of ELISA. The
stimulation
3 0 index is the ratio of cytokine measured in the supernatants of cells
cultured with envelope
antigen versus the ones cultured without antigen. Results are expressed as the
geometric
mean of pg cytokine/ml secreted of all 19 volunteers. Samples with a cytokine
amount
below detection limit were assigned the value of the detection limit.
Similarly samples with
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extremely high concentrations of cytokine out of the linear range of the assay
were
assigned the value of the limit of the linear range of the assay.
Example 18: Fine mapping of cellular response against E1 in vaccinated healthy
volunteers.
In order to map the E1 specific responses a series of 20-mer peptides was
synthesized,
using standard Fmoc chemistry, with 8 amino acids overlap and covering the
entire
sequence of Els. All peptides were C-terminally amidated and N-terminally
acetylated,
with the exception of IGP 1626 which has a free amino-terminus.
IGP1626: YEVRNVSGIYHVTNDCSNSS (amino acid 192-211)(SEQ ID N0:112)
IGP1627: TNDCSNSSIVYEAADMIMHT (amino acid 204-223)(SEQ ID N0:113)
IGP1628: AADMIMHTPGCVPCVRENNS (amino acid 216-235)(SEQ ID N0:114)
IGP1629: PCVRENNSSRCWVALTPTLA (amino acid 228-247)(SEQ ID NO:115)
IGP1630: VALTPTLAARNASVPTTTIR (amino acid 240-259)(SEQ ID N0:116)
IGP1631: SVPTTTIRRHVDLLVGAAAF (amino acid 252-271)(SEQ ID N0:117)
IGP1632: LLVGAAAFCSAMYVGDLCGS (amino acid 264-283)(SEQ ID N0:118)
IGP1633: YVGDLCGSVFLVSQLFTISP (amino acid 276-295)(SEQ ID N0:119)
IGP1634: SQLFTISPRRHETVQDCNCS (amino acid 288-307)(SEQ ID N0:120)
IGP1635: TVQDCNCSIYPGHITGHRMA (amino acid 300-319)(SEQ ID N0:121)
IGP1636: HITGHRMAWDMMMNWSPTTA (amino acid 312-331)(SEQ ID N0:122)
PBMC from 14 different healthy donors not vaccinated with Els or 10 donors
vaccinated
with Els were cultured in the presence of 25 pg/ml (non vaccinated persons) or
10 pg/ml
(vaccinated persons, samples taken after the third or booster injection) of
each peptide
separately. As can be judged from figure 53 the peptides IGP 1627, 1629, 1630,
1631,
1633, 1635 and 1635 all induced significantly higher responses in vaccinated
persons
compared to non-vaccinated persons. Using a stimulation index of 3 as cut-off
the peptides
IGP 1627, 1629, 1631 and 1635 were the most frequently recognized (i.e.
recognized by at
3 0 least half of the vaccinated persons tested).
This experiment proofs that the T-cell responses induced by Els derived from
mammalian
cell culture are specific against E1 since these responses can not only be
recalled by the
same Els derived from mammalian cell culture but also by synthetic peptides.
In addition
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this experiment delineates the most immunogenic T-cell domains in E1 are
located
between amino acids 204-223, 228-271, 276-295, 300-331 and more particularly
even
between amino acids 204-223, 228-247, 252-271 and 300-319.
Legend to figure 53: The stimulation index (cellular immune response) was
obtained by
culturing PBMC (3 x105 cells), in the presence or absence of peptides and
determining the
amount of tritiated thymidine incorporated in these cells during a pulse after
5-6 days of
culture. The stimulation index is the ratio of thymidine incorporated in the
cells cultured
with peptide versus the ones cultured without peptide. Results are expressed
as individual
values for vaccinated persons (top panel) or non vaccinated or controls (lower
panel).
The present invention also provides therefor, the following E1 peptides,
proteins,
compisitions and kits containing the same, nucleic acid sequences coding for
these peptides
and proteins containing the same, and methods of their manufacture and use, as
are
generally described herein for other El and related peptides of the present
invention.
IGP 1626 spanning positions 192-211 of the E1 region
(SEQ ID NO:I 12),
IGP 1627 spanning positions 204-223 of the E1 region
(SEQ ID N0:113),
IGP 1628 spanning positions 216-235 of the E1 region
(SEQ ID N0:114),
IGP 1629 spanning positions 228-247 of the E1 region
(SEQ ID NO:115),
2 0 IGP 1630 spanning positions 240-259 of the E1 region
(SEQ ID NO:116),
IGP 1631 spanning positions 252-271 of the E 1 region
(SEQ ID N0:117),
IGP 1632 spanning positions 264-283 of the E1 region
(SEQ ID N0:118),
IGP 1633 spanning positions 276-295 of the E1 region
(SEQ ID N0:119),
IGP 1634 spanning positions 288-307 of the E1 region
(SEQ ID N0:120),
2 5 IGP 1635 spanning positions 300-319 of the E1 region
(SEQ ID N0:121),
IGP 1636 spanning positions 312-331 of the E1 region
(SEQ ID N0:122).
Example 19: Exemplification of therapeutic utility of E1-vaccination in man
The HCV Els protein (amino acids 192-326 (SEQ 10 NO: 123:
30 YEVRNVSGMYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRENNSSRCWVALTPT
LAARNASVPTTTIRRHVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPRRHETV
QDCNCSIYPGHITGHRMAWDMMMNW)) was expressed in Vero cells using
recombinant vaccinia virus HCV 11 B. This vaccinia virus is essentially
identical to
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vvHCVI l A (described in, for example, PCT/EP95/03031 and U.S. Patent No.
6,510,134,
the entire contents of each of which are incorporated herein by reference) but
has been
passaged from RK13 to Vero cells. The protein was purified (by means of lentil
chromatography, reduction-alkylation and size exclusion chromatography)
essentially as
described in Example 9 of PCT/E99/04342 (the entire contents of each of which
are
incorporated herein by reference), making use of iodoacetamide as alkylating
agent for the
cysteines. After purification the 3% Empigen-BB (N-Dodecyl-N,N-
dimethylglycine) was
exchanged to 3% betain by size exclusion chromatography as described in
Example 1 of
PCT/E99/04342 this process allows to recover Els as a particle. Finally the
material was
desalted to PBS containing 0.5% betain and an Els concentration of 400 pg/mL.
This El
was mixed with an equal volume of Alhydrogel 1.3% (Superfos, Denmark) and
finally
further diluted with 8 volumes of 0.9% NaCI to yield alum-adjuvanted E1 at a
concentration of 40 ~g E1/mL and 0.13% of Alhydrogel.
In order to demonstrate a therapeutic effect of E1 immunization in man, an
immune
response was induced in chronically infected patients. This immune response
was both
quantitatively and qualitatively different from the base line response against
E1 which is
present in such patients.
Twenty-six chronically genotype 1 infected HCV patients were vaccinated with 5
doses of
pg Els formulated on 0.13% Alhydrogel in 0.5 mL. All immunizations were given
2 0 intramuscularly. Immunizations were performed at week 0, 4, 8, 12 and 24.
In addition, 9
patients received an identical number of placebo injections consisting of alum
only.
As evidenced in the following Table 14, a T -cell response is normally absent
in chronic
HCV earners (only 4%, 1 out of 26, have a detectable T -cell reactivity). Upon
immunization this increases to about 70% (18 out of 26) already after 4
immunizations.
2 5 This observation demonstrates that the immune response has been
qualitatively changed
from a T -cell non-responsiveness to a clear response. Furthermore this immune
response
can be sustained, by boosting with larger intervals since after the fifth
immunization 3
monhts later, the cellular immune response is still of the same level or
slightly increased.
No significant changes were observed in the placebo group.
Table 14: The stimulation index (SI; cellular immune response) was obtained by
culturing
PBMC, drawn from the individuals four weeks (W16) after the fourth
immunization and
two weeks (W26) after the fifth immunizationt in the presence or absence of 3
pg of Els
and determining the amount of tritiated thymidine incorporated in these cells
during a pulse
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of 18 hours after 5 days of culture. The stimulation index is the ratio of
thymidine
incorporated in the cells cultured with envelope antigen versus the ones
cultured without
antigen. A stimulation index of >3 is considered a positive signal.
T-cell response
WO W 16 W26
SI>3 1 /26 18/26 17/25
Mean SI 1.5 13.7 19.3
In addition the antibody titers were determined by ELISA prior to the first
immunization
(this was done for each patient on three samples taken at different time
points before
immunization) and after the fifth immunization (this was done for each patient
on two
samples: W26 and W28). A serial dilution of the sample was compared to an in-
house
standard (this in-house standard is, as previously described, defined as
having 1000
mU/mL of E1 antibody is a mixture of three sera from HCV chronic carriers
selected based
on a high anti-envelope titer). From this analysis it was concluded that, on
average, the titer
doubled from 331 to 715 mU/mL. This result demonstrates that the humoral arm
of the
immune response has at least been quantitatively altered.
An E1 vaccine formulated on alum significantly changes both the qualitative
and
quantitative immune response against E1. Such an E1 based vaccine and/or any
of the
vaccines described herein may be additionally useful if administered in
conjunction with
antiviral therapy such but not limited to interferon and alternatively with
its combination
with ribavirin (i.e., prior to, after or with a composition of the present
invention).
The following as well as all previously and subsequently identified text,
references,
2 0 patents, publication, etc., are hereby incorporated herein in their
entirety by reference.
Example 20: Effects of therapeutic E1-vaccination in chronicall infected
patients.
In a first course of this study, 26 patients received 5 doses of 20 pg Els as
described in
Example 19. In a second course, 25 of said 26 patients received a further 6
intramuscular
doses of 20 ~g Els formulated on 0.13% Alhydrogel in 0.5 mL (as in Example
19). The
immunizations of this second series were administered with 3-week intervals
and the first
injection was given at week 50. Four weeks after the last injection of the
second course, a
liver biopsy was performed in 24 out of the 25 patients who completed the two
courses of
therapeutic E1 s vaccination. A liver biopsy was also performed in all
patients prior to the
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onset of the therapeutic vaccine treatment (i.e. prior to course 1 vaccination
scheme). The
mean time elapsed between pre-treatment and post-treatment biopsies was 17
months.
Liver histology slides thus obtained before and after therapeutic vaccination
were scored
centrally in a blind way by two expert pathologists for fibrosis and
inflammation, this
according to the Ishak and Metavir scoring systems (Ishak et al. 1995 which is
a
modification of the scoring system of Knodell et al. 1981; Bedossa and Poynard
1996), as
well as for anti-E2 immunostain using the IGH222 murine anti-E2 HVRI
monoclonal
antibody as described in International Patent Application W099/50301.
Perisinusoidal
fibrosis was assessed based on staining of collagen with Sirius Red (on liver
histology
slides).
The Ishak scores range from 0 to 18 for grading of inflammation and from 0 to
6 for
staging of fibrosis/cirrhosis. The sum of the Ishak inflammation and fibrosis
scores comes
closest to the Histological Activity Index , (HAI; Knodell et al. 1981 ) which
has been
widely used. The change in Ishak fibrosis score in the 24 patients who
followed the two
courses of therapeutic E 1 s vaccination is -0.04 (with a 95% confidence
interval of -0.60 to
+0.68) and with a change from baseline- to end-value from 2.54 (baseline) to
2.50 (end).
An overview of the different assessed necro-inflammatory intensities (Ishak
scoring) is
given in Table 15.
2 0 The Ishak equivalent of the HAI scores for the treated patients showed a
mean absolute
change from baseline of-0.17 (with a 95% confidence interval of-1.36 to 1.03)
and with a
change from baseline- to end-value from 8.88 (baseline) to 8.71 (end).
Furthermore, nine
(9) out of the 24 patients (38%) improved 2 points or more on the sum of Ishak
inflammation- and Ishak fibrosis- scores whereas ten (10) out of the 24
patients remained
2 5 stable (no change or a change of +1 or -1 ) and five (5) patients evolved
to a worse
condition (worsening of 2 points or more).
The Metavir scores range from 0 to 3 for grading of inflammation and from 0 to
4 for
staging of fibrosis/cirrhosis. The overall progression rate of the Metavir
score in an
3 0 untreated patient is estimated to be 0.133 per year (Poynard et al. 1997).
For the patients of the current study, the average progression over the 17-
month period
based on the linear extension of the baseline score and the estimated duration
of the
infection (which is reported for 19 out of the 24 patients who followed the
two courses of
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therapeutic Els vaccination) would be 0.20. This correlates well with the
published overall
rate of progression which would be 0.19 for the 17 month period.
The change in Metavir score observed for the treated patients is, however,
0.00 (with a
95% confidence interval of-0.43 to +0.43) and with a mean baseline- an end-
score of 1.67.
An overview of Metavir scores at baseline and at the end of the second course
treatment is
given in Table 16. Unblinded comparison of pre- and post treatment slides
revealed that l0
patients had diminished perisinusoidal fibrosis based on Sirius Red staining.
