U.S. patent application number 11/678513 was filed with the patent office on 2008-06-12 for purified hepatitis c virus envelope proteins for diagnostic and therapeutic use.
This patent application is currently assigned to INNOGENETICS N.V.. Invention is credited to Fons Bosman, Marie-Ange Buyse, Guy De Martynoff, Geert Maertens.
Application Number | 20080138894 11/678513 |
Document ID | / |
Family ID | 8218662 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138894 |
Kind Code |
A1 |
Maertens; Geert ; et
al. |
June 12, 2008 |
PURIFIED HEPATITIS C VIRUS ENVELOPE PROTEINS FOR DIAGNOSTIC AND
THERAPEUTIC USE
Abstract
The present invention relates to a method for purifying
recombinant HCV single or specific oligomeric envelope proteins
selected from the group consisting of E1 and/or E1/E2 characterized
in that upon lysing the transformed host cells to isolate the
recombinantly expressed protein a disulphide bond cleavage or
reduction step is carried out with a disulphide bond cleavage
agent. The present invention also relates to a composition isolated
by such a method. The present invention also relates to the
diagnostic and therapeutic application of these compositions.
Furthermore, the invention relates to the use of HCV E1 protein and
peptides for prognosing and monitoring the clinical effectiveness
and/or clinical outcome of HCV treatment.
Inventors: |
Maertens; Geert; (Brugge,
BE) ; Bosman; Fons; (Opwijk, BE) ; De
Martynoff; Guy; (Waterloo, BE) ; Buyse;
Marie-Ange; (Merelbeke, BE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
INNOGENETICS N.V.
Ghent
BE
|
Family ID: |
8218662 |
Appl. No.: |
11/678513 |
Filed: |
February 23, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09899303 |
Jul 6, 2001 |
|
|
|
11678513 |
Feb 23, 2007 |
|
|
|
08928017 |
Sep 11, 1997 |
|
|
|
09899303 |
Jul 6, 2001 |
|
|
|
08612973 |
Mar 11, 1996 |
6150134 |
|
|
PCT/EP95/03031 |
Jul 31, 1995 |
|
|
|
08928017 |
Sep 11, 1997 |
|
|
|
Current U.S.
Class: |
435/325 ;
435/243; 435/320.1; 536/23.72 |
Current CPC
Class: |
C07K 16/109 20130101;
C12N 2770/24243 20130101; C12N 2770/24222 20130101; A61K 39/00
20130101; Y10S 435/803 20130101; Y10S 435/81 20130101; C07K 2317/34
20130101; Y10S 435/975 20130101; A61K 39/12 20130101; C12N
2770/24234 20130101; C07K 14/005 20130101; Y10S 435/915 20130101;
C12N 2710/24143 20130101; A61K 2039/5256 20130101; A61P 1/16
20180101; A61K 39/29 20130101; A61P 31/12 20180101 |
Class at
Publication: |
435/325 ;
435/243; 435/320.1; 536/023.72 |
International
Class: |
C12N 15/00 20060101
C12N015/00; C12N 1/00 20060101 C12N001/00; C12N 15/11 20060101
C12N015/11; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 29, 1994 |
EP |
94870132.1 |
Claims
1. A recombinant vector comprising a vector sequence and an:
appropriate eukaryotic or viral promoter sequence operably linked
to a nucleotide sequence comprising an HCV signal sequence attached
before a sequence encoding an HCV envelope E2 protein starting at
amino acid position 384 and ending in the region between amino acid
positions 600 and 820 and wherein said HCV envelope E2 protein is
produced by recombinant expression in lower eukaryotic or mammalian
host cells.
2. The recombinant vector according to claim 1, with said
nucleotide sequence being characterized further in that it ends at
any of amino acid positions 623, 650, 673, 710, 715, 720, 746 or
809.
3. The recombinant vector according to claim 1, wherein said
nucleotide sequence is comprising a sequence encoding an HCV signal
sequence starting at amino acid position 347 or 364 and ending at
amino acid position 383 and a sequence encoding an HCV envelope E2
protein starting at amino acid position 384.
4. The recombinant vector according to claim 1, with said
nucleotide sequence being characterized further in that a
5'-terminal ATG codon and a 3'-terminal stop codon have been added
to it.
5. The recombinant vector according to claim 1, with said
nucleotide sequence being characterized further in that a factor Xa
cleavage site and/or 3 to 10, histidine codons have been added.
6. The recombinant vector according to claim 5, wherein said
histidine codons are added 3'-terminally to the coding region.
7. The recombinant vector according to claim 1, with said HCV E2
encoding sequence being chosen from the group as represented in SEQ
ID NO 35, 37, 39, 41, 43, and 45.
8. The recombinant vector according to claim 1, with said
nucleotide sequence encoding an HCV protein spanning amino acid
positions 384-746, 384-809, 384-673, 347-746, 347-809, 347-673,
347-683, 364-673, 364-746 or 384-673.
9. Recombinant nucleic acid comprising any of the HCV E2 encoding
sequences as represented in SEQ ID NO 35, 37, 39, 41, 43, 45.
10. Recombinant nucleic acid encoding an HCV E2 protein spanning
amino acid positions 384-746, 384-809, 384-673, 347-746, 347-809,
347-673, 347-683, 364-673, 364-746 or 384-673.
11. Recombinant vector according to claim 1, further characterized
in that at least one of the glycosylation sites present in said HCV
protein has been removed by site-directed mutagenesis of the
nucleotide sequence encoding a glycosylation site.
12. An isolated host cell transformed with at least one recombinant
vector according to claim 1.
13. A composition comprising a recombinant vector according to
claim 1.
Description
[0001] The present application is a divisional of Ser. No.
09/899,303, filed Jul. 6, 2001 (pending), which is a divisional of
Ser. No. 08/928,017, filed Sep. 11, 1997 (abandoned), which is a
divisional of Ser. No. 08/612,973, filed Mar. 11, 1996 (now U.S.
Pat. No. 6,150,134), which is a 371 U.S. national phase of
PCT/EP95/03031, filed Jul. 31, 1995 and claims benefit of EP
94870132.1 filed Jul. 29, 1994, the entire contents of each of
which is incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the general fields of
recombinant protein 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.
[0003] 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
[0004] The E2 protein purified from cell lysates according to the
methods described in the 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. 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 (Inoue 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).
[0005] About 75% of HCV sera (and 95% of chronic patients) are
anti-s positive using the purified, vaccinia-expressed recombinant
E1 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
14150 (28%) sera using baculovirus-expressed E1.
[0006] These results show that nor 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.
AIMS OF THE INVENTION
[0007] 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.
[0008] It is another aim of the present invention to provide
compositions comprising purified (single or specific oligomeric)
recombinant E1 and/or E2 and/or E1/E2 glycoproteins comprising
conformational epitopes from the E1 and/or E2 domains of HCV.
[0009] 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.
[0010] It is also an aim of the present invention to provide a
method for producing and purifying recombinant HCV E1 and/or E2
and/or E1/E2 proteins.
[0011] 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 E1/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 E1/E2
proteins of the present invention.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] All the aims of the present invention are considered to have
been met by the embodiments as set out below.
DEFINITIONS
[0019] The following definitions serve to illustrate the different
terms and expressions used in the present invention.
[0020] 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 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 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 topically 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.
[0021] The HCV antigens of the present invention comprise
conformational epitopes from the E1 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,
preferably 748, 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 810-1025), 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.
[0022] 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.
[0023] The E1 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
(polygonal 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.
[0024] The HCV antigens of the present invention can be made by any
recombinant method that provides the epitope of interest. 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 mutant yeast strains (e.g.
mnn 9 mutant (Kniskern et al., 1194) or glycosylation mutants
derived by means of vanadate resistance 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 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.
[0025] The term `fusion polypeptide` intends a polypeptide in which
the HCV antigen(s) 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 intervening amino acid sequences. The fusion
polypeptides may also contain amino acid sequences exogenous to
HCV.
[0026] 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 means such as
hydrophobic adsorption.
[0027] 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.
[0028] 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.
[0029] The term `immune complex` intends the combination formed
when an antibody binds to an epitope on an antigen.
[0030] `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 membranes. In most natural HCV strains, the E1
protein is encoded in the viral polyprotein following the C (core)
protein. The E1 protein extends from approximately amino acid (aa)
192 to about aa 383 of the full-length polyprotein.
[0031] The term `E1` as used herein also includes analogs and
truncated forms that are immunologically cross-reactive with
natural E1, and includes E1 proteins of genotypes 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or any other newly identified HCV type or subtype.
[0032] `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 NS1 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 E1 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 well as deletions of amino acids 384-387 have
been reported by Kato et al. (1992).
[0033] `E1/E2` as used herein refers to an oligomeric form of
envelope proteins containing at least one E1 component and at least
one E2 component.
[0034] The term `specific oligomeric` E1 and/or E2 and/or E1/E2
envelope proteins refers to all possible oligomeric forms of
recombinantly expressed E1 and/or E2 envelope proteins which are
not aggregates. E1 and/or E2 specific oligomeric envelope proteins
are also referred to as homo-oligomeric E1 or E2 envelope proteins
(see below).
[0035] 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 E1/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) (E2), wherein x can be a number
between 0 and 100, and y can be a number between 0 and 100,
provided that x and y are not both 0. With x=1 and y=0 said
envelope proteins include monomeric E1.
[0036] The term `homo-oligomer` as used herein refers to a complex
of E1 and/or E2 containing more than one E1 or E2 monomer, e.g.
E1/E1 dimers, E1/E1E1 trimers or E1/E1/E1/E1, tetramers and E2/E2
dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, E1 pentamers and
hexamers, E2 pentamers and hexamers or any higher-order
homo-oligomers of E1 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.
[0037] 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 at least about
95% of the total protein component. The composition may contain
other compounds such as carbohydrates, salts, lipids, solvents, and
the like, without affecting the determination of the percentage
purity as used herein. An `isolated` HCV protein intends an HCV
protein composition that is at least 35% pure.
[0038] 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 to pure, preferably, 90%, more preferably
95%, more preferably 97%, more preferably 98%, more preferably 99%,
even more preferably 99%, and most preferably the contaminating
proteins should be undetectable by conventional methods like
SDS-PAGE and silver staining.
[0039] 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.
[0040] The term `lower eukarycote` 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,
Kluveromyces, 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.
[0041] The term `prokaryotes` refers to hosts such as E. coli,
Lactobacillus. Lactococcus, Salmonella, Streptococcus, Bacillus
subtilis or Streptomyces. Also these hosts are contemplated within
the present invention.
[0042] The term `higher eukaryote` refers to host cells derived
from higher animals, such as mammals, reptiles, insects, and the
like. Presently preferred higher eukaryote host cells are derived
from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells),
baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13
cells (RK13), the human osteosarcoma cell line 143 B, the human
cell line HeLa and human hepatoma cell lines like Hep G2, and
insect cell lines (e.g. Spodoptera 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.
[0043] 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 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] The term `vector` is a replicon further comprising sequences
providing replication and/or expression of a desired open reading
frame.
[0048] 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 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.
[0049] 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 a; the normal transcription initiation site
for the adjacent structural gene.
[0050] 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.
[0051] 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.
[0052] 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 or the coding sequence are determined by a translation
star codon at the 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.