The anti-E2 immunostain scores range from 0 to 4, with 0 = no E2 anitgen
detectable, 1 =
occasional cell with detectable E2 antigen, 2= clusters of E2 antigen positive
cells but less
than 25% of cells positive, 3 = clusters of E2 antigen positive cells and 25-
50% of cells
positive, 4 = clusters of E2 antigen positive cells and more than SO% of cells
positive. The
anti-E2 immunostain scores for the treated patients showed a mean absolute
change from
baseline of -0.75 (with a 95% confidence interval of -1.64 to 0.14) and with a
change from
baseline- to end-value from 2.54 (baseline) to 1.79 (end). The reduction seen
for anti-E2
immoreactivity in the liver using blind scoring was confimed by unblinded
comparison of
paired slides per patient. Eleven patients showed negativation or a marked
reduction in
HCV E2 immunostaining, while only three patients had a stronger immunostain
after
treatment. Of these 11 patients, 3 also showed a reduction in steatosis.
Serum HCV RNA levels were determined using the Amplicor HCV Monitor kit
(Roche,
Basel, Switzerland). The serum HCV RNA levels remained unchanged or did not
change
more than one log from baseline except in 1 patient. This concerned a 37-year
old
treatment-naive female patient, infected with a genotype 1 a virus and with
flu-like
symptoms at baseline. HCV-RNA dropped 3 logs from week 8 (after two injections
with
E1) to reach levels below 3000 IU/ml at weeks 16 and 20, in parallel ALT
dropped from a
single peak value of 400 U/ml at week 8 to normal ALT values at weeks 16, 20
and 24.
This was accompanied by disappearance of the flu-like symptoms. After this
period both
ALT and HCV-RNA increased again, and symptoms re-appeared. The sequence of the
3 0 virus present during the emerging viral load increase was compared with
the baseline
sequence. No evidence for the presence of escape mutant virus was found.
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Serum ALT (alanine aminotransferase activity) levels in the treated patients
decreased on
average 22% (with a 95% confidence interval of -15% to -30%) as compared to
the
baseline levels. A positive correlation between % change from baseline in ALT
levels and
absolute change from baseline in fibrosis scores was noticed: a Spearman rank
of p=0.0007
for Ishak fibrosis score and of p=0.002 for Metavir fibrosis score were
obtained as
correlations between the change from baseline in serum ALT and the absolute
change from
baseline Ishak and Metavir fibrosis scores, respectively.
A further outcome of the current study is the predictive character of the
immune response (in
terms of increase in serum anti-Els antibody levels) to treatment for
improvement in
histological scores (fibrosis and overall) as well as serum ALT levels.
Antibody titers were determined by ELISA. A serial dilution of a serum sample
was
compared to an in house standard (this in house standard defined as having
1000 mU/mL of
Els antibody is a mixture of three sera from HCV chronic carriers selected
based on a high
anti-envelope titer). The detection limit for this assay is 5 mU/mL.
A Spearman rank of p=0.007 was obtained as correlation between the change from
baseline
in serum anti-El antibody level and the absolute change from baseline in total
Ishak
(inflammation plus fibrosis) score. Pearson p-values of p=0.06 and of p=0.009
were obtained
as correlation between the change from baseline in serum anti-E1 antibody
level and the
2 0 absolute (p= 0.06) and relative (p=0.009) change from baseline in Ishak
fibrosis score,
respectively. A Pearson p-value of p=0.01 was obtained as correlation between
the change
from baseline in serum anti-E1 antibody level and the relative change from
baseline in ALT
value.
These associations remain after correction for baseline liver histology
scores, ALT, serum
2 5 HCV-RNA viral load, age and sex, and IFN exposure and thus as add to the
likelihood of a
real positive effect induced by the therapeutic EI s vaccination treatment.
The seven patients with the highest increase in serum antibody levels in
response to the
therapeutic vaccine candidate (i.e., patients with an increase in anti-E1
antibody level of at
30 least 700 mU/mL) had on average a significant decrease of -0.9 points (
with a 95%
confidence interval of -0.2 to -1.5) on Metavir liver fibrosis score. In this
subgroup, serum
ALT on average decreased 37% (with a 95% confidence interval of -25 to - 49%).
Table 17
provides an overview of the correlation between immune response (in terms of
anti-Els
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antibody levels induced by treatment) and overall change (relative to
baseline) in Ishak
scores. Figure 54 illustrates the correlation between changes in Ishak
fibrosis score, changes
in ALT-levels and changes in anti-Els antibody levels. Figure 55 illustrates
the correlation
between changes in Metavir fibrosis score, changes in ALT-levels and changes
in anti-Els
antibody levels. Figure 56 illustrates Ishak fibrosis score versus ALT levels
both at the start
of the first treatment course (panel A) and at the end of the second treatment
course (panel B).
Figure 57 illustrates patient's age versus Ishak fibrosis score both at the
start of the first
treatment course (panel A) and at the end of the second treatment course
(panel B). Both
Figures 56 and 57 further indicate the seven patients with the highest
increase in anti-Els
antibody levels induced by the therapeutic vaccination treatment.
A positive correlation between serum anti-Els antibody levels and T cell
proliferation index
values at the end of the second treatment course was observed, with a Pearson
p-value of
p=0.009.
Concludingly, the increase in antibody levels to the Els vaccination
significantly predicted
improvement in liver fibrosis (Metavir and Ishak scores), sum of inflammation
and fibrosis
scores (Ishak), and in ALT levels, even after correcting for any other
baseline prognostic
variables. The increase in anti-E1 antibodies after vaccination was also
significantly
correlated with an increase in T cell proliferation index to E1.
This study is clearly supportive for a El-based therapeutic vaccination
strategy to have the
2 0 potential to halt disease progression towards liver cirrhosis.
Table 15. Ishak grading of necro-inflammatory intensities for periportal
hepatitis,
confluent necrosis, focal inflammation, portal inflammation and the overall
total
inflammation grading. Scores are indicated as the change from baseline (mean
and 95
2 5 confidence interval) and the mean baseline- to end-values.
Score for Change of score from Change of score
baseline from
mean 95% confidence intervalbaseline to end
mean
eri ortal he atitis-0.21 -0.62 to 0.20 1.42 to 1.21
confluent necrosis 0.38 -0.17 to 0.92 0.00 to 0.38
focal inflammation -0.17 -0.59 to 0.26 2.63 to 2.46
ortal inflammation -0.13 -0.51 to 0.26 2.29 to 2.17
total inflammation -0.13 -1.18 to 0.93 6.33 to 6.21
adin
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Table 16. Overview of frequencies (given as number of patients) of changes of
a given
baseline Metavir score (given in top row; Baseline 0 to 4) to a given Metavir
score at the
end of the second course of therapeutic Els vaccination (given in left row;
EOT 0 to 4).
For instance, the "5" marked with a "*" (i.e., "5*") means that S patients had
a baseline
Metavir score of 1 and a Metavir score of 0 at the end of the second course
treatment (EOT
= end of treatment).
BaselineBaselineBaselineBaselineBaselineTotal
0 1 2 3 4
EOT 0 5 * 1 0 0 6
0
EOT 1 3 2 0 0 6
1
EOT 0 5 1 0 0 6
2
EOT 0 0 0 1 1 2
3
EOT4 0 0 1 2 1 4
Total 1 13 5 3 2 24
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Table 17. Correlation between serum anti-El antibody levels induced by
therapeutic Els
vaccination and change in overall Ishak scores. Given are the number of
patients
corresponding to the possible criteria as outlined in the Table.
Overall change of Ishak < -1, 0, or > +1
score -1 +1
chan a from baseline
Hi h anti-Els antibod 5 2 0
res onse
Low anti-Els antibod 4 8 5
res onse
Example 21: Effects of therapeutic E1-vaccination regimen.
In a first course of this study, 26 patients received 5 doses of 20 pg Els
("Els patients")
and 9 patients received an identical number of placebo injections ("placebo
patients")
consisting of alum only, as described in Example 19. In a second course, 25 of
said 26
patients that received Els during the first course and 9 of said 9 patients
that received a
placebo were all immunized with a further 6 intramuscular doses of 20 pg Els
formulated
on 0.13% Alhydrogel in 0.5 mL (as in Example 19), thus giving rise to two
groups of
patiens: the "E 1 s/E 1 s patients" and "placebo/E 1 s patients". The
immunizations of this
second series, i.e. course 2, were administered with 3-week intervals
(compared to 4-week
intervals during the first series, i.e. course 1) and the first injection was
given at week 50.
The median values for anti-E1 antibodies after 4 Els injections were 195 mU/mL
for 4-week
intervals (as deduced from the E1s patients) and 274 mU/mL for 3-week
intervals (as
2 0 deduced from the placebo/E 1 s patients), thus indicating that a 3-week
interval regimen
induces a stronger humoral immune response in a shorter period of time.
In placebo/E 1 s patients E 1 s-specific T cell proliferation increased from
0/9 patients at
baseline to 9/9 patients after 6 injections with 3-week intervals at week 69.
If the humoral and cellular immune response to Els in healthy male volunteers
(see
2 5 Examples 16 and 17) are compared with those in the patient group, a
somewhat slower
increase in anti-El antibody levels and T cell response is observed. The
booster effect seen in
healthy volunteers was almost absent in patients (median titer of 165 mU/mL
after the fifth
immunization administered to the E 1 s/E 1 s patients as a booster 12 weeks
after the 4°'
immunization), thus suggesting a somewhat impaired build up of immunological
memory to
30 Els in patients. Repeated intramuscular injections at 3-week intervals seem
to overcome this
impairment (median titer of 530 mU/mL after 6 consecutive immunizations with a
3-week
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interval in the placebo/Els patients), thus indicating another advantage of an
Els vaccination
regimen with 3-week intervals and/or administering more than 4 doses in the
first series of
immunizations.
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Shan, S. & Wong (1993) CRC-press p. 30-33.
Shiffman ML (1999) Viral Hepatitis Reviews 5(1):27-43.
Spaete, R., Alexander, D., Rugroden, M., Choo, Q., Berger, K., Crawford, K.,
Kuo, C.,
Leng, S., Lee, C., Ralston, R., et al. (1992) Virology 188(2):819-30.
Skehel, J., (1984) Proc. Natl. Acad. Sci. USA 81, 1179-1783.
Stunnenberg, H., Lange, H., Philipson, L., Miltenburg, R. & van der Vliet, R.
(1988) Nucl.
2 0 Acids Res. 16, 2431-2444.
Stuyver, L., Van Arnhem, W., Wyseur, A., DeLeys, R. & Maertens, G. (1993a)
Biochem.
Biophys. Res. Commun. 192, 635-641.
Stuyver, L., Rossau, R., Wyseur, A., Duhamel, M., Vanderborght, B., Van
Heuverswyn,
H., & Maertens, G. (1993b) J. Gen. Virol. 74, 1093-I 102.
2 5 Stuyver, L., Van Arnhem, W., Wyseur, A., Hernandez, F., Delaporte, E.,
Maertens, G.
(1994) Proc. Natl. Acad. Sci. USA 91, 10134-10138.
Takimoto M, Ohkoshi S, Ichida T, Takeda Y, Nomoto M, Asakura H, Naito A, Mori
S,
Hata K, Igarashi K, Hara H, Ohta H, Soga K, Watanabe T, Kamimura T (2002) Dig
Dis
Sci. 47(1):170-176.
30 Weil, L. & Seibler, S. (1961) Arch. Biochem. Biophys. 95, 470.
Yokosuka, O., Ito, Y., Imazeki, F., Ohto, M. & Omata, M. (1992) Biochem.
Biophys. Res.
Commun. 189:565-571.
WO 96/04385 (PCT/EP95/03031) - Purified Hepatitis C Virus Envelope Proteins
for
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121
Diagnostic and Therapeutic Use.
All references cited herein are incorporated in their entirety by reference.
CA 02468690 2004-05-28
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Sequence Listing
<110> Innogenetics N.V.
<120> Purified hepatitis C virus envelope proteins for diagnostic and
therapeutic use.