[0053] 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 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
1a, 1b, 1c, 1d, 1e, 1f, 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, 4l,
5a, 5b, 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 amino
acids constituting the epitope need not be par, of a linear
sequence, but may be interspersed by any number of amino acids,
thus forming a conformational epitope.
[0054] 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 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.
[0055] The term `therapeutic` refers to a composition capable of
treating HCV infection.
[0056] 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 .mu.g/dose, preferably
0.1 to 50 .mu.g/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
[0057] 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 transformed host cells to isolate the recombinantly
expressed protein a disulphide bond cleavage or reduction step is
carried out with a disulphide bond cleaving agent.
[0058] 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.
[0059] 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
as de-ailed in the Examples section.
[0060] 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 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
resistance (Ballou et al., 1991).
[0061] 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
WO92/08734 that `aggregates` were obtained after purification, but
it was not described which protein interactions were involved. In
patent application WO 92/08734, recombinant E1/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.
[0062] 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 (>959% 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-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
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.
[0063] Said disulfide bond cleavage may also be achieved by:
(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 .fwdarw.2 R--SOC.sup.2-.sub.3) for
example by means of sulphite (SO.sup.2-.sub.3) together with a
proper oxidant such as Cu.sup.2- in which case the cysteine is
modified into S-sulpho-cysteine (Bailey and Cole, 1959).
[0064] (3) Reduction by means of mercaptans, such as dithiothreitol
(DDT), .beta.-mercapto-ethanol, cysteine, glutathione Red,
.epsilon.-mercapto-ethylamine, or thioglycolic acid, of which DTT
and .beta.-mercapto-ethanol are commonly used (Cleland, 1964), is
the preferred method of this invention because the method can be
performed in a water environment and because the cysteine remains
unmodified.
(4) Reduction by means of a phosphine (e.g. Bu.sub.3P) (Ruegg and
Rudinger, 1977).
[0065] All these compounds are thus to be regarded as agents or
means for cleaving disulphide bonds according to the present
invention.
[0066] Said disulphide bond cleavage (or reducing) step of the
present invention is preferably a partial disulphide bend cleavage
(reducing) step (carried out under partial cleavage or reducing
conditions).
[0067] 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.
[0068] Said disulphide bond cleavage step may also be carried out
in the presence or a 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 DecylPEG,
EMPIGEN-BB, NP-40, sodium cholate, Triton X-100.
[0069] Said reduction or cleavage step (preferably a partial
reduction or cleavage step) is carried out preferably in in the
presence of (with) a detergent. A preferred detergent 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.
[0070] 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 (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 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.
[0071] It is, however, to be understood that also any other
combination of any reducing agent 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.
[0072] Apart from reducing the disulphide bonds, a disulphide bond
cleaving means 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: R1S-S R2/R3SH.fwdarw.R1S-S R3/R2SH
[0073] R1, R2: compounds of protein aggregates
[0074] R3 SH: competitive agent (organic, proteinaeous)
[0075] The term `disulphide bridge exchanging agent` is to be
interpretated as including disulphide bond reforming as well as
disulphide bond blocking agents.
[0076] The present invention also relates to methods for purifying
or isolating HCV single or specific oligomeric envelope proteins as
set out above further including the use of any SH group blocking or
binding reagent known in the art such as chosen from the following
list: [0077] Glutathion [0078] 5,5'-dithiobis-(2-nitrobenzoic acid)
or bis-(3-carboxy-4-nitrophenyl)-disulphide (DTNB or Ellman's
reagent) (Elmann. 1959) [0079] N-ethylmaleimide (NEM; Benesch et
al., 1956) [0080] N-(4-dimethylamino-3,5-dinitrophenyl) maleimide
or Tuppy's maleimide which provides a color to the protein [0081]
P-chloromercuribenzoate (Grassetti et al., 1969) [0082]
4-vinylpyridine (Friedman and Krull, 1969) can be liberated after
reaction by acid hydrolysis [0083] acrylonitrile, can be liberated
after reaction by acid hydrolysis (Weil and Seibles, 1961) [0084]
NEM-biotin (e.g. obtained from Sigma B1267) [0085]
2,2'-dithiopyridine (Grassetti and Murray, 1967) [0086]
4,4'-dithiopyridine (Grassetti and Murray, 1967) [0087]
6,6'-dithiodinicontinic acid (DTDNA; Brown and Cunnigham, 1970)
[0088] 2,2'-dithiobis-(5'-nitropyridine) (DTNP: U.S. Pat. No.
3,597,160) or other dithiobis (heterocyclic derivative) compounds
(Grassetti and Murray, 1969)
[0089] A survey of the publications cited shows that often
different reagents for sulfhydryl 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
hydrochloride 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 DTNE, 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.
[0090] Alternatively, conditions such as low pH (preferably lower
than pH 6) for preventing 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.
[0091] A preferred SH group blocking reagent according to the
present invention is N-ethylmaleimide (NEM). Said SH croup blocking
reagent may be administrated during lysis of the recombinant host
cells and after 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.
[0092] Methods for cleaving cysteine bridges and blocking free
cysteines have also been described in Darbre (1987), Mears and
Feeney (1971), and by Wong (1993).
[0093] 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: [0094] lysing recombinant E1 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. [0095] 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 anti-E2 specific
monoclonal antibodies, followed by, [0096] 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, [0097] recovering the reduced HCV
E1 and/or E2 and/or E1/E2 envelope proteins for instance by
gelfiltration (size exclusion chromatography or molecular sieving)
and possibly also by an additional Ni.sup.2--IMAC chromatography
and desalting step.
[0098] 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.
[0099] 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 agglutinin (PSA),
or Allium ursinum agglutinin (AUA).
[0100] Preferably said method is usable to purify single or
specific oligomeric HCV envelope protein produced intracellularly
as detailed above.
[0101] For secreted E1 or E2 or E1/E2 oligomers, lectins binding
complex sugars such as Ricinus communis agglutinin I (RCA I), are
preferred lectins.
[0102] 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.
[0103] The present invention more particularly relates to the
purification or isolation of recombinant envelope proteins which
are expressed from recombinant mammalian cells such as
vaccinia.
[0104] The present invention also relates to the purification or
isolation of recombinant envelope proteins which are expressed from
recombinant yeast cells.
[0105] The present invention equally relates to the purification or
isolation of recombinant envelope proteins which are expressed from
recombinant bacterial (prokaryotic) cells.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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 a-mating factor
sequence for expression into yeast cells, but particularly
preferred constructs according to the present invention 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 domain(s) as
illustrated in the examples section, or of the E2 hypervariable
region 1.
[0110] 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 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 vector encompasses a recombinant nucleic acid
having a HCV cDNA segment 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 hydrophobic domain
deleted (positions 264 to 293 plus or minus 8 amino acids), or
forms to which a 5'-terminal ATC 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.
[0111] 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 320 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 segment 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.
[0112] 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 transformed with a recombinant virus. The vectors
may replicate within the host independently, or may integrate into
the host cell genome.
[0113] 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
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 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.
[0114] Also known are insect expression transfer vectors derived
from baculovirus 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
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.
[0115] 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 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 carrying such a mutated E1 and/or E2 protein
encoding nucleic acid are also within the scope of the present
invention.
[0116] The present invention also relates to recombinant E1 and/or
E2 and/or E1/E2 proteins 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 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 transformed with such a recombinant vector.
[0117] 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.
[0118] 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
E1 and/or E2 and/or E1/E2 protein.
[0119] 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, including
HeLa cells, Chinese hamster ovary (COC) cells. Baby hamster kidney
(BHK) cells. PK15, RK13 and a number of other cell lines.
[0120] The present invention relates particularly to a recombinant
E1 and/or E2 and/or E1/E2 protein expressed by a host cell as
defined above containing a recombinant vector as defined above.
These recombinant proteins are particularly purified according to
the method of the present invention.
[0121] A preferred method for isolating or purifying HCV envelope
proteins as defined above is further characterized as comprising at
least the following steps: [0122] 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 E1/E2 HCV envelope proteins in a suitable culture
medium, [0123] causing expression of said vector sequence as
defined above under suitable conditions, and, [0124] 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 Empigen-BE, [0125] 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, [0126]
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-NECM, and,
[0127] isolating the HCV single or specific oligomeric E1 and/or E2
and/or E1/E2 proteins such as by means of gelfiltration and
possibly also by a subsequent Ni.sup.2--IMAC chromatography
followed by a desalting step.
[0128] As a result of the above-mentioned process, E1 and/or 2 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 avoid volume of the gelfiltration column
or 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.
[0129] Ni.sup.2--IMAC chromatography followed by a desalting step
is preferably used for constructs bearing a (His).sub.5 as
described by Janknecht et al. 1991, and Hochuli et al., 1988.
[0130] 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.
[0131] The present invention further relates to a composition
comprising at least one of the following E1 peptides as listed in
Table 3: [0132] E1-31 (SEQ ID NO 56) spanning amino acids 181 to
200 of the Core/E1 V1 region, [0133] E1-33 (SEQ ID NO 57) spanning
amino acids 193 to 212 of the E1 region. [0134] E1-35 (SEQ ID NO
58) spanning amino acids 205 to 224 of the E1 V2 region (epitope
B), [0135] E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of
the E1 V2 region (epitope B). [0136] 1bE1 (SEQ ID NO 53) spanning
amino acids 192 to 228 of E1 regions (V1, C1, and V2 regions
(containing epitope B)), [0137] E1-51 (SEC ID NO 66) spanning amino
acids 301 to 320 of the E1 region, [0138] E1-53 (SEQ ID NO 67)
spanning amino acids 313 to 332 of the E1 C4 region (epitope A),
[0139] E1-55 (SEC ID NO 68) spanning amino acids 325 to 344 of the
E1 region.
[0140] The present invention also relates to a composition
comprising at least one of the following E2 peptides as listed in
Table 3: [0141] Env 67 or E2-67 (SEQ ID NO 72) spanning amino acid
positions 397 to 416 of [0142] the E2 region (epitope A, recognized
by monoclonal antibody 2-10H10. see FIG. 19), [0143] Env 69 or
E2-69 (SEQ ID NO 73) spanning amino acid positions 409 to 423 of
the E2 region (epitope A), [0144] Env 23 or 2-23 (SEQ ID NO 86)
spanning positions 583 to 602 of the E2 region (epitope E). [0145]
Env 25 or E2-25 (SEQ ID NO 87) spanning positions 595 to 614 of the
2 region (epitope E). [0146] Env 27 or E2-27 (SEQ ID NO 88)
spanning positions 607 to 626 of the E2 region (epitope E), [0147]
Env 17B or E2-17B (SEQ ID NO 83) spanning positions 5417 to 566 of
the E2 region (epitope D), [0148] Env 13B or E2-13B (SEQ ID NO 82)
spanning positions 523 to 542 of the E2 region (epitope C;
recognized by monoclonal antibody 16A6E7, see FIG. 19).
[0149] The present invention also relates to a composition
comprising at least one of the following E2 conformational
epitopes: [0150] epitope F recognized by monoclonal antibodies
15C8C1, 12D11F1 and 8G10D1H9, [0151] epitope G recognized by
monoclonal antibody 9G3E6. [0152] epitope H (or C) recognized by
monoclonal antibody 10D3C4 and 4H.sub.6B2, or, [0153] epitope I
recognized by monoclonal antibody 17F2C2.
[0154] 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.