<140> PCT/EP02/00219
<160> 123
<170> PatentIn 3.1
<210> 1
<211> 21
<212> DNA
<213> Hepatitis C virus
<400> 1
ggcatgcaag cttaattaat t 21
<210> 2
<211> 68
<212> DNA
<213> Hepatitis C virus
<400> 2
ccggggaggc ctgcacgtga tcgagggcag acaccatcac caccatcact aatagttaat 60
taactgca 68
<210> 3
<211> 642
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..639
<220>
<221> mat_peptide
<222> 1..636
<400> 3
atg ccc ggt tgc tct ttc tct atc ttc ctc ttg get tta ctg tcc tgt 48
Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
ctg acc att cca get tcc get tat gag gtg cgc aac gtg tcc ggg atg 96
Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Met
20 25 30
CA 02468690 2004-05-28
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tac cat gtc acg aac gac tgc tcc aac tca agc att gtg tat gag gca 144
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 40 45
gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc gtt cgg gag 192
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
50 55 60
aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg ctc gca get 290
Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
agg aac gcc agc gtc ccc acc acg aca ata cga cgc cac gtc gat ttg 288
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
ctc gtt ggg gcg get get ctc tgt tcc get atg tac gtg ggg gat ctc 336
Leu Val Gly Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
tgc gga tct gtc ttc ctc gtc tcc cag ctg ttc acc atc tcg cct cgc 384
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile Ser Pro Arg
115 120 125
cgg cat gag acg gtg cag gac tgc aat tgc tca atc tat ccc ggc cac 932
Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
ata aca ggt cac cgt atg get tgg gat atg atg atg aac tgg tcg cct 480
Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro
145 150 155 160
aca acg gcc ctg gtg gta tcg cag ctg ctc cgg atc cca caa get gtc 528
Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val
165 170 175
gtg gac atg gtg gcg ggg gcc cat tgg gga gtc ctg gcg ggc ctc gcc 576
Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala
180 185 190
tac tat tcc atg gtg ggg aac tgg get aag gtt ttg att gtg atg cta 624
Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile Val Met Leu
195 200 205
ctc ttt get ctc taatag 692
Leu Phe Ala Leu
210
<210> 4
<211> 212
<212> PRT
<213> Hepatitis C virus
<900> 9
Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
CA 02468690 2004-05-28
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Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Met
20 25 30
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 ' 40 45
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
50 55 60
Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
Leu Val Gly Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile Ser Pro Arg
115 120 125
Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro
145 150 155 160
Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val
165 170 175
Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala
180 185 190
Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile Val Met Leu
195 200 205
Leu Phe Ala Leu
210
<210> 5
<211> 795
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..792
<220>
<221> mat_peptide
<222> 1..789
<400> 5
atg ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
CA 02468690 2004-05-28
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20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 194
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
acaggg aatttgccc ggttgctct ttctctatc ttcctcttgget ttg 192
ThrGly AsnLeuPro GlyCysSer PheSerIle PheLeuLeuAla Leu
50 55 60
ctgtcc tgtctgacc gttccaget tccgettat gaagtgcgcaac gtg 240
LeuSer CysLeuThr ValProAla SerAlaTyr GluValArgAsn Val
65 70 75 80
tccggg atgtaccat gtcacgaac gactgctcc aactcaagcatt gtg 288
SerGly MetTyrHis ValThrAsn AspCysSer AsnSerSerIle Val
85 90 95
tatgag gcagcggac atgatcatg cacaccccc gggtgcgtgccc tgc 336
TyrGlu AlaAlaAsp MetIleMet HisThrPro GlyCysValPro Cys
100 105 110
gttcgg gagaacaac tcttcccgc tgctgggta gcgctcaccccc acg 384
ValArg GluAsnAsn SerSerArg CysTrpVal AlaLeuThrPro Thr
115 120 125
ctcgca getaggaac gccagcgtc cccaccacg acaatacgacgc cac 432
LeuAla AlaArgAsn AlaSerVal ProThrThr ThrIleArgArg His
130 135 140
gtcgat ttgctcgtt ggggcgget getttctgt tccgetatgtac gtg 480
ValAsp LeuLeuVal GlyAlaAla AlaPheCys SerAlaMetTyr Val
145 150 155 160
ggggac ctctgcgga tctgtcttc ctcgtctcc cagctgttcacc atc 528
GlyAsp LeuCysGly SerValPhe LeuValSer GlnLeuPheThr Ile
165 170 175
tcgcct cgccggcat gagacggtg caggactgc aattgctcaatc tat 576
SerPro ArgArgHis GluThrVal GlnAspCys AsnCysSerIle Tyr
180 185 190
cccggc cacataacg ggtcaccgt atggettgg gatatgatgatg aac 624
ProGly HisIleThr GlyHisArg MetAlaTrp AspMetMetMet Asn
195 200 205
tggtcg cctacaacg gccctggtg gtatcgcag ctgctccggatc cca 672
TrpSer ProThrThr AlaLeuVal ValSerGln LeuLeuArgIle Pro
210 215 220
caaget gtcgtggac atggtggcg ggggcccat tggggagtcctg gcg 720
GlnAla ValValAsp MetValAla GlyAlaHis TrpGlyValLeu Ala
225 230 235 240
ggtctc gcctactat tccatggtg gggaactgg getaaggttttg att 768
GlyLeu AlaTyrTyr SerMetVal GlyAsnTrp AlaLysValLeu Ile
245 250 255
gtgatg ctactcttt getccc-taatag 795
ValMet LeuLeuPhe AlaPro
260
<210> 6
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<211> 263
<212> PRT
<213> Hepatitis C virus
<400> 6
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val
145 150 155 160
Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile
165 170 175
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro
210 215 220
Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala
225 230 235 240
Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile
245 250 255
Val Met Leu Leu Phe Ala Pro
260
<210> 7
CA 02468690 2004-05-28
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<211> 633
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..630
<220>
<221> mat_peptide
<222> 1..627
<400> 7
atg ttg ggt aag gtc atc gat acc ctt acg tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
atg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggt get gcc aga 96
Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gaa gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 90 45
aca ggg aat ttg cct ggt tgc tct ttc tct atc ttc ctc ttg get tta 192
Thr Gly Asn Leu .Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc att cca get tcc get tat gag gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gtt cgg gag aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg 384
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
ctcgcagetaggaac gccagcgtc cccactacg acaatacga cgccac 432
LeuAlaAlaArgAsn AlaSerVal ProThrThr ThrIleArg ArgHis
130 135 140
gtcgatttgctcgtt ggggcgget getttctgt tccgetatg tacgtg 480
ValAspLeuLeuVal GlyAlaAla AlaPheCys SerAlaMet TyrVal
145 150 155 160
ggggatctctgcgga tctgtcttc ctcgtctcc cagctgttc accatc 528
GlyAspLeuCysGly SerValPhe LeuValSer GlnLeuPhe ThrIle
165 170 175
tcgcctcgccggcat gagacggtg caggactgc aattgctca atctat 576
SerProArgArgHis GluThrVal GlnAspCys AsnCysSer IleTyr
180 185 190
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ccc ggc cac ata aca ggt cac cgt atg get tgg gat atg atg atg aac 624
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
tgg taatag 633
Trp
210
<210> 8
<211> 209
<212> PRT
<213> Hepatitis C virus
<400> 8
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 90 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val
145 150 155 160
Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile
165 170 175
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
Pro G1y His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
Trp
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<210> 9
<211> 983
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..480
<220>
<221> mat_peptide
<222> 1..477
<400> 9
atg ccc ggt tgc tct ttc tct atc ttc ctc ttg gcc ctg ctg tcc tgt 48
Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
ctg acc ata cca get tcc get tat gaa gtg cgc aac gtg tcc ggg gtg 96
Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val
20 25 30
tac cat gtc acg aac gac tgc tcc aac tca agc ata gtg tat gag gca 144
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 40 95
gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc gtt cgg gag 192
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
50 55 60
ggc aac tcc tcc cgt tgc tgg gtg gcg ctc act ccc acg ctc gcg gcc 240
Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
agg aac gcc agc gtc ccc aca acg aca ata cga cgc cac gtc gat ttg 288
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
ctc gtt ggg get get get ttc tgt tcc get atg tac gtg ggg gat ctc 336
Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
tgc gga tct gtt ttc ctt gtt tcc cag ctg ttc acc ttc tca cct cgc 384
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg
115 120 125
cgg cat caa aca gta cag gac tgc aac tgc tca atc tat ccc ggc cat 432
Arg His Gln Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
gta tca ggt cac cgc atg get tgg gat atg atg atg aac tgg tcc taatag 483
Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser
195 150 155 160
<210> 10
<211> 159
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
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<400> 10
Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val
20 25 30
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 40 45
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
50 55 60
Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg
115 120 125
Arg His Gln Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser
145 150 155
<210> 11
<211> 480
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..477
<220>
<221> mat_peptide
<222> 1..474
<400> 11
atg tcc ggt tgc tct ttc tct atc ttc ctc ttg gcc ctg ctg tcc tgt 98
Met Ser Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
ctg acc ata cca get tcc get tat gaa gtg cgc aac gtg tcc ggg gtg 96
Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val
20 25 30
tac cat gtc acg aac gac tgc tcc aac tca agc ata gtg tat gag gca 144
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 90 95
gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc gtt cgg gag 192
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
CA 02468690 2004-05-28
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50 55 60
ggc aac tcc tcc cgt tgc tgg gtg gcg ctc act ccc acg ctc gcg gcc 240
Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
agg aac gcc agc gtc ccc aca acg aca ata cga cgc cac gtc gat ttg 288
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
ctc gtt ggg get get get ttc tgt tcc get atg tac gtg ggg gat ctc 336
Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
tgc gga tct gtt ttc ctt gtt tcc cag ctg ttc acc ttc tca cct cgc 384
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg
115 120 125
cgg cat caa aca gta cag gac tgc aac tgc tca atc tat ccc ggc cat 432
Arg His Gln Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
gta tca ggt cac cgc atg get tgg gat atg atg atg aac tgg taatag 480
Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
145 150 155
<210> 12
<211> 158
<212> PRT
<213> Hepatitis C virus
<400> 12
Met Ser Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys
1 5 10 15
Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val
20 25 30
Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala
35 40 45
Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu
50 55 60
Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala
65 70 75 80
Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu
85 90 95
Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu
100 105 110
Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg
115 120 125
Arg His Gln Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His
130 135 140
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Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
145 150 155
<210> 13
<211> 636
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..633
<220>
<221> mat_peptide
<222> 1..630
<400> 13
atg ctg ggt aag gcc atc gat acc ctt acg tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gaa gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
aca ggg aat ttg cct ggt tgc tct ttc tct atc ttc ctc ttg get tta 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt cta acc att cca get tcc get tac gag gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gttcgggagaac aactcttcc cgctgctgg gtagcgctcacc cccacg 384
ValArgGluAsn AsnSerSer ArgCysTrp ValAlaLeuThr ProThr
115 120 125
ctcgcggetagg aacgccagc atccccact acaacaatacga cgccac 932
LeuAlaAlaArg AsnAlaSer IleProThr ThrThrIleArg ArgHis
130 135 140
gtcgatttgctc gttggggcg getgetttc tgttccgetatg tacgtg 480
ValAspLeuLeu ValGlyAla AlaAlaPhe CysSerAlaMet TyrVal
145 150 155 160
ggggatctctgc ggatctgtc ttcctcgtc tcccagctgttc accatc 528
GlyAspLeuCys GlySerVal PheLeuVal SerGlnLeuPhe ThrIle
165 170 175
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tcg cct cgc cgg cat gag acg gtg cag gac tgc aat tgc tca atc tat 576
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
ccc ggc cac ata acg ggt cac cgt atg get tgg gat atg atg atg aac 624
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
tgg tac taatag 640
Trp Tyr
210
<210> 14
<211> 210
<212> PRT
<213> Hepatitis C virus
<400> 14
Met Leu Gly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Ile Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val
145 150 155 160
Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile
165 170 175
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
Trp Tyr
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
210
<210> 15
<211> 26
<212> DNA
<213> Hepatitis C virus
<400> 15
atgcccggtt gctctttctc tatctt 26
<210> 16
<211> 26
<212> DNA
<213> Hepatitis C virus
<400> 16
atgttgggta aggtcatcga taccct 26
<210> 17
<211> 30
<212> DNA
<213> Hepatitis C virus
<220>
<221> misc_feature
<223>/note="antisense"
<400> 17
ctattaggac cagttcatca tcatatccca 30
<210> 18
<211> 27
<212> DNA
<213> Hepatitis C virus
<900> 18
ctattaccag ttcatcatca tatccca 27
<210> 19
<211> 36
<212> DNA
<213> Hepatitis C virus
<400> 19
atacgacgcc acgtcgattc ccagctgttc accatc 36
<210> 20
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<211> 36
<212> DNA
<213> Hepatitis C virus
<220>
<221> misc_feature
<223>/note="antisense"
<400> 20
gatggtgaac agctgggaat cgacgtggcg tcgtat 36
<210> 21
<211> 723
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..720
<220>
<221> mat_peptide
<222> 1..717
<400> 21
atg ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
aca ggg aat ttg ccc ggt tgc tct ttc tct atc ttc ctc ttg get ttg 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc gtt cca get tcc get tat gaa gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tccgggatgtac catgtcacgaac gactgc tccaactca agcattgtg 288
SerGlyMetTyr HisValThrAsn AspCys SerAsnSer SerIleVal
85 90 95
tatgaggcagcg gacatgatcatg cacacc cccgggtgc gtgccctgc 336
TyrGluAlaAla AspMetIleMet HisThr ProGlyCys ValProCys
100 105 110
gttcgggagaac aactcttcccgc tgctgg gtagcgctc acccccacg 389
ValArgGluAsn AsnSerSerArg CysTrp ValAlaLeu ThrProThr
115 120 125
ctcgcagetagg aacgccagcgtc cccacc acgacaata cgacgccac 432
LeuAlaAlaArg AsnAlaSerVal ProThr ThrThrIle ArgArgHis
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
130 135 140
gtcgattcccag ctgttcaccatc tcgcctcgc cggcatgag acggtg 480
ValAspSerGln LeuPheThrIle SerProArg ArgHisGlu ThrVal
145 150 155 160
caggactgcaat tgctcaatctat cccggccac ataacgggt caccgt 528
GlnAspCysAsn CysSerIleTyr ProGlyHis IleThrGly HisArg
165 170 175
atggettgggat atgatgatgaac tggtcgcct acaacggcc ctggtg 576
MetAlaTrpAsp MetMetMetAsn TrpSerPro ThrThrAla LeuVal
180 185 190
gtatcgcagctg ctccggatccca caagetgtc gtggacatg gtggcg 624
ValSerGlnLeu LeuArgIlePro GlnAlaVal ValAspMet ValAla
195 200 205
ggggcccattgg ggagtcctggcg ggtctcgcc tactattcc atggtg 672
GlyAlaHisTrp GlyValLeuAla GlyLeuAla TyrTyrSer MetVal
210 215 220
gggaactggget aaggttttgatt gtgatgcta ctctttget ccctaatag 723
GlyAsnTrpAla LysValLeuIle ValMetLeu LeuPheAla Pro
225 230 235 240
<210>
22
<211> 239
<212> PRT
<213> Hepatitis C virus
<400> 22
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
145 150 155 160
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val
180 185 190
Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala
195 200 205
Gly Ala His Trp Gly Val Leu.Ala Gly Leu Ala Tyr Tyr Ser Met Val
210 215 220
Gly Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Pro
225 230 235
<210> 23
<211> 561
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..558
<220>
<221> mat_peptide
<222> 1..555
<400> 23
atg ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
aca ggg aat ttg ccc ggt tgc tct ttc tct atc ttc ctc ttg get ttg 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc gtt cca get tcc get tat gaa gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gtt cgg gag aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg 384
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
115 120 125
ctc gca get agg aac gcc agc gtc ccc acc acg aca ata cga cgc cac 432
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
gtc gat tcc cag ctg ttc acc atc tcg cct cgc cgg cat gag acg gtg 480
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
cag gac tgc aat tgc tca atc tat ccc ggc cac ata acg ggt cac cgt 528
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
atg get tgg gat atg atg atg aac tgg taatag 561
Met Ala Trp Asp Met Met Met Asn Trp
180 185
<210> 24
<211> 185
<212> PRT
<213> Hepatitis C virus
<400> 24
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 190
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
Met Ala Trp Asp Met Met Met Asn Trp
180 185
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<210> 25
<211> 606
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..603
<220>
<221> mat_peptide
<222> 1..600
<400> 25
atg ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly,Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
aca ggg aat ttg ccc ggt tgc tct ttc tct atc ttc ctc ttg get ttg 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc gtt cca get tcc get tat gaa gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gtt cgg gag aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg 384
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
ctc gca get agg aac gcc agc gtc ccc acc acg aca ata cga cgc cac 432
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
gtc gat tcc cag ctg ttc acc atc tcg cct cgc cgg cat gag acg gtg 480
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
cag gac tgc aat tgc tca atc tat ccc ggc cac ata acg ggt cac cgt 528
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
atg get tgg gat atg atg atg aac tgg tcg cct aca acg gcc ctg gtg 576
Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val
CA 02468690 2004-05-28
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180 185 190
gta tcg cag ctg ctc cgg atc ctc taatag 606
Val Ser Gln Leu Leu Arg Ile Leu
195 200
<210> 26
<211> 200
<212> PRT
<213> Hepatitis C virus
<400> 26
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val
180 185 190
Val Ser Gln Leu Leu Arg Ile Leu
195 200
<210> 27
<211> 636
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..633
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<220>
<221> mat_peptide
<222> 1..630
<400> 27
atg ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 95
aca ggg aat ttg ccc ggt tgc tct ttc tct atc ttc ctc ttg get ttg 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc gtt cca get tcc get tat gaa gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gtt cgg gag aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg 384
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
ctc gca get agg aac gcc agc gtc ccc acc acg aca ata cga cgc cac 432
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
gtc gat tcc cag ctg ttc acc atc tcg cct cgc cgg cat gag acg gtg 480
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
cag gac tgc aat tgc tca atc tat ccc ggc cac ata acg ggt cac cgt 528
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
atg get tgg gat atg atg atg aac tgg tcg cct aca acg gcc ctg gtg 576
Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val
180 185 190
gta tcg cag ctg ctc cgg atc gtg atc gag ggc aga cac cat cac cac 624
Val Ser Gln Leu Leu Arg Ile Val Ile Glu Gly Arg His His His His
195 200 205
cat cac taatag 636
His His
210
ctc gca get agg aac gcc agc gtc ccc acc acg aca
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<210> 28
<211> 210
<212> PRT
<213> Hepatitis C virus
<400> 28
Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile' Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val
145 150 155 160
Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
165 170 175
Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val
180 185 190
Val Ser Gln Leu Leu Arg Ile Val Ile Glu Gly Arg His His His His
195 200 205
His His
210
<210> 29
<211> 630
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..627
<220>
<221> mat peptide
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<222> 1..624
<400> 29
atg ggt aag gtc atc gat acc ctt acg tgc gga ttc gcc gat ctc atg 48
Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met
1 5 10 15
ggg tac atc ccg ctc gtc ggc get ccc gta gga ggc gtc gca aga gcc 96
Gly Tyr Ile Pro Leu Val Gly Ala Pro Val Gly Gly Val Ala Arg Ala
20 25 30
ctt gcg cat ggc gtg agg gcc ctt gaa gac ggg ata aat ttc gca aca 144
Leu Ala His Gly Val Arg Ala Leu Glu Asp Gly Ile Asn Phe Ala Thr
35 40 45
ggg aat ttg ccc ggt tgc tcc ttt tct att ttc ctt ctc get ctg ttc 192
Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Phe
50 55 60
tct tgc tta att cat cca gca get agt cta gag tgg cgg aat acg tct 240
Ser Cys Leu Ile His Pro Ala Ala Ser Leu Glu Trp Arg Asn Thr Ser
65 70 75 80
ggc ctc tat gtc ctt acc aac gac tgt tcc aat agc agt att gtg tac 288
Gly Leu Tyr Val Leu Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr
85 90 95
gag gcc gat gac gtt att ctg cac aca ccc ggc tgc ata cct tgt gtc 336
Glu Ala Asp Asp Val Ile Leu His Thr Pro Gly Cys Ile Pro Cys Val
100 105 110
cag gac ggc aat aca tcc acg tgc tgg acc cca gtg aca cct aca gtg 384
Gln Asp Gly Asn Thr Ser Thr Cys Trp Thr Pro Val Thr Pro Thr Val
115 120 125
gcagtcaag tacgtcggagca accaccget tcgatacgc agtcatgtg 432
AlaValLys TyrValGlyAla ThrThrAla SerIleArg SerHisVal
130 135 140
gacctatta gtgggcgcggcc acgatgtgc tctgcgctc tacgtgggt 480
AspLeuLeu ValGlyAlaAla ThrMetCys SerAlaLeu TyrValGly
145 150 155 160
gacatgtgt ggggetgtcttc ctcgtggga caagccttc acgttcaga 528
AspMetCys GlyAlaValPhe LeuValGly GlnAlaPhe ThrPheArg
165 170 175
cctcgtcgc catcaaacggtc cagacctgt aactgctcg ctgtaccca 576
ProArgArg HisGlnThrVal GlnThrCys AsnCysSer LeuTyrPro
180 185 190
ggc cat ctt tca gga cat cga atg get tgg gat atg atg atg aac tgg 624
Gly His Leu Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
195 200 205
taatag 634
<210> 30
<211> 208
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<400> 30
Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met
1 5 10 15
Gly Tyr Ile Pro Leu Val Gly Ala Pro Val Gly Gly Val Ala Arg Ala
20 25 30
Leu Ala His Gly Val Arg Ala Leu Glu Asp Gly Ile Asn Phe Ala Thr
35 90 45
Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Phe
50 55 60
Ser Cys Leu Ile His Pro Ala Ala Ser Leu Glu Trp Arg Asn Thr Ser
65 70 75 80
Gly Leu Tyr Val Leu Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr
85 90 95
Glu Ala Asp Asp Val Ile Leu His Thr Pro Gly Cys Ile Pro Cys Val
100 105 110
Gln Asp Gly Asn Thr Ser Thr Cys Trp Thr Pro Val Thr Pro Thr Val
115 120 125
Ala Val Lys Tyr Val Gly Ala Thr Thr Ala Ser Ile Arg Ser His Val
130 135 140
Asp Leu Leu Val Gly Ala Ala Thr Met Cys Ser Ala Leu Tyr Val Gly
145 150 155 160
Asp Met Cys Gly Ala Val Phe Leu Val Gly Gln Ala Phe Thr Phe Arg
165 170 175
Pro Arg Arg His Gln Thr Val Gln Thr Cys Asn Cys Ser Leu Tyr Pro
180 185 190
Gly His Leu Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
195 200 205
<210> 31
<211> 630
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..627
<220>
<221> mat_peptide
<222> 1..624
<400> 31
atg ggt aag gtc atc gat acc cta acg tgc gga ttc gcc gat ctc atg 48
Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met
1 5 10 15
CA 02468690 2004-05-28
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ggg tat atc ccg ctc gta ggc ggc ccc att ggg ggc gtc gca agg get 96
Gly Tyr Ile Pro Leu Val Gly Gly Pro Ile Gly Gly Val Ala Arg Ala
20 25 30
ctc gca cac ggt gtg agg gtc ctt gag gac ggg gta aac tat gca aca 144
Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr
35 40 95
ggg aat tta ccc ggt tgc tct ttc tct atc ttt att ctt get ctt ctc 192
Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Ile Leu Ala Leu Leu
50 55 60
tcg tgt ctg acc gtt ccg gcc tct gca gtt ccc tac cga aat gcc tct 240
Ser Cys Leu Thr Val Pro Ala Ser Ala Val Pro Tyr Arg Asn Ala Ser
65 70 75 80
ggg att tat cat gtt acc aat gat tgc cca aac tct tcc ata gtc tat 288
Gly Ile Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr
85 90 95
gag gca gat aac ctg atc cta cac gca cct ggt tgc gtg cct tgt gtc 336
Glu Ala Asp Asn Leu Ile Leu His Ala Pro Gly Cys Val Pro Cys Val
100 105 110
atg aca ggt aat gtg agt aga tgc tgg gtc caa att acc cct aca ctg 384
Met Thr Gly Asn Val Ser Arg Cys Trp Val Gln Ile Thr Pro Thr Leu
115 120 125
tca gcc ccg agc ctc gga gca gtc acg get cct ctt cgg aga gcc gtt 432
Ser Ala Pro Ser Leu Gly Ala Va1 Thr Ala Pro Leu Arg Arg Ala Val
130 135 140
gac tac cta gcg gga ggg get gcc ctc tgc tcc gcg tta tac gta gga 480
Asp Tyr Leu Ala Gly Gly Ala Ala Leu Cys Ser Ala Leu Tyr Val Gly
145 150 155 160
gac gcg tgt ggg gca cta ttc ttg gta ggc caa atg ttc acc tat agg 528
Asp Ala Cys Gly Ala Leu Phe Leu Val Gly Gln Met Phe Thr Tyr Arg
165 170 175
cct cgc cag cac get acg gtg cag aac tgc aac tgt tcc att tac agt 576
Pro Arg Gln His Ala Thr Val Gln Asn Cys Asn Cys Ser Ile Tyr Ser
180 185 190
ggc cat gtt acc ggc cac cgg atg gca tgg gat atg atg atg aac tgg 624
Gly His Val Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
195 200 205
taatag 630
<210> 32
<211> 208
<212> PRT
<213> Hepatitis C virus
<400> 32
Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met
1 5 10 15
CA 02468690 2004-05-28
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Gly Tyr Ile Pro Leu Val Gly Gly Pro Ile Gly Gly Val Ala Arg Ala
20 25 30
Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr
35 40 45
Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Ile Leu Ala Leu Leu
50 55 60
Ser Cys Leu Thr Val Pro Ala Ser Ala Val Pro Tyr Arg Asn Ala Ser
65 70 75 80
Gly Ile Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr
85 90 95
Glu Ala Asp Asn Leu Ile Leu His Ala Pro Gly Cys Val Pro Cys Val
100 105 110
Met Thr Gly Asn Val Ser Arg Cys Trp Val Gln Ile Thr Pro Thr Leu
115 120 125
Ser Ala Pro Ser Leu Gly Ala Val Thr Ala Pro Leu Arg Arg Ala Val
130 135 140
Asp Tyr Leu Ala Gly Gly Ala Ala Leu Cys Ser Ala Leu Tyr Val Gly
145 150 155 160
Asp Ala Cys Gly Ala Leu Phe Leu Val Gly Gln Met Phe Thr Tyr Arg
165 170 175
Pro Arg Gln His Ala Thr Val Gln Asn Cys Asn Cys Ser Ile Tyr Ser
180 185 190
Gly His Val Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
195 200 205
<210> 33
<211> 23
<212> DNA
<213> Hepatitis C virus
<400> 33
tgggatatga tgatgaactg gtc 23
<210> 34
<211> 30
<212> DNA
<213> Hepatitis C virus
<400> 34
ctattatggt ggtaagccac agagcaggag 30
<210> 35
<211> 1476
<212> DNA
<213> Hepatitis C virus
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<220>
<221> CDS
<222> 1..1473
<220>
<221> mat_peptide
<222> 1..1470
<400> 35
tgg gat atg atg atg aac tgg tcg cct aca acg gcc ctg gtg gta tcg 48
Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser
1 5 10 15
cag ctg ctc cgg atc cca caa get gtc gtg gac atg gtg gcg ggg gcc 96
Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala
20 25 30
cat tgg gga gtc ctg gcg ggc ctc gcc tac tat tcc atg gtg ggg aac 144
His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn
35 40 45
tgg get aag gtt ttg gtt gtg atg cta ctc ttt gcc ggc gtc gac ggg 192
Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly
50 55 60
cat acc cgc gtg tca gga ggg gca gca gcc tcc gat acc agg ggc ctt 290
His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu
65 70 75 80
gtg tcc ctc ttt agc ccc ggg tcg get cag aaa atc cag ctc gta aac 288
Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn
85 90 95
acc aac ggc agt tgg cac atc aac agg act gcc ctg aac tgc aac gac 336
Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp
100 105 110
tcc ctc caa aca ggg ttc ttt gcc gca cta ttc tac aaa cac aaa ttc 389
Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe
115 120 125
aac tcg tct gga tgc cca gag cgc ttg gcc agc tgt cgc tcc atc gac 432
Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp
130 135 140
aag ttc get cag ggg tgg ggt ccc ctc act tac act gag cct aac agc 480
Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser
145 150 155 160
tcg gac cag agg ccc tac tgc tgg cac tac gcg cct cga ccg tgt ggt 528
Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly
165 170 175
att gta ccc gcg tct cag gtg tgc ggt cca gtg tat tgc ttc acc ccg 576
Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro
180 185 190
agc cct gtt gtg gtg ggg acg acc gat cgg ttt ggt gtc ccc acg tat 624
Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr
195 200 205
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
aac tgg ggg gcg aac gac tcg gat gtg ctg att ctc aac aac acg cgg 672
Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg
210 215 220
ccg ccg cga ggc aac tgg ttc ggc tgt aca tgg atg aat ggc act ggg 720
Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly
225 230 235 240
ttc acc aag acg tgt ggg ggc ccc ccg tgc aac atc ggg ggg gcc ggc 768
Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly
245 250 255
aac aac acc ttg acc tgc ccc act gac tgt ttt cgg aag cac ccc gag 816
Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu
260 265 270
gcc acc tac gcc aga tgc ggt tct ggg ccc tgg ctg aca cct agg tgt 864
Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys
275 280 285
atg gtt cat tac cca tat agg ctc tgg cac tac ccc tgc act gtc aac 912
Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn
290 295 300
ttc acc atc ttc aag gtt agg atg tac gtg ggg ggc gtg gag cac agg 960
Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
305 310 315 320
ttc gaa gcc gca tgc aat tgg act cga gga gag cgt tgt gac ttg gag 1008
Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu
325 330 335
gac agg gat aga tca gag ctt agc ccg ctg ctg ctg tct aca aca gag 1056
Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu
340 345 350
tgg cag ata ctg ccc tgt tcc ttc acc acc ctg ccg gcc cta tcc acc 1104
Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr
355 360 365
ggc ctg atc cac ctc cat cag aac atc gtg gac gtg caa tac ctg tac 1152
Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr
370 375 380
ggt gta ggg tcg gcg gtt gtc tcc ctt gtc atc aaa tgg gag tat gtc 1200
Gly Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp Glu Tyr Val
385 390 395 400
ctg ttg ctc ttc ctt ctc ctg gca gac gcg cgc atc tgc gcc tgc tta 1248
Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu
405 410 415
tgg atg atg ctg ctg ata get caa get gag gcc gcc tta gag aac ctg 1296
Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu
420 425 430
gtg gtc ctc aat gcg gcg gcc gtg gcc ggg gcg cat ggc act ctt tcc 1344
Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly Thr Leu Ser
435 440 445
ttc ctt gtg ttc ttc tgt get gcc tgg tac atc aag ggc agg ctg gtc 1392
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val
950 455 460
cct ggt gcg gca tac gcc ttc tat ggc gtg tgg ccg ctg ctc ctg ctt 1440
Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu
465 470 475 480
ctg ctg gcc tta cca cca cga get tat gcc tagtaa 1976
Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala
985 490
<210> 36
<211> 990
<212> PRT
<213> Hepatitis C virus
<400> 36
Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser
1 5 10 15
Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala
20 25 30
His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn
35 90 95
Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly
50 55 60
His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu
65 70 75 80
Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn
85 90 95
Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp
100 105 110
Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe
115 120 125
Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp
130 135 140
Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser
145 150 155 160
Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly
165 170 175
Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro
180 185 190
Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr
195 200 205
Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg
210 215 220
Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
225 230 235 240
Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly
245 250 255
Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu
260 265 270
Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys
275 280 285
Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn
290 295 300
Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg
305 310 315 320
Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu
325 330 335
Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu
340 345 350
Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr
355 360 365
Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr
370 375 380
Gly Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp Glu Tyr Val
385 390 395 400
Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu
405 410 415
Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu
420 425 430
Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly Thr Leu Ser
435 440 945
Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val
450 455 460
Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu
465 470 475 480
Leu Leu Ala Leu.Pro Pro Arg Ala Tyr Ala
485 490
<210> 37
<211> 1021
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 2..1018
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<220>
<221> mat_peptide
<222> 2..1015
<400> 37
g atc cca caa get gtc gtg gac atg gtg gcg ggg gcc cat tgg gga 46
Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly
1 5 10 15
gtc ctg gcg ggc ctc gcc tac tat tcc atg gtg ggg aac tgg get aag 94
Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys
20 25 30
gtt ttg gtt gtg atg cta ctc ttt gcc ggc gtc gac ggg cat acc cgc 192
Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg
35 40 95
gtg tca gga ggg gca gca gcc tcc gat acc agg ggc ctt gtg tcc ctc 190
Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu
50 55 60
ttt agc ccc ggg tcg get cag aaa atc cag ctc gta aac acc aac ggc 238
Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly
65 70 75
agt tgg cac atc aac agg act gcc ctg aac tgc aac gac tcc ctc caa 286
Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln
80 85 90 95
aca ggg ttc ttt gcc gca cta ttc tac aaa cac aaa ttc aac tcg tct 334
Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser
100 105 110
gga tgc cca gag cgc ttg gcc agc tgt cgc tcc atc gac aag ttc get 382
Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala
115 120 125
cag ggg tgg ggt ccc ctc act tac act gag cct aac agc tcg gac cag 430
Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln
130 135 140
agg ccc tac tgc tgg cac tac gcg cct cga ccg tgt ggt att gta ccc 478
Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro
195 150 155
gcg tct cag gtg tgc ggt cca gtg tat tgc ttc acc ccg agc cct gtt 526
Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val
160 165 170 175
gtg gtg ggg acg acc gat cgg ttt ggt gtc ccc acg tat aac tgg ggg 574
Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly
180 185 190
gcg aac gac tcg gat gtg ctg att ctc aac aac acg cgg ccg ccg cga 622
Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg
195 200 205
ggc aac tgg ttc ggc tgt aca tgg atg aat ggc act ggg ttc acc aag 670
Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys
210 215 220
acg tgt ggg ggc ccc ccg tgc aac atc ggg ggg gcc ggc aac aac acc 718
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr
225 230 235
ttg acc tgc ccc act gac tgt ttt cgg aag cac ccc gag gcc acc tac 766
Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr
240 245 250 255
gcc aga tgc ggt tct ggg ccc tgg ctg aca cct agg tgt atg gtt cat 814
Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His
260 265 270
tac cca tat agg ctc tgg cac tac ccc tgc act gtc aac ttc acc atc 862
Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile
275 280 285
ttc aag gtt agg atg tac gtg ggg ggc gtg gag cac agg ttc gaa gcc 910
Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala
290 295 300
gca tgc aat tgg act cga gga gag cgt tgt gac ttg gag gac agg gat 958
Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp
305 310 315
aga tca gag ctt agc ccg ctg ctg ctg tct aca aca gag tgg cag agt 1006
Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Ser
320 325 330 335
ggc aga get taatta 1021
Gly Arg Ala
<210> 38
<211> 338
<212> PRT
<213> Hepatitis C virus
<400> 38
Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val
1 5 10 15
Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val
20 25 30
Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val
35 40 45
Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe
50 55 60
Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly Ser
65 70 75 80
Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr
85 90 95
Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly
100 105 110
Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gln
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
115 120 125
Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln Arg
130 135 140
Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala
145 150 155 160
Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val
165 170 175
Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala
180 185 190
Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg Gly
195 200 205
Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr
210 215 220
Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr Leu
225 230 235 240
Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala
245 250 255
Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His Tyr
260 265 270
Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe
275 280 285
Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala Ala
290 295 300
Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg
305 310 315 320
Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Ser Gly
325 330 335
Arg Ala
<210> 39
<211> 1034
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 2..1032
<220>
<221> mat_peptide
<222> 2..1029
<400> 39
g atc cca caa get gtc gtg gac atg gtg gcg ggg gcc cat tgg gga 46
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly
1 5 10 15
gtc ctg gcg ggc ctc gcc tac tat tcc atg gtg ggg aac tgg get aag 94
Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys
20 25 30
gtt ttg gtt gtg atg cta ctc ttt gcc ggc gtc gac ggg cat acc cgc 142
Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg
35 40 45
gtg tca gga ggg gca gca gcc tcc gat acc agg ggc ctt gtg tcc ctc 190
Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu
50 55 60
ttt agc ccc ggg tcg get cag aaa atc cag ctc gta aac acc aac ggc 238
Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly
65 70 75
agt tgg cac atc aac agg act gcc ctg aac tgc aac gac tcc ctc caa 286
Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln
80 85 90 95
aca ggg ttc ttt gcc gca cta ttc tac aaa cac aaa ttc aac tcg tct 334
Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser
100 105 110
gga tgc cca gag cgc ttg gcc agc tgt cgc tcc atc gac aag ttc get 382
Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala
115 120 125
cag ggg tgg ggt ccc ctc act tac act gag cct aac agc tcg gac cag 430
Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln
130 135 140
agg ccc tac tgc tgg cac tac gcg cct cga ccg tgt ggt att gta ccc 478
Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro
145 150 155
gcg tct cag gtg tgc ggt cca gtg tat tgc ttc acc ccg agc cct gtt 526
Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val
160 165 170 175
gtg gtg ggg acg acc gat cgg ttt ggt gtc ccc acg tat aac tgg ggg 574
Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly
180 185 190
gcg aac gac tcg gat gtg ctg att ctc aac aac acg cgg ccg ccg cga 622
Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg
195 200 205
ggc aac tgg ttc ggc tgt aca tgg atg aat ggc act ggg ttc acc aag 670
Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys
210 215 220
acg tgt ggg ggc ccc ccg tgc aac atc ggg ggg gcc ggc aac aac acc 718
Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr
225 230 235
ttg acc tgc ccc act gac tgt ttt cgg aag cac ccc gag gcc acc tac 766
Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr
240 245 250 255
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
gcc aga tgc ggt tct ggg ccc tgg ctg aca cct agg tgt atg gtt cat 814
Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His
260 265 270
tac cca tat agg ctc tgg cac tac ccc tgc act gtc aac ttc acc atc 862
Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile
275 280 285
ttc aag gtt agg atg tac gtg ggg ggc gtg gag cac agg ttc gaa gcc 910
Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala
290 295 300
gca tgc aat tgg act cga gga gag cgt tgt gac ttg gag gac agg gat 958
Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp
305 310 315
aga tca gag ctt agc ccg ctg ctg ctg tct aca aca ggt gat cga ggg 1006
Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gly Asp Arg Gly
320 325 330 335
cag aca cca tca cca cca tca cta at ag 1034
Gln Thr Pro Ser Pro Pro Ser Leu
340
<210> 40
<211> 343
<212> PRT
<213> Hepatitis C virus
<400> 40
Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val
1 5 10 15
Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val
20 25 30
Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val
35 40 45
Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe
50 55 60
Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly Ser
65 70 75 80
Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr
85 90 95
Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly
100 105 110
Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gln
115 120 125
Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln Arg
130 135 190
Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
145 150 155 160
Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val
165 170 175
Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala
180 185 190
Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg Gly
195 200 205
Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr
210 215 220
Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr Leu
225 230 235 240
Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala
245 250 255
Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His Tyr
260 265 270
Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe
275 280 285
Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala Ala
290 295 300
Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg
305 310 315 320
Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gly Asp Arg Gly Gln
325 330 335
Thr Pro Ser Pro Pro Ser Leu
340
<210> 41
<211> 945
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..942
<220>
<221> mat_peptide
<222> 1..939
<400> 41
atg gtg ggg aac tgg get aag gtt ttg gtt gtg atg cta ctc ttt gcc 98
Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala
1 5 10 15
ggc gtc gac ggg cat acc cgc gtg tca gga ggg gca gca gcc tcc gat 96
Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp
20 25 30
acc agg ggc ctt gtg tcc ctc ttt agc ccc ggg tcg get cag aaa atc 144
Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
35 40 45
cag ctc gta aac acc aac ggc agt tgg cac atc aac agg act gcc ctg 192
Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu
50 55 60
aac tgc aac gac tcc ctc caa aca ggg ttc ttt gcc gca cta ttc tac 240
Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr
65 70 75 80
aaa cac aaa ttc aac tcg tct gga tgc cca gag cgc ttg gcc agc tgt 288
Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys
85 90 95
cgc tcc atc gac aag ttc get cag ggg tgg ggt ccc ctc act tac act 336
Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr
100 105 110
gag cct aac agc tcg gac cag agg ccc tac tgc tgg cac tac gcg cct 389
Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro
115 120 125
cga ccg tgt ggt att gta ccc gcg tct cag gtg tgc ggt cca gtg tat 432
Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr
130 135 140
tgc ttc acc ccg agc cct gtt gtg gtg ggg acg acc gat cgg ttt ggt 480
Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly
145 150 155 160
gtc ccc acg tat aac tgg ggg gcg aac gac tcg gat gtg ctg att ctc 528
Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu
165 170 175
aac aac acg cgg ccg ccg cga ggc aac tgg ttc ggc tgt aca tgg atg 576
Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met
180 185 190
aat ggc act ggg ttc acc aag acg tgt ggg ggc ccc ccg tgc aac atc 624
Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile
195 200 205
ggg ggg gcc ggc aac aac acc ttg acc tgc ccc act gac tgt ttt cgg 672
Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg
210 215 220
aag cac ccc gag gcc acc tac gcc aga tgc ggt tct ggg ccc tgg ctg 720
Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu
225 230 235 240
aca cct agg tgt atg gtt cat tac cca tat agg ctc tgg cac tac ccc 768
Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro
245 250 255
tgc act gtc aac ttc acc atc ttc aag gtt agg atg tac gtg ggg ggc 816
Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly
260 265 270
gtg gag cac agg ttc gaa gcc gca tgc aat tgg act cga gga gag cgt 864
Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
275 280 285
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
tgt gac ttg gag gac agg gat aga tca gag ctt agc ccg ctg ctg ctg 912
Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu
290 295 300
tct aca aca gag tgg cag agc tta att aat tag 945
Ser Thr Thr Glu Trp Gln Ser Leu Ile Asn
305 310
<210> 42
<211> 314
<212> PRT
<213> Hepatitis C virus
<400> 42
Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala
1 5 10 15
Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp
20 25 30
Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile
35 90 45
Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu
50 55 60
Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr
65 70 75 80
Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys
85 90 95
Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr
100 105 110
Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro
115 120 125
Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr
130 135 140
Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly
145 150 155 160
Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu
165 170 175
Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met
180 185 190
Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile
195 200 205
Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg
210 215 220
Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu
225 230 235 240
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro
245 250 255
Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly
260 265 270
Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
275 280 285
Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu
290 295 300
Ser Thr Thr Glu Trp Gln Ser Leu Ile Asn
305 310
<210> 43
<211> 961
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..958
<220>
<221> mat_peptide
<222> 1..955
<400> 43
atg gtg ggg aac tgg get aag gtt ttg gtt gtg atg cta ctc ttt gcc 48
Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala
1 5 10 15
ggc gtc gac ggg cat acc cgc gtg tca gga ggg gca gca gcc tcc gat 96
Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp
20 25 30
acc agg ggc ctt gtg tcc ctc ttt agc ccc ggg tcg get cag aaa atc 144
Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile
35 90 45
cag ctc gta aac acc aac ggc agt tgg cac atc aac agg act gcc ctg 192
Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu
50 55 60
aac tgc aac gac tcc ctc caa aca ggg ttc ttt gcc gca cta ttc tac 240
Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr
65 70 75 80
aaa cac aaa ttc aac tcg tct gga tgc cca gag cgc ttg gcc agc tgt 288
Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys
85 90 95
cgc tcc atc gac aag ttc get cag ggg tgg ggt ccc ctc act tac act 336
Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr
100 105 110
gag cct aac agc tcg gac cag agg ccc tac tgc tgg cac tac gcg cct 384
Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro
115 120 125
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
cga ccg tgt ggt att gta ccc gcg tct cag gtg tgc ggt cca gtg tat 432
Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr
130 135 140
tgc ttc acc ccg agc cct gtt gtg gtg ggg acg acc gat cgg ttt ggt 480
Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly
145 150 155 160
gtc ccc acg tat aac tgg ggg gcg aac gac tcg gat gtg ctg att ctc 528
Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu
165 170 175
aac aac acg cgg ccg ccg cga ggc aac tgg ttc ggc tgt aca tgg atg 576
Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met
180 185 190
aat ggc act ggg ttc acc aag acg tgt ggg ggc ccc ccg tgc aac atc 624
Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile
195 200 205
ggg ggg gcc ggc aac aac acc ttg acc tgc ccc act gac tgt ttt cgg 672
Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg
210 215 220
aag cac ccc gag gcc acc tac gcc aga tgc ggt tct ggg ccc tgg ctg 720
Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu
225 230 235 240
aca cct agg tgt atg gtt cat tac cca tat agg ctc tgg cac tac ccc 768
Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro
295 250 255
tgc act gtc aac ttc acc atc ttc aag gtt agg atg tac gtg ggg ggc 816
Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly
260 265 270
gtg gag cac agg ttc gaa gcc gca tgc aat tgg act cga gga gag cgt 864
Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
275 280 285
tgt gac ttg gag gac agg gat aga tca gag ctt agc ccg ctg ctg ctg 912
Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu
290 295 300
tct aca aca ggt gat cga ggg cag aca cca tca cca cca tca cta a 958
Ser Thr Thr Gly Asp Arg Gly Gln Thr Pro Ser Pro Pro Ser Leu
305 310 315
tag 961
<210> 44
<211> 319
<212> PRT
<213> Hepatitis C virus
<400> 44
Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala
1 5 10 15
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp
20 25 30
Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile
35 40 45
Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu
50 55 60
Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr
65 70 75 80
Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys
85 90 95
Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr
100 105 110
Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro
115 120 125
Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr
130 135 140
Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly
145 150 155 160
Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu
165 170 175
Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met
180 185 190
Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile
195 200 205
Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg
210 215 220
Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu
225 230 235 240
Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro
245 250 255
Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly
260 265 270
Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
275 280 285
Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu
290 295 300
Ser Thr Thr Gly Asp Arg Gly Gln Thr Pro Ser Pro Pro Ser Leu
305 310 315
<210> 45
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<211> 1395
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..1392
<220>
<221> mat_peptide
<222> 1..1389
<400> 45
atg gtg gcg ggg gcc cat tgg gga gtc ctg gcg ggc ctc gcc tac tat 48
Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr
1 5 10 15
tcc atg gtg ggg aac tgg get aag gtt ttg gtt gtg atg cta ctc ttt 96
Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe
20 25 30
gcc ggc gtc gac ggg cat acc cgc gtg tca gga ggg gca gca gcc tcc 144
Ala Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser
35 40 45
gat acc agg ggc ctt gtg tcc ctc ttt agc ccc ggg tcg get cag aaa 192
Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys
50 55 60
atc cag ctc gta aac acc aac ggc agt tgg cac atc aac agg act gcc 240
Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala
65 70 75 80
ctg aac tgc aac gac tcc ctc caa aca ggg ttc ttt gcc gca cta ttc 288
Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe
85 90 95
tac aaa cac aaa ttc aac tcg tct gga tgc cca gag cgc ttg gcc agc 336
Tyr Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser
100 105 110
tgt cgc tcc atc gac aag ttc get cag ggg tgg ggt ccc ctc act tac 384
Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr
115 120 125
act gag cct aac agc tcg gac cag agg ccc tac tgc tgg cac tac gcg 432
Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala
130 135 140
cct cga ccg tgt ggt att gta ccc gcg tct cag gtg tgc ggt cca gtg 480
Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val
145 150 155 160
tat tgc ttc acc ccg agc cct gtt gtg gtg ggg acg acc gat cgg ttt 528
Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe
165 170 175
ggt gtc ccc acg tat aac tgg ggg gcg aac gac tcg gat gtg ctg att 576
Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile
180 185 190
ctc aac aac acg cgg ccg ccg cga ggc aac tgg ttc ggc tgt aca tgg 624
CA 02468690 2004-05-28
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Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp
195 200 205
atg aat ggc act ggg ttc acc aag acg tgt ggg ggc ccc ccg tgc aac 672
Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn
210 215 220
atc ggg ggg gcc ggc aac aac acc ttg acc tgc ccc act gac tgt ttt 720
Ile Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe
225 230 235 240
cgg aag cac ccc gag gcc acc tac gcc aga tgc ggt tct ggg ccc tgg 768
Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp
245 250 255
ctg aca cct agg tgt atg gtt cat tac cca tat agg ctc tgg cac tac 816
Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr
260 265 270
ccc tgc act gtc aac ttc acc atc ttc aag gtt agg atg tac gtg ggg 864
Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly
275 280 285
ggc gtg gag cac agg ttc gaa gcc gca tgc aat tgg act cga gga gag 912
Gly Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu
290 295 300
cgt tgt gac ttg gag gac agg gat aga tca gag ctt agc ccg ctg ctg 960
Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu
305 310 315 320
ctg tct aca aca gag tgg cag ata ctg ccc tgt tcc ttc acc acc ctg 1008
Leu Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu
325 330 335
ccg gcc cta tcc acc ggc ctg atc cac ctc cat cag aac atc gtg gac 1056
Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp
340 345 350
gtg caa tac ctg tac ggt gta ggg tcg gcg gtt gtc tcc ctt gtc atc 1104
Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val Val Ser Leu Val Ile
355 360 365
aaa tgg gag tat gtc ctg ttg ctc ttc ctt ctc ctg gca gac gcg cgc 1152
Lys Trp Glu Tyr Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg
370 375 380
atc tgc gcc tgc tta tgg atg atg ctg ctg ata get caa get gag gcc 1200
Ile Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala
385 390 395 400
gcc tta gag aac ctg gtg gtc ctc aat gcg gcg gcc gtg gcc ggg gcg 1248
Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala
405 910 415
cat ggc act ctt tcc ttc ctt gtg ttc ttc tgt get gcc tgg tac atc 1296
His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile
420 425 930
aag ggc agg ctg gtc cct ggt gcg gca tac gcc ttc tat ggc gtg tgg 1344
Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
435 440 445
ccg ctg ctc ctg ctt ctg ctg gcc tta cca cca cga get tat gcc tagtaa 1395
Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala
950 955 460
<210> 46
<211> 463
<212> PRT
<213> Hepatitis C virus
<400> 46
Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr
1 5 10 15
Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe
20 25 30
Ala Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser
35 40 45
Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys
50 55 60
Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala
65 70 75 80
Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe
85 90 95
Tyr Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser
100 105 110
Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr
115 120 125
Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala
130 135 140
Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val
145 150 155 160
Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe
165 170 175
Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile
180 185 190
Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp
195 200 205
Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn
210 215 220
Ile Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe
225 230 235 240
Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp
245 250 255
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr
260 265 270
Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly
275 280 285
Gly Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu
290 295 300
Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu
305 310 315 320
Leu Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu
325 330 335
Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp
340 345 350
Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val Val Ser Leu Val Ile
355 360 365
Lys Trp Glu Tyr Val Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg
370 375 380
Ile Cys Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala
385 390 395 400
Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala
405 910 415
His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile
420 425 430
Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp
435 440 445
Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala
450 455 460
<210> 47
<211> 2082
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..2079
<220>
<221> mat_peptide
<222> 1..2076
<400> 47
aat ttg ggt aag gtc atc gat acc ctt aca tgc ggc ttc gcc gac ctc 48
Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
gtg ggg tac att ccg ctc gtc ggc gcc ccc cta ggg ggc get gcc agg 96
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac ggc gtg aac tat gca 144
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 45
aca ggg aat ttg ccc ggt tgc tct ttc tct atc ttc ctc ttg get ttg 192
Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
ctg tcc tgt ctg acc gtt cca get tcc get tat gaa gtg cgc aac gtg 240
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
tcc ggg atg tac cat gtc acg aac gac tgc tcc aac tca agc att gtg 288
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
tat gag gca gcg gac atg atc atg cac acc ccc ggg tgc gtg ccc tgc 336
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
gtt cgg gag aac aac tct tcc cgc tgc tgg gta gcg ctc acc ccc acg 384
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
ctc gca get agg aac gcc agc gtc ccc acc acg aca ata cga cgc cac 432
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
gtc gat ttg ctc gtt ggg gcg get get ttc tgt tcc get atg tac gtg 480
Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val
145 150 155 160
ggg gac ctc tgc gga tct gtc ttc ctc gtc tcc cag ctg ttc acc atc 528
Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile
165 170 175
tcg cct cgc cgg cat gag acg gtg cag gac tgc aat tgc tca atc tat 576
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
ccc ggc cac ata acg ggt cac cgt atg get tgg gat atg atg atg aac 624
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