[0155] The present invention also relates to an E1 or E2 specific
antibody screened from a 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.
[0156] The E1 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 animal, particularly
from a mouse or rat, immunizes 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 hard, 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.
[0157] The antibodies involved in the invention can be labelled by
an appropriate label of the enzymatic, fluorescent, or radioactive
type.
[0158] The monoclonal antibodies according to this preferred
embodiment of the invention may be humanized versions of mouse
monoclonal antibodies made by means of recombinant 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.
[0159] 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).
[0160] 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).
[0161] 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 E1 or E2 antigen), for prognosing/monitoring of HCV
disease, or as therapeutic agents.
[0162] 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 for detecting
the presence of E1 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.
[0163] 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 [0164] (i)
contacting said biological sample with any of the E1 and/or E2
specific monoclonal antibodies as defined above, preferably in an
immobilized form under appropriate conditions which allow the
formation of an immune complex, [0165] (ii) removing unbound
components. [0166] (iii) incubating the immune complexes formed
with heterologous antibodies, 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, [0167] (iv) detecting the presence of
said immune complexes visually or mechanically (e.g. by means of
densitometry, fluorimetry, colorimetry).
[0168] The present invention also relates to a kit for in vitro
diagnosis of HCV antigen present in a biological sample,
comprising: [0169] at least one monoclonal antibody as defined
above, with said antibody being preferentially immobilized on a
solid substrate. [0170] a buffer or components necessary for
producing the buffer enabling binding reaction between these
antibodies and the HCV antigens present in the biological sample,
[0171] a means for detecting the immune complexes formed in the
preceding binding reaction, [0172] possibly also including an
automated scanning and interpretation device for inferring the HCV
antigens present in the sample from the observed binding
pattern.
[0173] The present invention also relates to a composition
comprising E1 and/or E2 and/or 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.
[0174] 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 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.
[0175] 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.
[0176] 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.
[0177] Immunogenic compositions can be prepared according to
methods known in the art. The present compositions comprise an
immunogenic amount of a recombinant E1 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.
[0178] The single or specific oligomeric envelope proteins of the
present invention, either E1 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 E1 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.
[0179] 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.
[0180] Preferred adjuvants to enhance effectiveness of the
composition include, but are nor limited to: aluminim hydroxide
(alum), N-acetyl-muramyl-L-threonyl-O-isoglutamine (thr-MOP) as
found in U.S. Pat. No. 4,606,918,
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-aceylmuramyl-L-alanyl-O-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and R1E1,
which contains three 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, Mass.) or SAF-1 (Syntex) may be used.
Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's
Adjuvant (IFA) may be user for non-human applications and research
purposes.
[0181] 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.
[0182] 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 E1 and E2 proteins may also be incorporated into Immune
Stimulating Complexes together with saponins, for example Quil A
(ISCOMS).
[0183] 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 tall in a relatively broad range that can be determined
through routine trials. Usually, the amount will vary from 0.01 to
1000 .mu.g/dose, more particularly from 0.1 to 100 .mu.g/dose.
[0184] 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 NS5
regions) or heterologous (non-HCV) haptens, in the same manner as
Hepatitis 3 surface antigen (see European Patent Application 174,
444). In this use, envelope proteins 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 V1
region (encompassing amino acid positions 191 to 202), the V2
region (encompassing 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 V1
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
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.
[0185] The immunogenic compositions are conventionally administered
parenterally, 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.
[0186] 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.
[0187] 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.
[0188] 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 [0189] (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.
[0190] (ii) removing unbound components, [0191] (iii) incubating
the immune complexes formed with heterologous antibodies, with said
heterologous antibodies having conjugated to a detectable label
under appropriate conditions, [0192] (iv) detecting the presence of
said immune complexes visually or mechanically (e.g. by means of
densitometry, fluorimetry, colorimetry).
[0193] 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.
[0194] The present invention also relates to a kit for determining
the presence of HCV antibodies, in a biological sample, comprising
[0195] 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 solid substrate, more
preferably on different microwells of the same ELISA plate, and
even more preferentially on one and the same membrane strip, [0196]
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, [0197]
means for detecting the immune complexes formed in the preceding
binding reaction, [0198] possibly also including an automated
scanning and interpretation device for inferring the HCV genotypes
present in the sample from the observed binding pattern.
[0199] 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 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 strength using an excess
of antigen. The incubation of the antigen with the specimen is
followed by detection of immune complexes comprised of the
antigen.
[0200] 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 assays which utilize biotin and avidin or
streptavidin, and enzyme-labeled and mediated immunoassays, such as
LISA assays.
[0201] 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 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, polyvinylidine fluoride (known as
Immunolon.TM.), diazotized paper, nylon membranes, activated beads,
and Protein A beads. For example, Dynatech Immunolon.TM.1 or
Immunlon.TM.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 antibodies. Both standard and
competitive formats are know in the art.
[0202] In a homogeneous format, the rest 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.
[0203] 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 known amount
of labeled antibody (or other competing ligand) in the complex.
[0204] 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 enzyme label).
[0205] 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.
[0206] 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 REC. The addition of specific antigen antibodies
present in the body component, if any, causes the RSCs coated with
the purified antigen to agglutinate.
[0207] 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.
[0208] The HCV single or specific 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.
[0209] 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 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 no: exhibit antibodies to the native HCV antigens, E1 or
E2.
[0210] 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 VII, immunoglobulin, etc.) `screening` tests are
typically formatted to increase sensitivity (to insure no
contaminated blood passes) at the expense of specificity: i.e. the
false-positive rare is increased. Thus, it is typical to only defer
for further resting 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
confirmed) at the expense of sensitivity. Therefore the
purification method described in the present invention for E1 and
E2 will be very advantageous for including single or specific
oligomeric envelope proteins into HCV diagnostic assays.
[0211] 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.
[0212] The present invention further contemplates the use of E1
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: [0213] 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, [0214] removing unbound components. [0215] calculating the
anti-E1 titers present in said sample (for example at the start of
and/or during the course of (interferon) therapy), [0216]
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.
[0217] Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, or
preferably more than 20 times of the initial anti-E1 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-E1 assay may be very useful for
prognosing long-term response to IFN treatment, or to treatment of
Hepatitis C virus disease in general.
[0218] 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: [0219] E1-31 (SEQ ID NO 56) spanning
amino acids 181 to 200 of the Core/E1 V1 region, [0220] E1-33 (SEC
ID NO 57) spanning amino acids 193 to 212 of the E1 region. [0221]
E1-35 (SEQ ID NO 58) spanning amino acids 205 to 224 of the E1 V2
region (epitope B). [0222] E1-35A (SEQ ID NO 59) spanning amino
acids 208 to 227 of the E1 V2 region (epitope B). [0223] 1bE1 (SEQ
ID NC 53) spanning amino acids 192 to 228 of E1 regions (V1, C1,
and V2 regions (containing epitope B)), [0224] E1-51 (SEQ ID NO 66)
spanning amino acids 307 to 320 of the E1 region, [0225] E1-53 (SEQ
ID NO 67) spanning amino acids 313 to 332 of the E1 C4 region
(epitope A), [0226] E1-55 (SEQ ID NO 68) spanning amino acids 325
to 344 of the E1 region.
[0227] 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 rested for their ability to be used
in an assay as detailed above and in the Examples section.
[0228] 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:
[0229] at least one E1 protein or E1 peptide, more particularly an
E1 protein or E1 peptide as defined above, [0230] 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, [0231] means for
detecting the immune complexes formed in the preceding binding
reaction, [0232] possibly also an automated scanning and
interpretation device for inferring a decrease of anti-E1 titers
during the progression of treatment.
[0233] 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 above for the E1
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.
[0234] The present invention also relates to a stereotyping 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 [0235] (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/of E1/E2 protein compositions or at least
one of the E1 or E2 peptide compositions as defined above,
preferentially in an immobilized form under appropriate conditions
which allow the formation of an immune complex, [0236] (ii)
removing unbound components, [0237] (iii) incubating the immune
complexes formed with heterologous antibodies, with said
heterologous antibodies being conjugated to a detectable label
under appropriate conditions, [0238] (iv) detecting the presence of
said immune complexes visually or mechanically (e.g. by means of
densitometry, fluorimetry, colorimetry) and inferring the presence
of one or more HCV serological types present from the observed
binding pattern.
[0239] 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.
[0240] 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: [0241] at least one
E1 and/or E2 and/or E1/E2 protein or E1 or E2 peptide, as defined
above, [0242] 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,
[0243] means for detecting the immune complexes formed in the
preceding binding reaction, [0244] possibly also an automated
scanning and interpretation device for detecting the presence of
one or more serological types present from the observed binding
pattern.
[0245] 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 polyprotein regions also lies within the
scope of the present invention.
FIGURE AND TABLE LEGENDS
[0246] FIG. 1: Restriction map of plasmid pgpt ATA 18
[0247] FIG. 2: Restriction map of plasmid pgs ATA 18
[0248] FIG. 3: Restriction map of plasmid pMS 66
[0249] FIG. 4: Restriction map of plasmid pv HCV-11A
[0250] FIG. 5: Anti-1 levels in non-responders to IFN treatment
[0251] FIG. 6: Anti-E1 levels in responders to IFN treatment
[0252] FIG. 7: Anti-E1 levels in patients with complete response to
IFN treatment
[0253] FIG. 8: Anti-E1 levels in incomplete responders to IFN
treatment
[0254] FIG. 9: Anti-E2 levels in non-responders to IFN
treatment
[0255] FIG. 10 Anti-E2 levels in responders to IFN treatment
[0256] FIG. 11: Anti-E2 levels in incomplete responders to IFN
treatment
[0257] FIG. 12: Anti-E2 levels in complete responders to IFN
treatment
[0258] FIG. 13: Human anti-E1 reactivity competed with peptides
[0259] FIG. 14: Competition of reactivity of anti-E1 monoclonal
antibodies with peptides
[0260] FIG. 15: Anti-E1 (epitope 1) levels in non-responders to IFN
treatment
[0261] FIG. 16: Anti-E1 (epitope 1) levels in responders to IFN
treatment
[0262] FIG. 17: Anti-E1 (epitope 2) levels in non-responders to IFN
treatment
[0263] FIG. 18: Anti-E1 (epitope 2) levels in responders to IFN
treatment
[0264] FIG. 19: Competition of reactivity of anti-E2 monoclonal
antibodies with peptides
[0265] FIG. 20: Human anti-E2 reactivity competed with peptides
[0266] FIG. 21: FIGS. 21A-L provide 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.
[0267] FIG. 22: ELISA results obtained from lentil lectin
chromatography eluate fractions of 4 different E1 purifications of
cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b),
vvHCV62 type 3a), and vvHCV63 (type 5a).
[0268] FIG. 23: Elution profiles obtained from the lentil lectin
chromatography of the 4 different E1 constructs on the basis of the
values as shown in FIG. 22.
[0269] FIG. 24: ELISA results obtained from fractions obtained
after gelfiltration chromatography of 4 different E1 purifications
of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b),
vvHCCV62 (type 3a), and vvHCV63 (type 5a).
[0270] FIG. 25: Profiles obtained from purifications of E1 proteins
of type 1b (1), type 3a (2), and type 5a (3) (from RK13 cells
infected with vvHCV39, vvHCV62, and vvHCV63, 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 FIG. 24, E1 reactivity mainly in fractions 26 to
30).
[0271] FIG. 26: Silver staining of an SDS-PACE as described in
example 4 of a raw lysate of E1 vvHCV40 (type 1b) (lane 1), pool 1
of the gelfiltration of vvHCV40 representing fractions 10 to 17 as
shown in FIG. 25 (lane 2), pool 2 of the gelfiltration of vvHCV40
representing fractions 13 to 25 as shown in FIG. 25 (lane 3), and
E1 pool (fractions 25 to 30) (lane 4).
[0272] FIG. 27: Streptavidine-alkaline phosphatase blot of the
fractions of the gelfiltration of E1 constructs 39 (type 1b) and 62
(type 3a). The proteins were labelled with NEM-biotin. 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 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.
[0273] FIG. 28: Siver staining of an SOS-PAGE gel of the
gelfiltration fractions of vvHCV-39 (E1s, type 1b) and vvHCV-62
(E1s, type 3a) run under identical conditions as FIG. 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 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.
[0274] FIG. 29: Western Blot analysis with anti-cl mouse monoclonal
antibody 5E1A10 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).
[0275] FIG. 30: OD.sub.280 profile (continuous line) of the lentil
lectin chromatography of E2 protein from RK13 cells infected with
vvHCV44. The dotted line represents the 0.7 reactivity as detected
by ELISA (as in example 6).
[0276] FIG. 31A: OD.sub.290 profile (continuous line) of the
lentil-lectin gelfiltration chromatography E2 protein pool from
RK13 cells infected with vvHCV44 in which the 2 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).
[0277] FIG. 31B: OD.sub.280 profile (continuous line) of the
lentil-lectin gelfiltration chromatography E2 protein pool from
RK13 cells infected with vvpCV44 in which the 2 pool was reduced
and blocked according to Example 5.3 (reduced conditions). The
dotted line represents the 2 reactivity as detected by ELISA (as in
example 6).
[0278] FIG. 32: Ni.sup.2--IMAC chromatography and ELISA reactivity
of the E2 protein as expressed from vvHCV44 after gelfiltration
under reducing conditions as shown in FIG. 31B.
[0279] FIG. 33: Silver staining of an SOS-PAGE of 0.5 .mu.g of
purified E2 protein recovered by a 200 mM imidazole elution step
(lane 2) and a 30 mM imidazole wash (lane 1) of the Ni.sup.2--IMAC
chromatography as shown in FIG. 32.
[0280] FIG. 34: OD profiles of a desalting step of the purified E2
protein recovered by 200 mM immidazole as shown in FIG. 33,
intended to remove imidazole.
[0281] FIGS. 35A-1 to 35A-8: 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 months after
treatment determined by means of the LIAscan method. The average
values are indicated by the curves with the open squares.
[0282] FIGS. 35B-1 to 35B-8: Antibody levels to the different HCV
antigens (NS4, NS5, 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.
[0283] FIGS. 36A and 36B: Average E1 antibody (E1Ab) and E2
antibody (E2Ab) levels in the LTR and NR groups.
[0284] FIGS. 36A-D: Averages E1 antibody (E1Ab) levels for
non-responders (NR) and long term responders (LTR) for type 1b and
type 3a.
[0285] FIG. 37: Averages E1 antibody (E1Ab) levels for
non-responders (NR) and long term responders (LTR) for type 1b and
type 3a.
[0286] FIG. 38: Relative map positions of the anti-E2 monoclonal
antibodies.
[0287] FIG. 39: Partial deglycosylation of HCV E1 envelope protein.
The lysate of vvHCV10A-infected RK13 cells were incubated with
different concentrations of glycosidases according to the
manutacturer's instructions. Right panel: Glycopepidase F (PNC-ase
F). Left panel: Endoclycosidase H (Endo H).
[0288] FIG. 40: Partial deglycosylation of HCV E2 envelope
proteins. The lysate of vvHCV64-infected (E2) and vvHCV41-infected
(E2s)RK13 cells were incubated with different concentrations of
Glycopeptidase F (PNGase F) according to the manufacturer's
instructions.
[0289] FIG. 41: in vitro mutagenesis of HCV E1 glycoproteins. Map
of the mutated sequences and the creation of new restriction
sites.
[0290] FIG. 42A: In vitro mutagenesis of HCV E1 glycoprotein (part
1). First step of PCR amplification.
[0291] FIG. 42B: In vitro mutagensis of HCV E1 glycoprotein (part
2). Overlap extension and nested PCR.
[0292] FIG. 43: In vitro mutagenesis of HCV E1 glycoproteins. Map
of the PCR mutated fragments (GLY-# and OVR-#) synthesized during
the first step of amplification.
[0293] FIG. 44A: Analysis of E1 glycoprotein mutants by Western
blot expressed in HeLa (left) and RK13 (right) cells. Lane 1: wild
type VV (vaccinia virus), Lane 2:original E1 protein (vvHCV-10A).
Lane 3: E1 mutant Gly-1 (vvHCV-81). Lane 4: E1 mutant Gly-2
(vvHCV-82), Lane 5: E1 mutant Gly-3 (vvHCV-83), Lane 6: E1 mutant
Gly-4 (vvHCV-g8). Lane 7: E1 mutant Gly-5 (vvHCV-85). Lane 8: E1
mutant Gly-6 (vvHCV-86).
[0294] FIG. 44B: Analysis of E1 glycosylation mutant vaccinia
viruses by PCR amplification/restriction. Lane 1: E1 (vvHCV-10A),
BspE I. Lane 2: E1.GLY-1 (vvHCV-81). BspE1. Lane 4: E1 (vvHCV-10A),
Sac Lane 5: E1.GLY-2 (vvHCV-82), Sac I, Lane 7: E1 (vvHCV-10A), Sac
I, Lane 8: E1.GLY-3 (vvHCV-82), Sac Lane 10: E1 (vvHCV-10A), Stu I,
Lane 11: E1.GLY-4 (vvHCV-84). Stu I, Lane 13: E1 (vvHCV-10A), Sma
I, Lane 14: E1.GLY-5 (vvHCV-8E). Sma I, Lane 16: E1 (vvHCV-10A),
Stu I, Lane 17: E1.GLY-6 (vvHCV-86), Stu I, Lane 3-6-9-12-15: Low
Molecular Weight Marker, pBluescript SK/Msp 1
[0295] FIG. 45: SDS polyacrylamide gel electrophoresis of
recombinant E2 expressed in S. cerevisiae. Innoculates were crown
in leucine selective medium for 72 hrs. and diluted 1/15 in
complete medium. After 10 days of culture at 28.degree. C., medium
samples were taken. The equivalent of 200 .mu.l of culture
supernatant concentrated by speedHvc was loaded on the gel. Two
independent transformants were analysed.
[0296] FIG. 46: SDS polyacrylamide gel electrophoresis of
recombinant E2 expressed in a glycosylation deficient S. cerevisiae
mutant. Innoculate were grown in leucine selective medium for 72
hrs. and diluted 1/15 in complete medium. After 10 days of culture
at 28.degree. C. medium samples were taken. The equivalent of 350
.mu.l of culture supernatant, concentrated by ion exchange
chromatography, was loaded on the gel.
[0297] 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.
[0298] Table 2: Summary of Anti-E1 tests
[0299] Table 3: Synthetic peptides for competition studies
[0300] Table 4: Changes of envelope antibody levels over time.
[0301] Table 5: Difference between LTR and NR
[0302] Table 6: Competition experiments between murine E2
monoclonal antibodies
[0303] Table 7: Primers for construction of E1 glycosylation
mutants
[0304] Table 8: Analysis of E1 glycosylation mutants by ELISA
EXAMPLE 1
Cloning and Expression of the Hepatitis C Virus E1 Protein
1. Construction of Vaccinia Virus Recombination Vectors
[0305] The pgptATA18 vaccinia recombination plasmid is a modified
version of pATA18 (Stunnenberg et al, 1988) with an additional
insertion containing the E. coli xanthine guanine phosphoribosyl
transferase gene under the control of the vaccinia virus 13
intermediate promoter (FIG. 1). The plasmid pgsATA18 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 pATA18 vector. This created an extra Pac I
restriction site (FIG. 2). The original HindIII site was not
restored. TABLE-US-00001 Oligonucleotide linker with SEQ ID NO
1/94: 5' G GCATGC AAGCTT AATTAATT 3' 3' ACGTC CGTACG TTCGAA
TTAATTAA TCGA 5' PstI SphI HindIII Pac I (HindIII)
[0306] In order to facilitate rapid and efficient purification by
means of Ni.sup.2- chelation of engineered histidine stretches
fused to the recombinant proteins, the vaccinia recombination
vector pMSc6 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
restriction enzymes generating blunt ends (Sma I, Stu I and Pml
I/Bbr 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 SEC ID NO 2/95 was introduced between the Xma
I and Pst I sites of pgptATA18 (FIG. 3). TABLE-US-00002
Oligonucleotide linker with SEQ ID NO 2/95: '5' CCGGG
GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCACCATCACTAATAGTTAATTAA CTGCA3
3' C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGTAGTGATTATCAATTAATT G
XmaI PstI
EXAMPLE 2
Construction of HCV Recombinant Plasmids
2.1. Constructs Encoding Different Forms of the E1 Protein
[0307] Polymerase Chain Reaction (PCB) 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 recombination
vectors: HCCl9A (SEQ ID NO 31. HCCl10A (SEQ ID NO 5), HCCl11A (SEQ
ID NO 7). HCCl12A (SEQ ID NO 9), HCCl13A (SEQ ID NO 11), and
HCCl17A (SEQ ID NO 13) as depicted in FIG. 21. cDNA fragments
containing the E1-coding regions were cleaved by EcoI and HindIII
restriction from the respective pS-q72 plasmids and inserted into
the EcoRI/HindIII-cut pgptATA-18 vaccinia recombination vector
(described in example 1), downstream of the 11 K 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-11A is shown in FIG. A,
2.2. Hydrophobic Region E1 Deletion Mutants
[0308] Clone HCCl-37, containing a deletion of codons Asp264 to
Val2S7 (nucleotides 790 to 861, region encoding hydrophobic domain
1) was generated as follows: 2 PCR fragments were generated from
clone HCCl10A with primer sets HCPr52 (SEQ ID NO 16)/HCPr107 (SEQ
ID NO 19) and HCPr108 (SEQ ID NO 20)/HCPR54 (SEQ ID NO 12). These
primers are shown in FIG. 21. The two PCR fragments were purified
from agarose gel after electrophoresis and 1 ng of each fragment
was used together as template for PCR by means of primers HCPr52
(SEQ ID NO 16) and HCPr54 (SEC ID NO 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 pSP72HCCl37 containing clone HCCl37
(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 SamH 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 R1 and Bst
Ell-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 HCCl38 is represented by
SEQ ID NO 23.
[0309] 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 HCCl39 (SEQ ID NO 21). 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 HCCl40 (SEQ ID NO 27) in plasmid pvHCV-40, containing a 6
histidine tail at its carboxy-terminal end.
2.3. E1 of Other Genotypes
[0310] Clone HCCl62 (SEQ ID NO 29) was derived from a type
3a-infected patient with chronic hepatitis C (serum BR6, clone
BR36-9-13, SEQ ID NO 19 in WO 94/25601, and see also Stuyver et al.