tgg tcg cct aca acg gcc ctg gtg gta tcg cag ctg ctc cgg atc cca 672
Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro
210 215 220
caa get gtc gtg gac atg gtg gcg ggg gcc cat tgg gga gtc ctg gcg 720
Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala
225 230 235 240
ggc ctc gcc tac tat tcc atg gtg ggg aac tgg get aag gtt ttg gtt 768
Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Val
245 250 255
gtg atg cta ctc ttt gcc ggc gtc gac ggg cat acc cgc gtg tca gga 816
Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val Ser Gly
260 265 270
ggg gca gca gcc tcc gat acc agg ggc ctt gtg tcc ctc ttt agc ccc 864
CA 02468690 2004-05-28
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Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro
275 280 285
ggg tcg get cag aaa atc cag ctc gta aac acc aac ggc agt tgg cac 912
Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His
290 295 300
atc aac agg act gcc ctg aac tgc aac gac tcc ctc caa aca ggg ttc 960
Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe
305 310 315 320
ttt gcc gca cta ttc tac aaa cac aaa ttc aac tcg tct gga tgc cca 1008
Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly Cys Pro
325 330 335
gag cgc ttg gcc agc tgt cgc tcc atc gac aag ttc get cag ggg tgg 1056
Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp
340 345 350
ggt ccc ctc act tac act gag cct aac agc tcg gac cag agg ccc tac 1104
Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr
355 360 365
tgc tgg cac tac gcg cct cga ccg tgt ggt att gta ccc gcg tct cag 1152
Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln
370 375 380
gtg tgc ggt cca gtg tat tgc ttc acc ccg agc cct gtt gtg gtg ggg 1200
Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly
385 390 395 400
acg acc gat cgg ttt ggt gtc ccc acg tat aac tgg ggg gcg aac gac 1248
Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp
405 . 410 415
tcg gat gtg ctg att ctc aac aac acg cgg ccg ccg cga ggc aac tgg 1296
Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp
420 425 930
ttc ggc tgt aca tgg atg aat ggc act ggg ttc acc aag acg tgt ggg 1344
Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly
935 940 445
ggc ccc ccg tgc aac atc ggg ggg gcc ggc aac aac acc ttg acc tgc 1392
Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys
450 455 460
ccc act gac tgt ttt cgg aag cac ccc gag gcc acc tac gcc aga tgc 1440
Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys
465 470 975 480
ggt tct ggg ccc tgg ctg aca cct agg tgt atg gtt cat tac cca tat 1988
Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr
485 990 995
agg ctc tgg cac tac ccc tgc act gtc aac ttc acc atc ttc aag gtt 1536
Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val
500 505 510
agg atg tac gtg ggg ggc gtg gag cac agg ttc gaa gcc gca tgc aat 1584
Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala Ala Cys Asn
515 520 525
CA 02468690 2004-05-28
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tgg act cga gga gag cgt tgt gac ttg gag gac agg gat aga tca gag 1632
Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu
530 535 540
ctt agc ccg ctg ctg ctg tct aca aca gag tgg cag ata ctg ccc tgt 1680
Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys
545 550 555 560
tcc ttc acc acc ctg ccg gcc cta tcc acc ggc ctg atc cac ctc cat 1728
Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His
565 570 575
cag aac atc gtg gac gtg caa tac ctg tac ggt gta ggg tcg gcg gtt 1776
Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val
580 585 590
gtc tcc ctt gtc atc aaa tgg gag tat gtc ctg ttg ctc ttc ctt ctc 1824
Val Ser Leu Val Ile Lys Trp Glu Tyr Val Leu Leu Leu Phe Leu Leu
595 600 605
ctg gca gac gcg cgc atc tgc gcc tgc tta tgg atg atg ctg ctg ata 1872
Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu Trp Met Met Leu Leu Ile
610 615 620
get caa get gag gcc gcc tta gag aac ctg gtg gtc ctc aat gcg gcg 1920
Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala
625 630 635 640
gcc gtg gcc ggg gcg cat ggc act ctt tcc ttc ctt gtg ttc ttc tgt 1968
Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys
645 650 655
get gcc tgg tac atc aag ggc agg ctg gtc cct ggt gcg gca tac gcc 2016
Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala
660 665 670
ttc tat ggc gtg tgg ccg ctg ctc ctg ctt ctg ctg gcc tta cca cca 2064
Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro
675 680 685
cga get tat gcc tagtaa 2082
Arg Ala Tyr Ala
690
<210> 48
<211> 692
<212> PRT
<213> Hepatitis C virus
<400> 48
Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu
1 5 10 15
Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg
20 25 30
Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala
35 40 95
CA 02468690 2004-05-28
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Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu
50 55 60
Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val
65 70 75 80
Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val
85 90 95
Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys
100 105 110
Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr
115 120 125
Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His
130 135 140
Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val
145 150 155 160
Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile
165 170 175
Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr
180 185 190
Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn
195 200 205
Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro
210 215 220
Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala
225 230 235 240
Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Val
245 250 255
Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val Ser Gly
260 265 270
Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro
275 280 285
Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His
290 295 300
Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly Phe
305 310 315 320
Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly Cys Pro
325 330 335
Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp
340 345 350
Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr
355 360 365
Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln
CA 02468690 2004-05-28
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370 375 380
Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly
385 390 395 400
Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp
405 410 415
Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp
420 425 430
Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly
435 940 445
Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys
450 455 460
Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys
465 470 475 480
Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr
485 490 495
Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val
500 505 510
Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala Ala Cys Asn
515 520 525
Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu
530 535 540
Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys
545 550 555 560
Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu His
565 570 575
Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val
580 585 590
Val Ser Leu Val Ile Lys Trp Glu Tyr Val Leu Leu Leu Phe Leu Leu
595 600 605
Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu Trp Met Met Leu Leu Ile
610 615 620
Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala
625 630 635 640
Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys
645 650 655
Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala
660 665 670
Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro
675 680 685
Arg Ala Tyr Ala
690
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<210> 49
<211> 2433
<212> DNA
<213> Hepatitis C virus
<220>
<221> CDS
<222> 1..2430
<220>
<221> mat_peptide
<222> 1..2427
<400> 49
atg agc acg aat cct aaa cct caa aga aaa acc aaa cgt aac acc aac 48
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
cgc cgc cca cag gac gtc aag ttc ccg ggc ggt ggt cag atc gtt ggt 96
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
gga gtt tac ctg ttg ccg cgc agg ggc ccc agg ttg ggt gtg cgc gcg 144
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
act agg aag act tcc gag cgg tcg caa cct cgt ggg agg cga caa cct 192
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60 '
atc ccc aag get cgc cga ccc gag ggt agg gcc tgg get cag ccc ggg 240
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly
65 70 75 80
tac cct tgg ccc ctc tat ggc aat gag ggc atg ggg tgg gca gga tgg 288
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp
85 90 95
ctc ctg tca ccc cgc ggc tct cgg cct agt tgg ggc cct aca gac ccc 336
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
cgg cgt agg tcg cgt aat ttg ggt aag gtc atc gat acc ctt aca tgc 384
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
ggc ttc gcc gac ctc gtg ggg tac att ccg ctc gtc ggc gcc ccc cta 432
Gly Phe Ala Asp Leu Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
ggg ggc get gcc agg gcc ctg gcg cat ggc gtc cgg gtt ctg gag gac 480
Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
195 150 155 160
ggc gtg aac tat gca aca ggg aat ttg ccc ggt tgc tct ttc tct atc 528
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
ttc ctc ttg get ttg ctg tcc tgt ctg acc gtt cca get tcc get tat 576
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr
180 185 190
gaagtgcgc aacgtgtcc gggatgtaccat gtcacgaac gactgctcc 624
GluValArg AsnValSer GlyMetTyrHis ValThrAsn AspCysSer
195 200 205
aactcaagc attgtgtat gaggcagcggac atgatcatg cacaccccc 672
AsnSerSer IleValTyr GluAlaAlaAsp MetIleMet HisThrPro
210 215 220
gggtgcgtg ccctgcgtt cgggagaacaac tcttcccgc tgctgggta 720
GlyCysVal ProCysVal ArgGluAsnAsn SerSerArg CysTrpVal
225 230 235 290
gcgctcacc cccacgctc gcagetaggaac gccagcgtc cccaccacg 768
AlaLeuThr ProThrLeu AlaAlaArgAsn AlaSerVal ProThrThr
245 250 255
acaatacga cgccacgtc gatttgctcgtt ggggcgget getttctgt 816
ThrIleArg ArgHisVal AspLeuLeuVal GlyAlaAla AlaPheCys
260 265 270
tccgetatg tacgtgggg gacctctgcgga tctgtcttc ctcgtctcc 864
SerAlaMet TyrValGly AspLeuCysGly SerValPhe LeuValSer
275 280 285
cagctgttc accatctcg cctcgccgg catgagacggtg caggactgc 912
GlnLeuPhe ThrIleSer ProArgArg HisGluThrVal GlnAspCys
290 295 300
aattgctca atctatccc ggccacata acgggtcaccgt atggettgg 960
AsnCysSer IleTyrPro GlyHisIle ThrGlyHisArg MetAlaTrp
305 310 315 320
gatatgatg atgaactgg tcgcctaca acggccctggtg gtatcgcag 1008
AspMetMet MetAsnTrp SerProThr ThrAlaLeuVal ValSerGln
325 330 335
ctgctccgg atc'ccacaa getgtcgtg gacatggtggcg ggggcccat 1056
LeuLeuArg IleProGln AlaValVal AspMetValAla GlyAlaHis
340 345 350
tggggagtc ctggcgggc ctcgcctac tattccatggtg gggaactgg 1104
TrpGlyVal LeuAlaGly LeuAlaTyr TyrSerMetVal GlyAsnTrp
355 360 365
getaaggtt ttggttgtg atgctactc tttgccggcgtc gacgggcat 1152
AlaLysVal LeuValVal MetLeuLeu PheAlaGlyVal AspGlyHis
370 375 380
acccgcgtg tcaggaggg gcagcagcc tccgataccagg ggccttgtg 1200
ThrArgVal SerGlyGly AlaAlaAla SerAspThrArg GlyLeuVal
385 390 395 400
tccctcttt agccccggg tcggetcag aaaatccagctc gtaaacacc 1248
SerLeuPhe SerProGly SerAlaGln LysIleGlnLeu ValAsnThr
405 410 415
aacggcagt tggcacatc aacaggact gccctgaactgc aacgactcc 1296
AsnGlySer TrpHisIle AsnArgThr AlaLeuAsnCys AsnAspSer
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
420 425 430
ctc caa aca ggg ttc ttt gcc gca cta ttc tac aaa cac aaa ttc aac 1344
Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn
435 490 445
tcg tct gga tgc cca gag cgc ttg gcc agc tgt cgc tcc atc gac aag 1392
Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys
450 455 460
ttc get cag ggg tgg ggt ccc ctc act tac act gag cct aac agc tcg 1440
Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser
465 470 475 480
gac cag agg ccc tac tgc tgg cac tac gcg cct cga ccg tgt ggt att 1488
Asp Gln-Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile
485 490 495
gta ccc gcg tct cag gtg tgc ggt cca gtg tat tgc ttc acc ccg agc 1536
Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser
500 505 510
cct gtt gtg gtg ggg acg acc gat cgg ttt ggt gtc ccc acg tat aac 1584
Pro Val Val,Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn
515 520 525
tgg ggg gcg aac gac tcg gat gtg ctg att ctc aac aac acg cgg ccg 1632
Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro
530 535 540
ccg cga ggc aac tgg ttc ggc tgt aca tgg atg aat ggc act ggg ttc 1680
Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe
545 550 555 560
acc aag acg tgt ggg ggc ccc ccg tgc aac atc ggg ggg gcc ggc aac 1728
Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn
565 570 575
aac acc ttg acc tgc ccc act gac tgt ttt cgg aag cac ccc gag gcc 1776
Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala
580 585 590
acc tac gcc aga tgc ggt tct ggg ccc tgg ctg aca cct agg tgt atg 1824
Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met
595 600 605
gtt cat tac cca tat agg ctc tgg cac tac ccc tgc act gtc aac ttc 1872
Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe
610 615 620
acc atc ttc aag gtt agg atg tac gtg ggg ggc gtg gag cac agg ttc 1920
Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe
625 630 635 640
gaa gcc gca tgc aat tgg act cga gga gag cgt tgt gac ttg gag gac 1968
Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp
645 650 655
agg gat aga tca gag ctt agc ccg ctg ctg ctg tct aca aca gag tgg 2016
Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp
660 665 670
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
cagatactg ccctgttccttc accaccctg ccggcccta tccaccggc 2064
GlnIleLeu ProCysSerPhe ThrThrLeu ProAlaLeu SerThrGly
675 680 685
ctgatccac ctccatcagaac atcgtggac gtgcaatac ctgtacggt 2112
LeuIleHis LeuHisGlnAsn IleValAsp ValGlnTyr LeuTyrGly
690 695 700
gtagggtcg gcggttgtctcc cttgtcatc aaatgggag tatgtcctg 2160
ValGlySer AlaValValSer LeuValIle LysTrpGlu TyrValLeu
705 710 715 720
ttgctcttc cttctcctggca gacgcgcgc atctgcgcc tgcttatgg 2208
LeuLeuPhe LeuLeuLeuAla AspAlaArg IleCysAla CysLeuTrp
725 730 735
atg atg ctg ctg ata get caa get gag gcc gcc tta gag aac ctg gtg 2256
Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val
740 745 750
gtc ctc aat gcg gcg gcc gtg gcc ggg gcg cat ggc act ctt tcc ttc 2304
Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe
755 760 765
ctt gtg ttc ttc tgt get gcc tgg tac atc aag ggc agg ctg gtc cct 2352
Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro
770 775 780
ggt gcg gca tac gcc ttc tat ggc gtg tgg ccg ctg CtC Ctg ctt ctg 2400
Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu
785 790 795 800
ctg gcc tta cca cca cga get tat gcc tagtaa 2433
Leu Ala Leu Pro Pro Arg Ala Tyr Ala
805 810
<210> 50
<211> 809
<212> PRT
<213> Hepatitis C virus
<400> 50
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly
65 70 75 80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
Gly Phe Ala Asp Leu Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Va1 Leu Glu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr
180 185 190
Glu Val Arg Asn Val Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser
195 200 205
Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro
210 215 220
Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val
225 230 235 240
Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr
245 250 255
Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys
260 265 270
Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser
275 280 285
Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys
290 295 300
Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp
305 310 315 320
Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln
325 330 335
Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His
340 345 350
Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp
355 360 365
Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His
370 375 380
Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val
385 390 395 400
Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr
405 410 415
CA 02468690 2004-05-28
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Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser
420 425 430
Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn
435 440 445
Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys
450 455 460
Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser
465 470 475 480
Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile
485 490 495
Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser
500 505 510
Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn
515 520 525
Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro
530 535 540
Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe
545 550 555 560
Thr~Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn
565 570 575
Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala
580 585 590
Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met
595 600 605
Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe
610 615 620
Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe
625 630 635 640
Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp
645 650 655
Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp
660 665 670
Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr Gly
675 680 685
Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln Tyr Leu Tyr Gly
690 695 700
Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp Glu Tyr Val Leu
705 710 715 720
Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu Trp
725 730 735
Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu Glu Asn Leu Val
CA 02468690 2004-05-28
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740 745 750
Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe
755 760 765
Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro
770 775 780
Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu
785 790 795 800
Leu Ala Leu Pro Pro Arg Ala Tyr Ala
805
<210> 51
<211> 17
<212> PRT
<213> Hepatitis C virus
<220>
<221> Modified-site
<222> 1..17
<400> 51
Ser Asn Ser Ser Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys
1 5 10 15
Val
<210> 52
<211> 22
<212> PRT
<213> Hepatitis C virus
<220>
<221> Modified-site
<222> 1..22
<400> 52
Gly Gly Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
1 5 10 15
Ser Pro Thr Thr Ala Leu
<210> 53
<211> 37
<212> PRT
<213> Hepatitis C virus
<220>
<221> Modified-site
<222> 1..37
<400> 53
Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys
1 5 10 15
CA 02468690 2004-05-28
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Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr
20 25 30
Pro Gly Cys Gly Lys
<210> 54
<211> 25
<212> PRT
<213> Hepatitis C virus
<220>
<221> Modified-site
<222> 1..25
<400> 54
Gly Gly Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr
1 5 10 15
Gln Leu Arg Arg His Ile Asp Leu Leu
20 25
<210> 55
<211> 25
<212> PRT
<213> Hepatitis C virus
<220>
<221> Modified-site
<222> 1..25
<400> 55
Gly Gly Thr Pro Thr Leu Ala Ala Arg Asp Ala Ser Val Pro Thr Thr
1 5 10 15
Thr Ile Arg Arg His Val Asp Leu Leu
20 25
<210> 56
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 56
Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gln Val Arg Asn
1 5 10 15
Ser Thr Gly Leu
<210> 57
<211> 20
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<400> 57
Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr Asn Asp Cys Pro
1 5 10 15
Asn Ser Ser Ile
<210> 58
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 58
Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Ala His Asp Ala Ile
1 5 10 15
Leu His Thr Pro
<210> 59
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 59
Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr
1 5 10 15
Pro Gly Cys Val
<210> 60
<211> 19
<212> PRT
<213> Hepatitis C virus
<400> 60
His Asp Ala Ile Leu His Thr Pro Gly Val Pro Cys Val Arg Glu Gly
1 5 10 15
Asn Val Ser
<210> 61
<211> 20
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<400> 61
Cys Val Arg Glu Gly Asn Val Ser Arg Cys Trp Val Ala Met Thr Pro
1 5 10 15
Thr Val Ala Thr
<210> 62
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 62
Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr
1 5 10 15
Gln Leu Arg Arg
<210> 63
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 63
Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser
1 5 10 15
Ala Thr Leu Cys
<210> 64
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 69
Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu
1 5 10 15
Cys Gly Ser Val
<210> 65
<211> 20
CA 02468690 2004-05-28
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<212> PRT
<213> Hepatitis C virus
<400> 65
Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Gly Cys
1 5 10 15
Asn Cys Ser Ile
<210> 66
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 66
Thr Gln Gly Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His
1 5 10 15
Arg Met Ala Trp
<210> 67
<211> 20
<212> PRT
<213> Hepatitis C virus
<900> 67
Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro
1 5 10 15
Thr Ala Ala Leu
<210> 68
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 68
Asn Trp Ser Pro Thr Ala Ala Leu Val Met Ala Gln Leu Leu Arg Ile
1 5 10 15
Pro Gln Ala Ile
<210> 69
CA 02468690 2004-05-28
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<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 69
Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His
1 5 10 15
Trp Gly Val Leu
<210> 70
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 70
Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met
1 5 10 15
Val Gly Asn Met
<210> 71
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 71
Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr Ile Val Ser
1 5 10 15
Gly Gly Gln Ala
<210> 72
<211> 20
<212> PRT
<213> Hepatitis C virus
<900> 72
Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln
1 5 10 15
Leu Ile Asn Thr
CA 02468690 2004-05-28
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<210> 73
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 73
Gln Asn Ile Gln Leu Ile Asn Thr Asn Gly Gln Trp His Ile Asn Ser
1 5 10 15
Thr Ala Leu Asn
<210> 74
<211> 21
<212> PRT
<213> Hepatitis C virus
<900> 74
Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Trp Leu Ala Gly Leu
1 5 10 15
Ile Tyr Gln His Lys
<210> 75
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 75
Ala Gly Leu Ile Tyr Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu
1 5 10 15
Arg Leu Ala Ser
<210> 76
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 76
Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp Phe Asp
1 5 10 15
CA 02468690 2004-05-28
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Gln Gly Trp Gly
<210> 77
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 77
Thr Asp Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser
1 5 10 15
Gly Pro Asp Gln
<210> 78
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 78
Ala Asn Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro
1 5 10 15
Pro Lys Pro Cys
<210> 79
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 79
Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val
1 5 10 15
Cys Gly Pro Val
<210> 80
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 80
CA 02468690 2004-05-28
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Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val
1 5 10 15
Val Val Gly Thr
<210> 81
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 81
Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr
1 5 10 15
Tyr Ser Trp Gly
<210> 82
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 82
Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn Asp Thr Asp Val Phe Val
1 5 10 15
Leu Asn Asn Thr
<210> 83
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 83
Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys
1 5 10 15
Val Cys Gly Ala
<210> 84
<211> 20
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<400> 84
Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Val Cys Ile Gly Gly Ala
1 5 10 15
Gly Asn Asn Thr
<210> 85
<211> 19
<212> PRT
<213> Hepatitis C virus
<400> 85
Ile Gly Gly Ala Gly Asn Asn Thr Leu His Cys Pro Thr Asp Cys Arg
1 5 10 15
Lys His Pro
<210> 86
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 86
Thr Asp Cys Phe Arg Lys His Pro Asp Ala Thr Tyr Ser Arg Cys Gly
1 5 10 15
Ser Gly Pro Trp
<210> 87
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 87
Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Leu Val Asp
1 5 10 15
Tyr Pro Tyr Arg
<210> 88
<211> 20
<212> PRT
<213> Hepatitis C virus
CA 02468690 2004-05-28
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<400> 88
Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile
1 5 10 15
Asn Tyr Thr Ile
<210> 89
<211> 20
<212> PRT
<213> Hepatitis C virus
<900> 89
Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly
1 5 10 15
Gly Val Glu His
<210> 90
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 90
Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp
1 5 10 15
Thr Pro Gly Glu
<210> 91
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 91
Ala Cys Asn Trp Thr Pro Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp
1 5 10 15
Arg Ser Glu Leu
<210> 92
<211> 20
CA 02468690 2004-05-28
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<212> PRT
<213> Hepatitis C virus
<400> 92
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr
1 5 10 15
Gln Trp Gln Val
<210> 93
<211> 9
<212> PRT
<213> Hepatitis C virus
<400> 93
Tyr Gln Val Arg Asn Ser Thr Gly Leu
1 5
<210> 94
<211> 29
<212> DNA
<213> Hepatitis C virus
<400> 94
agctaattaa ttaagcttgc atgcctgca 29
<210> 95
<211> 60
<212> DNA
<213> Hepatitis C virus
<400> 95
gttaattaac tattagtgat ggtggtgatg gtgtctgccc tcgatcacgt gcaggcctcc 60
<210> 96
<211> 19
<212> DNA
<213> Hepatitis C virus
<900> 96
gtttaaccac tgcatgatg 19
<210> 97
<211> 20
CA 02468690 2004-05-28
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<212> DNA
<213> Hepatitis C virus
<400> 97
gtcccatcga gtgcggctac 20
<210> 98
<211> 45
<212> DNA
<213> Hepatitis C virus
<400> 98
cgtgacatgg tacattccgg acacttggcg cacttcataa gcgga 45
<210> 99
<211> 42
<212> DNA
<213> Hepatitis C virus
<400> 99
tgcctcatac acaatggagc tctgggacga gtcgttcgtg ac 42
<210> 100
<211> 92
<212> DNA
<213> Hepatitis C virus
<400> 100
tacccagcag cgggagctct gttgctcccg aacgcagggc ac 42
<210> 101
<211> 42
<212> DNA
<213> Hepatitis C virus
<400> 101
tgtcgtggtg gggacggagg cctgcctagc tgcgagcgtg gg 42
<210> 102
<211> 48
<212>
<400> 102
cgttatgtgg cccgggtaga ttgagcactg gcagtcctgc accgtctc 48
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<210> 103
<211> 42
<212> DNA
<213> Hepatitis C virus
<400> 103
cagggccgtt ctaggcctcc actgcatcat catatcccaa gc 42
<210> 104
<211> 26
<212> DNA
<213> Hepatitis C virus
<400> 104
ccggaatgta ccatgtcacg aacgac 26
<210> 105
<211> 24
<212> DNA
<213> Hepatitis C virus
<400> 105
gctccattgt gtatgaggca gcgg 24
<210> 106
<211> 23
<212> DNA
<213> Hepatitis C virus
<400> 106
gagctcccgc tgctgggtag cgc 23
<210> 107
<211> 25
<212> DNA
<213> Hepatitis C virus
<400> 107
cctccgtccc caccacgaca atacg 25
<210> 108
CA 02468690 2004-05-28
WO 03/051912 PCT/EP02/14480
<211> 27
<212> DNA
<213> Hepatitis C virus
<400> 108
ctacccgggc cacataacgg gtcaccg 27
<210> 109
<211> 24
<212> DNA
<213> Hepatitis C virus
<400> 109
ggaggcctac aacggccctg gtgg 24
<210> 110
<211> 22
<212> DNA
<213> Hepatitis C virus
<400> 110
ttctatcgat taaatagaat tc 22
<210> 111
<211> 23
<212> DNA
<213> Hepatitis C virus
<400> 111
gccatacgct cacagccgat ccc 23
<210> 112
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 112
Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys
1 5 10 15
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Ser Asn Ser Ser
<210> 113
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 113
Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp
1 5 10 15
Met Ile Met His Thr
<210> 114
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 114
Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val
1 5 10 15
Arg Glu Asn Asn Ser
<210> 115
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 115
Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu
1 5 10 15
Thr Pro Thr Leu Ala
<210> 116
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 116
Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro
1 5 10 15
Thr Thr Thr Ile Arg
<210> 117
CA 02468690 2004-05-28
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<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 117
Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val
1 5 10 15
Gly Ala Ala Ala Phe
<210> 118
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 118
Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly
1 5 10 15
Asp Leu Cys Gly Ser
<210> 119
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 119
Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu
1 5 10 15
Phe Thr Ile Ser Pro
<210> 120
<211> 20
<212> PRT
<213> Hepatitis C virus
<900> 120
Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln
1 5 10 15
Asp Cys Asn Cys Ser
<210> 121
<211> 20
<212> PRT
CA 02468690 2004-05-28
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<213> Hepatitis C virus
<400> 121
Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr
1 5 10 15
Gly His Arg Met Ala
<210> 122
<211> 20
<212> PRT
<213> Hepatitis C virus
<400> 122
His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp
1 5 10 15
Ser Pro Thr Thr Ala
<210> 123
<211> 135
<212> PRT
<213> hepatitis C virus
<400> 123
Tyr Glu Val Arg Asn Val Ser Gly Met Tyr His Val Thr Asn Asp Cys
1 5 10 15
Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr
20 25 30
Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser Arg Cys Trp
35 90 45
Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr
50 55 60
Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe
65 70 75 80
Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val
85 90 95
Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp
100 105 110
Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala
CA 02468690 2004-05-28
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115 120 125
Trp Asp Met Met Met Asn Trp
130 135