1993a) and HCCl63 (SEQ ID NO 31) was derived from a type
5a-infected child with post-transfusion hepatitis (serum BE95,
clone PC-4-1, SEQ ID NO 45 in WO 94125601).
2.4. E2 Constructs
[0311] The HCV E2 PCR fragment 22 was obtained from serum BE11
(genotype 1b) by means of primers HCPr109 (SEQ ID NO 33) and HCPr72
(SEQ ID NO 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 HCCl22A (SEQ ID NO 35) was cut with NcoI/AlwNI or by
BamHI/AlwNI and the sticky ends of the fragments were blunted (NcoI
and BamHI sites with Klenow DNA Polymerase I (Boehringer), and
AlwNI with T4 DNA polymerase (Boehringer)). The BamHI/AlwNI 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 Polymerase
(Boehringer). The resulting plasmid was named pvHCV-41 and encoded
the E2 region from amino acids Met347 to Gln673, including 37 amino
acids (from Met347 to Gly383) of the E1 protein that can serve as
signal sequence. The same HCV cDNA was inserted into the EcoR I and
Sbr PI-cut vector pMS66, that had subsequently been blunt ended
with Klenow DNA Polymerase. The resulting plasmid was named
pvHCV-42 and also encoded amino acids 347 to 683. The NcoI/AlwNI
fragment was inserted in a similar way into the same sites of
pgsA7A-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-14).
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 c73 of the mature E2
protein.
2.5. Generation of Recombinant HCV-Vaccinia Viruses
[0312] Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma
143B thymidine kinase deficient (TK) (ATC 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/l) for Hep
G2. The vaccinia virus WR strain (Western Reserve, ATTC VR119) was
routinely propagated in either 143B or RK13 cells, as described
previously (Panicali & Paoletti. 1982; Piccini et al., 1987;
Mackett et al., 1982, 1984, and 1986). A confluent monolayer of
1432 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 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
Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt)
protein were selected on rabbit kidney RK13 cells incubated in
selection medium (EMEM containing 25 .mu.g/ml mycophenolic acid
(MPA), 250 .mu.g/ml xanthine, and 15 .mu.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. Thymidine kinase deficient
(TK-) recombinant viruses were selected and then plaque purified on
fresh monolayers of human 1432 cells (TK-) in the presence of 25
.mu.g/ml 5-bromo-2'-deoxyuridine. Stocks of purified recombinant
HCV-vaccinia viruses were prepared by infecting either human 1433
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 all) 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-10A was derived from recombining the wild type WR strain with
the pvHCV-10A plasmid.
EXAMPLE 3
Infection of Cells with Recombinant Vaccinia Viruses
[0313] 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 (P--S) and the recombinant
vaccinia virus stock was diluted in MEM medium. Two hundred .mu.l
of the virus solution was added per 10.sup.5 cells such that the
m.o.i. was 3, and incubated for A5 min at 24.degree. C. The virus
solution was aspirated and 2 ml of complete growth medium (see
example 2) was added per 10.sup.5 cells. The cells were incubated
for 24 hr at 37.degree. C. during which expression of the HCV
proteins took place.
EXAMPLE 4
Analysis of Recombinant Proteins by Means of Western Blotting
[0314] The infected cells were washed two times with PBS, directly
lysed with lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl. 1%
Triton X-100, 5 mM MgCl.sub.2, 1 .mu.g/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 NaCl for 5 min, and collected by
centrifugation (5 min at 1000 g). The cell pellet was then
resuspended in 200 .mu.l lysis buffer (50 mM Tris.HCL pH 8.0, 2 mM
EDTA, 150 mM NaCl, 5 mM MgCl.sub.2 aprotinin; 1% Triton X-100) per
10.sup.8 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 .mu.l lysate were separated by means of sodium
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.degree. 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
phosphates substrate solution (100 mM Tris.HCl pH 9.5, 100 mM NaCl,
5 mM MgCl.sub.2, 0.38 .mu.g/ml nitroblue tetrazolium. 0.165
.mu.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
[0315] Infected RK13 cells (carrying E1 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 NaCl, 1% Triton X-100,
5 mM MgCl.sub.2, 1 .mu.g/ml aprotinin (Sigma, Bornem, Belgium)
containing 2 mM biotinylated N-ethylmaleimide (biotin-NEM) (Sigma)
was added per 10.sup.5 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 (30100 g at r.sub.max) for 1 hour at
4.degree. C.
5.2. Lectin Chromatography
[0316] The cleared cell lysate was loaded at a rate of 1 ml/min on
a 0.8 by 10 cm Lentil-lectin Sepharose 48 column (Pharmacia) that
had been equilibrated with 5 column volumes of lysis buffer at a
rate of 1 ml/min. The lentil-lectin column was washed with 5 to 10
column volumes of buffer 1 (0.1M potassium phosphate pH 7.3, 500 mM
KCl, 5% glycerol, 1 mM 6--NH.sub.2-hexanoic acid, 1 mM MgCl.sub.2,
and 1% DecylPEG (KWANT, Bedum, The Netherlands). In some
experiments, the column was subsequently washed with 10 column
volumes of buffer 1 containing 0.5% Empigen-BB (Calbiochem, San
Diego, Calif., USA) instead of 1% DecylPEGG. The bound material was
eluted by applying elution buffer (10 mM potassium phosphate pH
7.3, 59 glycerol, 1 mM hexanoic acid; 1 mM MgCl.sub.2, 0.5%
Empigen-BB, and 0.5 M .alpha.-methyl-mannopyranoside. The eluted
material was fractionated and fractions were screened for the
presence of E1 or 2 protein by means of ELISA as described in
example 6. FIG. 22 shows ELISA results obtained from lentil lectin
eluate fractions of 4 different E1 purifications of cell lysates
infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type
3a), and vvHCV53 (type 5a). FIG. 23 shows the profiles obtained
from the values shown in FIG. 22. These results show that the
lectin affinity column can be employed for envelope proteins of the
different types of HCV.
5.3. Concentration and Partial Reduction
[0317] 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.degree. C. In some
experiments the E1- or E2-positive fractions were pooled and
concentrated by nitrogen evaporation. An equivalent of 3.10.sup.8
cells was concentrated to approximately 200 .mu.l. For partial
reduction, 30% Empigen-BB (Calbiochem, San Diego, Calif. USA) was
added to this 200 .mu.l to a final concentration of 3.5%, and 1M
DTT in H.sub.2O was subsequently added to a final concentration of
1.5 to 7.5 mM and incubated for 30 min at 37.degree. C. NEM (1M in
dimethylsulphoxide) was subsequently added to a final concentration
of 50 mM and left to react for another 30 min at 37.degree. C. to
block the free sulfhydryl groups.
5.4 Gel Filtration Chromatography
[0318] A Superdex-200 HR 10/20 column (Pharmacia) was equilibrated
with 3 column volumes PSS/3% Empigen-BB. The reduced mixture was
injected in a 500 .mu.l sample loop of the Smart System (Pharmacia)
and PBS/3% Empigen-BB buffer was added for gelfiltration. Fractions
of 250 .mu.l were collected from V.sub.0 to V.sub.1. The fractions
were screened for the presence of E1 or E2 protein as described in
example 6.
[0319] FIG. 24 shows ELISA results obtained from fractions obtained
after gelfiltration chromatography of 4 different E1 purifications
of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b),
vvHCV62 (type 3a), and vvHCVS6 (type 5a). FIG. 25 shows the
profiles obtained from purifications of E1 proteins of types 1b, 3,
and 5a (from RK13 cells infected with vvHCV39, vvHCVS2, and
vvHCV63, respectively; purified on lentil lectin and reduced as in
the previous examples). The peaks indicated with `1`, `2`, and `3`,
represent sure 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 E1 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 FIG. 25 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 FIG. 27.
It can be readily observed that, amongst others, the 29 kDa and 45
kDa 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 65 kDa 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. FIG.
23 shows a silver stain of an SDS/PAGE gel run in identical
conditions as in FIG. 26. A complete overview of the purification
procedure is given in FIG. 29.
[0320] 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 1b E1 protein purified from vvHCV40-infected cells
according to the above scheme was aminoterminally sequenced on an
477 Perkins-Elmer sequencer and appeared to contain a tyrosine as
first residue. This confirmed that the E1 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.
5.5. Purification of the E2 Protein
[0321] The E2 protein (amino acids 384 to 673) was purified from
RK13 cells infected with vvHCVA4 as indicated in Examples 5.1 to
5.4. FIG. 30 shows the CCl.sub.2 profile (continuous line) of the
lentil lectin chromatography. The dotted line represents the E2
reactivity as detected by ELISA (see example 6). FIG. 31 shows the
same profiles part of obtained from gelfiltration chromatography of
the lentil-lectin E2 pool (see FIG. 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.sub.o 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. FIG. 32 shows an additional Ni.sup.2--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 vvHCV44. Contaminating proteins
either run through the column or can be removed by a 30 mM
imidazole wash. FIG. 33 shows a silver-stained SDS/PAGE of 0.5
.mu.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. FIG. 34 shows an additional desalting step intended to remove
imidazole and to be able to switch to the desired buffer, e.g. PES,
carbonate buffer, saline.
[0322] Starting from about 50,000 cm.sup.2 of RK13 cells infected
with vvHCV11A (or vvHCV40) for the production of E1 or vvHCV41,
vvHCV42, vvHCV 43, or vvHCV44 for production of E2 protein, the
procedures described in examples 5.1 to 5.5 allow the purification
of approximately 1.3 mg of E1 protein and 0.6 mg of 2 protein.
[0323] 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
[0324] Maxisorb microwell plates (Nunc. Roskilde, Denmark) were
coated with 1 volume (e.g. 50 .mu.l or 100 .mu.l or 200 .mu.l) per
well of a 5 .mu.g/ml solution of Streptavidin (Boehringer Mannheim)
in PBS for 10 hours at 4.degree. C. or for 1 hour at 37.degree. C.
Alternatively, the wells were coated with 1 volume of 5 .mu.g/ml of
Galanthus nivalis agglutinin (GNA) in 50 mM sodium carbonate buffer
pH 9.6 for 16 hours at 4.degree. C. or for 1 hour at 37.degree. C.
In the case of coating with GNA, the plates were washed 2 times
with 400 .mu.l of Washing Solution of the Innotest HCV Ab III kit
(Innogenetics, Zwijndrecht, Belgium). Unbound coating surfaces were
blocked with 1.5 to 2 volumes of blocking solution (0.1% casein and
0.1% NaN, in PBS) for 1 hour at 37.degree. C. or for 16 hours at
4.degree. C. Blocking solution was aspirated. Purified E1 or E was
diluted to 100-1000 ng/ml (concentration measured at A=280 nm) or
column fractions to be screened for E1 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.degree. 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, Zwijndrecht, 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.degree. C. The
microwells were washed 5 times with 400 .mu.l of Washing Solution
of the Innotest HCV Ab III kit (Innogenetics. Zwijndrecht,
Belgium). The bound antibodies were detected by incubating each
well for 1 hour at 37.degree. C. with a goat anti-human or
anti-mouse 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, Zwijndrecht,
Belgium), and color development was obtained by addition of
substrate of the Innotesz HCV Ab III kit (Innogenetics.
Zwijndrecht, Belgium) diluted 100 times in 1 volume of Substrate
Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht,
Belgium) for 30 min at 24.degree. C. after washing of the plates 3
times with 400 .mu.l of Washing Solution of the Innotest HCV Ab III
kit (Innogenerics, Zwijndrecht, Belgium).
EXAMPLE 7
Follow Up of Patient Groups with Different Clinical Profiles
7.1. Monitoring of Anti-E1 and Anti-E2 Antibodies
[0325] The current hepatitis C virus (HCV) diagnostic assays have
seen 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 disease. However, as is the case for hepatitis 2, 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-.alpha.
treated patients with long-term sustained response (defined as
patients with normal transaminase levels and negative HCV-RNA test
(PCR in the a 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.
[0326] A group of 8 IFN-.alpha. treated patients with long-term
sustained response (LTR, follow up 1 to 3.5 years, 3 type 3a and 5
type 1b) was compared with 9 patients showing non-complete
responses to treatment (NR, follow up 1 to 4 years, 6 type 1b and 3
type 3a). Type 1b (vvHCV-39, see example 2.5.) and 3a E1 (vvHCV-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 1b
hepatitis C virus were tested for reactivity with purified type 1b
E1 protein, while samples of a type 3a infection were tested for
reactivity of anti-type 3a E1 antibodies in an ELISA as described
in example 6. The genotypes of hepatitis C viruses infecting the
different patients were determined by means of the Inno-LiPA
genotyping assay (Innogenetics, Zwijndrecht, Belgium). FIG. 5 shows
the anti-E1 signal-to-noise ratios of these patients followed
during the course of 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-E1 titer could be
deduced from the signal to noise ratio as show in FIGS. 5, 6, 7,
and 8.
[0327] Already at the end of treatment, marked differences could be
observed between the 2 groups. Anti-Et antibody titers had
decreased 6.9 times in LTR but only 1.5 times in NR. At the end of
follow up, the anti-E1 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-.alpha. therapy correlates with
long-term, sustainer 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.
[0328] 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
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 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
FIGS. 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-E1 antibodies. FIG. 35 gives a complete
overview of the pilot study.
[0329] 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 become a useful
marker for clinical diagnosis of hepatitis C. Furthermore, the use
of more defined regions of the E1 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
[0330] The pilot study lead the inventors to conclude that, in case
infection was completely cleared, antibodies to the HCV envelope
proteins changed more rapidly than antibodies to the more
conventionally studied HCV antigens, with E1 antibodies changing
most vigorously. We therefore included more type 1b 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.
[0331] FIG. 36 depicts average E1 antibody (E1 Ab) 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-a therapy were associated with LTR (P<0.03).
Since much higher E1 antibody levels were observed in type
3a-infected patients compared with type 1b-infected patients (FIG.
37), the genotype was taken into account (Table 4). Within the type
1b-infected group, LTR also had higher E1 antibody levels than NR
at the initiation of treatment (P<0.0-1; the limited number of
type 3a-infected NR did not allow statistical analysis.
[0332] Of antibody levels monitored in LTR during the 1.5-year
follow up period, only E1 antibodies cleared rapidly compared with
levels measured at initiation of treatment [P=0.0058, end of
therapy; P=0.00-7 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<0.05). These data
confirmed the initial finding that E1Ab 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-E1 B
cell stimulation. Anti-E1 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
[0333] 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
.lamda.gt11 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
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 surveillance may significantly contribute to new
directions in HCV diagnosis and prophylaxis.
[0334] Several 20-mer peptides (Table 3) that overlapped each other
by 8 amino acids, were synthesized according to a previously
described method (EP-A-0 439 968) based on the HC-J1 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-E1 ELISA as described
in example 6, it was possible to discover additional epitopes as
follows: The anti-E1 ELISA as described in example 6 was modified
by mixing 50 .mu.g/ml of E1 peptide with the 1120 diluted human
serum in sample diluent. FIG. 13 shows the results of reactivity of
human sera to the recombinant E1 (expressed from vvHCV-40) protein,
in the presence of single or of a mixture of E1 peptides. While
only 2% of the sera could be detected by means of E1 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 E1 protein.
Some of the murine monoclonal antibodies obtained from Balb/C mice
after injection with purified E1 protein were subsequently competed
for reactivity to E1 with the single peptides (FIG. 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.
[0335] Therefore peptides were synthesized using technology
described by applicant previously (in WO 93/18054). The following
peptides were synthesized:
[0336] peptide env35A-biotin [0337]
NH.sub.2--SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO 51) [0338] spanning
amino acids 208 to 227 of the HCV polyprotein in the E1 region
peptide biotin-env53 (`epitope A`) [0339]
biotin-GG-ITCHRMAWDMMNWSPTTAL-COOH (SEQ ID NO 52) [0340] spanning
amino acids to 313 of 332 of the HCV polyprotein in the E1
region
[0341] peptide 1bE1 (`epitope B`) [0342]
H.sub.2N-YEVRNVSGIYHVTNDCSNSSIVYEAADMHTPGCGK-biotin (SEQ ID NO 53)
[0343] spanning amino acids 192 to 228 of the HCV polyprotein in
the E1 region and compared with the reactivities of peptides E1a-BB
(biotin-GG-TPTVATRDGKLPATQLRRHIDLL, SEQ ID NO 54) and E1b-BB
(biotin-GG-TPTLAARDASVPTTTIRRHVDLL, SEQ ID NO 55) which are derived
from the same region of sequences of genotype 1a and 1b
respectively and which have been described at the 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 6 and none reacted with env35A).
Reactivity towards epitopes A, B, and C was tested directly to the
biotinylated peptides (50 .mu.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 FIGS. 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-1bE1
antibodies (epitope B) were present almost exclusively in long term
responders at the start of IFN treatment. Therefore, anti-1bE1
(epitope B) antibodies and anti-env-53 (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 defection 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 5E1A10, the sequence
-GWD- 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 NO 93) and may be
useful for diagnosis. Env31 or E1-31 as shown in Table 3, is a part
of the peptide 1bE1. Peptides 1-33 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.
[0344] 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 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 single E1 and E2 proteins, was shown to be
useful.
7.4. Mapping of Anti-E2 Antibodies
[0345] Of the 24 anti-12 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 (FIG. 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 c) 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 (FIG.
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, 8G10D1H9, 10D3C4,
4H6B2, 17F2C2, 5H6A7, 15B7A2 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 presence of 100 times the amount of
non-biotinylated competing antibody and subsequently detected by
alkaline phosphatase labeled streptavidin.
[0346] Percentages of competition are given in Table 6. Based on
these results, 4 conformational anti-E2 epitope regions (epitopes
F, C, H and I) could be delineated (FIG. 38). Alternatively, these
Mabs may recognize mutant linear epitopes not represented by the
peptides used in this study. Mabs 4H6B2 and 10D3C4 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 chances conformation after binding
of 16A6E7 to the E2-13B region (epitope H).
EXAMPLE 8
E1 Glycosylation Mutants
8.1. Introduction
[0347] The E1 protein encoded by vvHCV10A, and the E2 protein
encoded by vvHCV41 to 44 expressed from mammalian cells contain 6
and 11 carbohydrate moieties, respectively. This could be shown by
incubating the lysate of vvHCV10A-infected or vvHCV44-infected RK13
cells with decreasing concentrations of glycosidases (PNGase F or
Endoglycosidase H, (Boehringer Mannheim Biochemical according to
the manufacturer's instructions), such that the proteins in the
lysate (including E1) are partially deglycosylated (FIGS. 39 and
40, respectively).
[0348] Mutants devoid of some of their glycosylation sires 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 the hemagglutinin
protein of an escape mutant of the A/Hong Kong/3/68
(H.sub.3N.sub.2) 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
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 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 at al., 1988; Doms et al., 1993;
Helenius, 1994).
[0349] After alignment of the different envelope protein sequences
of HCV genotypes, it may be inferred that not all 6 glycosylation
sites on the HCV subtype 1b E1 protein are required for proper
folding and reactivity, since some are absent in certain
(sub)types. The fourth carbohydrate motif (on Asn251), present in
types 1b, 6a, 7, 8, and 9, is absent in all other types know today.
This sugar-addition motif may be mutated to yield a type 1b E1
protein with improved reactivity. Also the type 2b sequences show
an extra glycosylation site in the V5 region (on Asn299). The
isolate S83, belonging to genotype 2c, even lacks the first
carbohydrate motif in the V1 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 PI 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
[0350] All mutations were performed on the E1 sequence of clone
HCCl10A (SEQ ID NO. 5). The first round of PCR was performed using
sense primer `GPT` (see Table 7) targetting the CPT sequence
located upstream of the vaccinia 11 K late promoter, and an
antisense primer (designated GLY#, with # representing the number
of the glycosylation size, see FIG. 41) containing the desired base
change to obtain the mutagenesis. The six GLYM# primers (each
specific for a given glycosylacion site) were designed such that:
[0351] 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 (one more --CH.sub.2-- group). [0352] 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 E1Gly5) 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
(pvHCV-10A) or from each other (FIG. 41). This additional
restriction site may also be useful for the construction of new
hybrid (double, triple, etc.) glycosylation mutants. [0353] 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 sequence of N-linked
glycosylation sites.
[0354] For site-directed mutagenesis, the `mispriming` or `overlap
extension` (Horton. 1993) was used. The concept is illustrated in
FIGS. 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 GLYP) was amplified with the 5' sense GPT
primer (see Table 7) and with the respective 3`antisense GLYM`
primers. The second fragment (product OVR#) was amplified with the
3' antisense TK.sub.R primer and the respective 5' sense primers
(OVR# primers, see Table 7, FIG. 43).
[0355] 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 FCR cycles created the full-length mutant
molecule E1 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 11 K
promoter (sense GPT-2-primer) and the 5' end of the vaccinia
thymidine kinase locus (antisense TK.sub.R-2 primer, see Table 7).
All PCR conditions were performed as described in Stuyver et al.
(1993).
[0356] Each of these PCR products was cloned by EcoRI/BamHI
cleavage into the EcoRI/BamHI-cut vaccinia vector containing the
original E1 sequence (pvHCV-10A).
[0357] The selected clones were analyzed for length of insert by
EcoRI/BamHI cleavage 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
[0358] Starting from the 6 plasmids containing the mutant E1
sequences as described in example 8.2, recombinant vaccinia viruses
were generated by recombination with wt vaccinia virus as described
in example 2.5. Briefly, 175 cm.sup.2-flasks of subconfluent RK13
cells were infected with the 6 recombinant vaccinia viruses
carrying the mutant E1 sequences, as well as with the vvHCV-10A
(carrying the non-mutated E1 sequence) and wt vaccinia viruses.
Cells were lysed after 24 hours of infection and analyzed on
western blot as described in example 4 (see FIG. 444A). All mutants
showed a faster mobility (corresponding to a smaller molecular
weight of approximately 2 to 3 kDa) on SDS-PAC-E than the original
E1 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.
FIG. 448 shows that all mutants (as shown in FIG. 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 (CD 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 E1 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 5, and by testing identical quantities of all the different
E1 proteins. However, the results shown in table 5 already indicate
that removal of the 1st (GLY1), 3rd (GLY3), and 6th (GLY6)
glycosylation motifs reduces reactivity of some sera, while removal
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.
EXAMPLE 9
Expression of HCV E2 Protein in Glycosylation-Deficient Yeasts
[0359] The E2 sequence corresponding to clone HCCL41 was provided
with the .alpha.-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 expression of such a
construct in S. cerevisiae strains (FIG. 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. 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 (FIG.
46).
EXAMPLE 10
General Utility
[0360] 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 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.
[0361] The purification method disclosed in the present invention
may also be used for `viral envelope proteins` in general. Examples
are those derived from Flaviviruses, the newly discovered GB-A.
GB-B and GB-C Hepatitis viruses, Pestiviruses (such as Bovine viral
Diarrhea Virus (BVDV). Hog Cholera Virus (HCV). Border Disease
Virus (BDV)), but also less related viruses such as Hepatitis B
Virus (mainly for the purification of HBsAg).
[0362] 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
prokaryotes as set out in the detailed description section.
TABLE-US-00003 TABLE 1 Recombinant vaccinia plasmids and viruses
cDNA subclone Vector used Plasmid name Name construction Length
(nt/aa) for insertion pvHCV-13A E1s EcoR I-Hind III 472/157
pgptATA-18 pvHCV-12A E1s EcoR I-Hind III 472/158 pgptATA-18
pvHCV-9A E1 EcoR I-Hind III 631/211 pgptATA-18 pvHCV-11A E1s EcoR
I-Hind III 625/207 pgptATA-18 pvHCV-17A E1s EcoR I-Hind III 625/208
pgptATA-18 pvHCV-10A E1 EcoR I-Hind III 783/262 pgptATA-18
pvHCV-18A COREs Acc I (Kl)-EcoR I (Kl) 403/130 pgptATA-18 pvHCV-34
CORE Acc I (Kl)-Fso I 595/197 pgptATA-18 pvHCV-33 CORE-E1 Acc I
(Kl) 1150/380 pgptATA-18 pvHCV-35 CORE-E1b.his EcoR I-BamH I (Kl)
1032/352 pMS-66 pvHCV-36 CORE-E1n.his EcoR I-Nco I (Kl) 1106/376
pMS-66 pvHCV-37 E1.DELTA. Xma I-BamH I 711/239 pvHCV-10A pvHCV-38
E1.DELTA.s EcoR I-BstE II 553/183 pvHCV-11A pvHCV-39 E1.DELTA.b
EcoR I-BamH I 960/313 pgsATA-18 pvHCV-40 E1.DELTA.b.his EcoR I-BamH
I (Kl) 960/323 pMS-66 pvHCV-41 E2bs BamH I (Kl)-AlwN I (T4)
1005/331 pgsATA-18 pvHCV-42 E2bs.his BamH I (Kl)-AlwN I (T4)
1005/341 pMS-66 pvHCV-43 E2ns Nco I (Kl)-AlwN I (T4) 932/314
pgsATA-18 pvHCV-44 E2ns.his Nco I (Kl)-AlwN I (T4) 932/321 pMS-66
pvHCV-62 E1s (type 3a) EcoR I-Hind III 625/207 pgsATA-18 pvHCV-63
E1s (type 5) EcoR I-Hind III 625/207 pgsATA-18 pvHCV-64 E2 BamH
I-Hind III 1410/463 pgsATA-18 pvHCV-65 E1-E2 BamH I-Hind III
2072/691 pvHCV-10A pvHCV-66 CORE-E1-E2 BamH I-Hind III 2427/809
pvHCV-33 HCV cDNA subclone Vector Plasmid Length used for Name Name
Construction (nt/aa) insertion pvHCV-81 E1*-GLY 1 EcoRI-BamH I
783/262 pvHCV-10A pvHCV-82 E1*-GLY 2 EcoRI-BamH I 783/262 pvHCV-10A
pvHCV-83 E1*-GLY 3 EcoRI-BamH I 783/262 pvHCV-10A pvHCV-84 E1*-GLY
4 EcoRI-BamH I 783/262 pvHCV-10A pvHCV-85 E1*-GLY 5 EcoRI-BamH I
783/262 pvHCV-10A pvHCV-86 E1*-GLY 6 EcoRI-BamH I 783/262 pvHCV-10A
nt: nucleotide aa: aminoacid Kl: Klenow DNA Pol filling T4: T4 DNA
Pol filling Position: aminoacid position in the HCV polyprotein
sequence
[0363] TABLE-US-00004 TABLE 2 Summary of anti-E1 tests S/N .+-. SD
(mean anti-E1 titer) Start of treatment End of treatment Follow-up
LTR 6.94 .+-. 2.29 4.48 .+-. 2.69 2.99 .+-. 2.69 (1:3946) (1:568)
(1:175) NR 5.77 .+-. 3.77 5.29 .+-. 3.99 6.08 .+-. 3.73 (1:1607)
(1:1060) (1:1978) LTR: Long-term, sustained response for more than
1 year NR: No response, response with relapse, or partial
response
[0364] TABLE-US-00005 TABLE 3 Synthetic peptides for competition
studies SEQ ID PROTEIN PEPTIDE AMINO ACID SEQUENCE POSITION NO E1
E1-31 LLSCLTVPASAYCVRNSTGL 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 E1-43 LPATQLRRHIDLLVGSATLC 253-272 63 E1-45
LVGSATLCSALYVGDLCGSV 265-284 64 E1-49 QLFTFSPRRHWTTQGCNCSI 289-308
65 E1-51 TQGCNCSIYPGHITGHRMAW 301-320 66 E1-53 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 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
[0365] TABLE-US-00006 TABLE 4 Change of Envelope Antibody levels
over time (complete study, 28 patients) Wilcoxon Signed E1Ab NR
E1Ab NR E1Ab NR E1Ab LTR E1Ab LTR E1Ab LTR E2Ab NR E1Ab LTR Rank
test (P values) All type 1b type 3a All type 1b type 3a All All End
of therapy* 0.1167 0.2604 0.285 0.0058** 0.043** 0.0499** 0.0186**
0.0640 6 months follow up* 0.86 0.7213 0.5930 0.0047** 0.043**
0.063 0.04326 0.0464** 12 months follow up* 0.7989 0.3105 1
0.0051** 0.0679 0.0277** 0.0869 0.0058** *Data were compared with
values obtained at initiation of therapy **P values < 0.05
[0366] TABLE-US-00007 TABLE 5 Difference between LTR and NR
(complete study) Mann-Withney E1Ab E1Ab E1Ab E1Ab E2Ab S/N titers
S/N S/N S/N U test (P values) All All type 1b type 3a All
Initiation of therapy 0.0257* 0.05* 0.68 0.1078 End of therapy
0.1742 0.1295 6 months follow up 1 0.6099 0.425 0.3081 12 months
follow up 0.67 0.23 0.4386 0.6629 *P values < 0.05
[0367] TABLE-US-00008 TABLE 6 Competition experiments between
murine E2 monoclonal antibodies Decrease (%) of anti-E2 reactivity
of biotinylated anti-E2 mabs 17H10F4D10 2F10H10 16A6EQ7 10D3C4
4H6B2 17C2F2 9G3E6 12D11F1 15C8C1 8G10D1H9 competitor 17H10F4D10 --
62 10 ND 11 ND 5 6 30 ND 2F10H10 90 -- 1 ND 30 ND 0 4 12 ND 16A6E7
ND ND -- ND ND ND ND ND ND ND 10D3C4 11 50 92 -- 94 26 28 43 53 30
4H6B2 ND ND 82 ND -- ND ND ND ND ND 17C2F2 2 ND 75 ND 56 -- 11 10 0
0 9G3E6 ND ND 68 ND 11 ND -- 60 76 ND 12D11F1 ND ND 26 ND 13 ND ND
-- 88 ND 15C8C1 ND ND 18 ND 10 ND ND ND -- ND 8G10D1H9 2 2 11 ND 15
ND 67 082 81 -- competitor controls 15B7A2 0 0 9 15 10 9 0 0 0 5
5H6A7 0 2 0 12 8 0 0 4 0 0 23C12H9 ND ND 2 12 ND 4 ND ND ND 2 ND,
not done
[0368] TABLE-US-00009 TABLE 7 Primers SEQ ID NO. 96 GPT
5'-GTTTAACCACTGCATGATG-3' SEQ ID NO. 97 TK.sub..parallel.
5'-GTCCCATCGAGTGCGGCTAC-3' SEQ ID NO. 98 GLY1
5'-CGTGACATGGTACATTCCGGACACTT GGCGCACTTCATAAGCGGA-3' SEQ ID NO. 99
GLY2 5'-TGCCTCATACACAATGGAGCTCTGGG ACGAGTCGTTCGTGAC-3' SEQ ID NO.
100 GLY3 5'-TACCCAGCAGCGGGAGCTCTGTTGCT CCCGAACGCAGGGCAC-3' SEQ ID
NO. 101 GLY4 5'-TGTCGTGGTGGGGACGGAGGCCTGCC TAGCTGCGAGCGTGGG-3' SEQ
ID NO. 102 GLY5 5'-CGTTATGTGGCCCGGGTAGATTGAGC
ACTGGCAGTCCTGCACCGTCTC-3' SEQ ID NO. 103 GLY6
5'-CAGGGCCGTTGTAGGCCTCCACTGCA TCATCATATCCCAAGC-3' SEQ ID NO. 104
OVR1 5'-CCGGAATGTACCATGTCACGAACG AC-3' SEQ ID NO. 105 OVR2
5'-GCTCCATTGTGTATGAGGCAGC GG-3' SEQ ID NO. 106 OVR3
5'-GAGCTCCCGCTGCTGGGTAGCGC-3' SEQ ID NO. 107 OVR4
5'-CCTCCGTCCCCACCACGACAATA CG-3' SEQ ID NO. 108 OVR5
5'-CTACCGGCCACATAACGGGTCAC CG-3' SEQ ID NO. 109 OVR6
5'-GGAGGCCTACAACGGCCCTGGT GG-3' SEQ ID NO. 110 GPT-2
5'-TTCTATCGATTAAATAGAATTC-3' SEQ ID NO. 111 TK.sub..parallel..2
5'-GCCATACGCTCACAGCCGATCCC-3' nucleotides underlined represent
additional restriction site nucleotides in bold represent mutations
with respect to the original HCCl10A sequence
[0369] TABLE-US-00010 TABLE 8 Analysis of E1 glycosylation mutants
by ELISA SERUM 1 2 3 4 5 6 7 8 9 LY1 1.802462 2.120971 1.403871
1.205597 2.120191 2.866913 1.950345 1.866183 1.730193 LY2 2.400795
1.76818 2.325495 2.639308 2.459019 5.043993 2.146302 1.595477
1.688973 LY3 1.642718 1.715477 2.261646 2.354748 1.591818 4.833742
1.96692 1.482099 1.602222 LY4 2.578154 3.824038 3.874605 1.499387
3.15 4.71302 4.198751 3.959542 3.710507 LY5 2.482051 1.793761
2.409344 2.627358 1.715311 4.964765 2.13912 1.576336 1.708937 LY6
2.031487 1.495737 2.131613 2.527925 2.494833 4.784027 2.02069
1.496489 1.704976 I 2.828205 2.227036 2.512792 2.790881 3.131579
4.869128 2.287753 1.954198 1.805556 10 11 12 13 14 15 16 17 18 LY1
2.468162 1.220654 1.629403 5.885561 3.233604 3.763498 1.985105
2.317721 6.675179 LY2 2.482212 1.467582 2.070524 7.556682 2.567613
3.621928 3.055649 2.933792 7.65433 LY3 2.191558 1.464216 1.721164
7.930538 2.763055 3.016099 2.945628 2.515305 5.775357 LY4 5.170841
4.250784 3.955153 8.176816 6.561122 5.707668 5.684498 5.604813
6.4125 LY5 3.021807 1.562092 2.07278 8.883408 2.940334 3.125561
3.338912 2.654224 5.424107 LY6 2.677757 1.529608 1.744221 8.005561
2.499952 2.621704 2.572385 2.363301 5.194107 I 2.616822 1.55719
2.593886 8.825112 3.183771 3.067265 3.280335 2.980354 7.191964 Sum
Average 19 20 21 22 23 24 S/N S/N LY1 1.93476 2.47171 4.378633
1.188748 2.158889 1.706992 59.88534 2.495223 LY2 2.127712 2.921288
4.680101 1.150781 1.661914 1.632785 69.65243 2.902185 LY3 1.980185
2.557384 4.268633 0.97767 1.336775 1.20376 62.09872 2.587447 LY4
3.813321 3.002535 4.293038 2.393011 3.68213 2.481585 102.6978
4.279076 LY5 2.442804 3.126761 4.64557 1.153656 1.817901 1.638211
69.26511 2.886046 LY6 1.506716 2.665433 2.781063 1.280743 1.475062
1.716423 61.32181 2.555075 I 2.771218 3.678068 5.35443 1.167286
2.083333 1.78252 76.54068 3.189195 1 2 3 4 5 6 7 8 9 E1 0.637316
0.952374 0.55869 0.431977 0.677036 0.588794 0.852516 0.954961
0.958261 E1 0.848876 0.793961 0.925463 0.94569 0.785233 1.035913
0.93817 0.816436 0.935431 E1 0.580834 0.770296 0.900053 0.84373
0.508312 0.992733 0.859761 0.758418 0.887385 E1 0.911587 1.717097
1.541952 0.537245 1.005882 0.967939 1.835317 2.026172 2.05505 E1
0.877607 0.805447 0.958831 0.941408 0.547746 1.019642 0.935031
0.806641 0.946488 E1 0.718296 0.671626 0.848305 0.90578 0.796669
0.982522 0.883264 0.765781 0.944294 10 11 12 13 14 15 16 17 18 E1
0.94319 0.783882 0.628171 0.644248 1.015652 1.226988 0.605153
0.777666 0.928144 E1 0.94856 0.942455 0.798232 0.85627 0.806469
1.180833 0.931505 0.984377 1.064289 E1 0.837488 0.940294 0.663547
0.898633 0.867856 0.983319 0.897966 0.843962 0.803029 E1 1.976
2.72978 1.524798 0.92654 2.060802 1.860833 1.732902 1.880587
0.89162 E1 1.154762 1.003148 0.799102 1.006606 0.923538 1.019006
1.017857 0.890574 0.75419 E1 1.023286 0.982288 0.672435 0.907134
0.785217 0.854737 0.784184 0.79296 0.72221 Sum Average 19 20 21 22
23 24 E1/GLY// E1/GLY// E1 0.698162 0.672013 0.817759 1.018386
1.036267 0.957628 19.36524 0.806885 E1 0.76779 0.794245 0.874061
0.98586 0.797719 0.915998 21.67384 0.903077 E1 0.714554 0.695306
0.797215 0.837558 0.641652 0.675314 19.19921 0.799967 E1 1.376045
0.816335 0.801773 2.050064 1.767422 1.392178 36.38592 1.51608 E1
0.881491 0.850109 0.867612 0.988323 0.872593 0.919042 21.78679
0.907783 E1 0.543702 0.724683 0.519395 1.097197 0.70803 0.962919
19.59691 0.816538
REFERENCES
[0370] Bailey. J. and Cole, R. (1959) J. Biol. Chem. 234,
1733-1739. [0371] Ballou, L., Hitzeman, R., Lewis, M. & Ballou,
C. (1991) PNAS 88, 3209-3212. [0372] Benesch, P., Benesch. R.,
Gutcho, M. & Lanfer, L. (1956) Science 123, 981. [0373] Cavins,
J. & Friedman. (1970) Anal. Biochem. 35, 489. [0374] Cleland,
W. (1964) Biochemistry 3, 480 [0375] Creighton, E. (1988) BioEssays
8, 57 [0376] Darbre, A., John Wiley & Sons Ltd. (1987)
Practical Protein Chemistry--A Handbook. [0377] Darbre, A., John
Wiley & Sons Ltd. (1987) Practical Proteinchemistry p.69-79.
[0378] Doms et al, (1993), Virology 193, 545-562. [0379] Ellman, G.
(1959) Arch. Biochem. Biophys. 82, 70. [0380] Falkner, F. &
Moss, B. (1988) J. Virol. 62, 1849-1854. [0381] Friedman, M. &
Krull. (1969) Biochem. Biophys. Res. Commun. 37, 630. [0382]
Gallagher J. (1988) J. Cell Biol. 107, 2059-2073. [0383] Glazer,
A., Delange, R., Sigman, D. (1975) North Holland publishing
company. [0384] Elsevier, Biomedical. Part: Modification of protein
(p. 116). [0385] Graham, F. & van der Eb, A. (1973) Virology
52, 456-467. [0386] Grakoui et al. (1993) Journal of Virology
67:1385-1395. [0387] Grassetti, D. & Murray. J. (1969) Analyt.
Chim. Acta. 46, 139. [0388] Grassetti. D. & Murray, J. (1967)
Arch. Biochem Biophys. 119, 41. [0389] Helenius, Mol. Biol. Cell
(1994), 5: 253-265. [0390] Hijikata, M., Kato, N., Ootsuyama. Y.,
Nakagawa, M. & Shimotohno, K. (1991) Proc. Natl. Acad. Sci.
U.S.A. 88(13):5547-51. [0391] Hochuli, E., Bannwarth, W., Dobeli.
H., Gentz. R., Stuber, D. (1988) Biochemistry 88, 8976. [0392] Hsu.
H., Donets, M., Greenberg, H. & Feinstone, S. (1993) Hepatology
17:763-771. [0393] Inoue, Y., Suzuki, R., Matsuura, Y., Harada, S.,
Chiba. J., Watanabe. Y., Saito, I. & Miyamura, T. (1992) J.
Gen. Virol. 73:215-2154. [0394] Janknecht, R., de Martynoff, G. et
al., (1991) Proc. Natl. Acad. Sci. USA 88, 8972-8976. [0395] Kayman
(1991) J. Virology 65, 5323-5332. [0396] Kato, N., Oostuyama. Y.,
Tanaka, T., Nakagawa, M., Muraiso, K., Ohkoshi, S., Hijikata, M.,
Shimitohno, K. (1992) Virus Res. 22:107-123. [0397] Kniskern, P.,
Hagopian, A., Burke, P., Schultz, L., Montgomery, D., Hurni, W. Yu
Ip. C., Schulman, C., Maigetter, R. Wampler, D., Kubek, D., Sitrin.
R., West, D., Ellis. R. Miller, W. (1994) Vaccine 12:1021-1025.
[0398] Kohara, M., Tsukiyama-Kohara. K., Maki, N. Asano, K.,
Yoshizawa. K. Miki, K. Tanaka. S. Hattori, N., Matsuura, Y., Saito.
I., Miyamura, T. & Nomoto. A. (1992) J. Gen. Virol.
73:2313-2318. [0399] Mackett, M., Smith, G. & Moss, S. (1985)
In: DNA cloning: a practical approach (Ed. Glover, D.) IRL Press,
Oxford. [0400] Mackett, M., & Smith, G. (1986) J. Gen. Virol.
67, 2067-2082. [0401] Mackett, M., Smith, G. & Moss, E. (1984)
J. Virol. 49, 857-864. [0402] Mackett, M., Smith, G. & Moss, B.
(1984) Proc. Natl. Acad. Sci. USA 79, 7415-7419. [0403] Means, G.
(1971) Holden Day, Inc. [0404] Means, G. & Feeney, R. (1971)
Holden Day p. 105 & p. 217. [0405] Mita, E. Hayashi, N., Ueda.
K., Kasahara. A., Fusamotc, H., Takamizawa, A., Matsubara, K.,
Okayama, H. & Kamada T. (1992) Biochem. Biophys. Res. Comm.
183:925-930. [0406] Moore, S. (1963) J. Biol. Chem. 238, 235-237.
[0407] Okamoto, H., Okada, S., Sugiyarna, Y., Yotsumoto, S.,
Tanaka, T., Yoshizawa, H. Tsuda, F., Miyakawa, Y. & Mayumi, M.
(1990) Jpn. J. Exp. Med. 60:167-177. [0408] Panicali & Paoletti
(1982) Proc. Natl. Acad. Sci. USA 79, 4927-4931. [0409] Piccini,
A., Perkus, M. & Paoletti, E. (1987) Meth. Enzymol. 153,
545-563. [0410] Rose (1988) Annu. Rev. Cell Biol. 1988, 4: 257-288:
[0411] Ruegg, V. and Rudinger, J. (1977) Methods Enzymol. 47,
111-116. [0412] Shan, S. & Wong (1993) CRC-press p. 30-33.
[0413] Spaete, R., Alexander, D., Rugroden, M., Choo, C. Berger,
K., Crawford. K. Kuo. C. Leng, S., Lee, C., Ralston, R., et al.
(1992) Virology 188(2):819-30. [0414] Skehel. J., (1984) Proc.
Natl. Acad. Sci. USA 81, 1179-1783. [0415] Scunnenberg. H. Lange.
H., Philipson. L. Miltenourg, R. & van der Vliet, R. (1988)
Nucl. Acids Res. 16, 2431-2444. [0416] Stuyver, L., Van Arnhem, W.
Wyseur. A., DeLeys, R. & Maertens, G. (1993a) Biochem. Biophys.
Res. Commun. 192, 635-641. [0417] Stuyver, L., Rossau. R., Wyseur,
A., Duhamel. M., Vanderborght. B., Van Heuverswyn, H., &
Maertens, G. (1993b) J. Gen. Virol. 74, 1093-1102. [0418] Stuyver,
L. Van Arnhem, W., Wyseur, A., Hernandez. F. Delaporte. E.,
Maertens. G. (1994), Proc. Natl. Acad. Sci. USA 91:10134-10138.
[0419] Weil, L. & Seibler, S. (1961) Arch. Biochem. Biophys.
95, 470. [0420] Yokosuka, O., Ito, Y., Imazek., F., Ohto, M. &
Omata, M. (1992) Biochem. Biophys. Res. Commun. 189:565-571. [0421]
Miller P, Yano J, Yano E, Carroll C, Jayaram K, Ts'o P (1979)
Biochemistry 18:5134-43. [0422] Nielsen P, Egholm M, Berg R,
Buchardt O (1991) Science 254:1497-500. [0423] Nielsen P, Egholm M,
Berg R, Buchardt O (1993) Nucleic-Acids-Res. 21:197-200. [0424]
Asseline U, Delarue M, Lancelot G, Toulme F. Thuong N (1984) Proc.
Natl. Acad. Sci. USA 81:3297-301. [0425] Matsukura M, Shinozuka K,
Zon G. Mitsuya H, Reitz M, Cohen J. Broder S (1987) Proc. Natl.
Acad. Sci. USA 84:7706-10.
Sequence CWU 1
1
* * * * *