U.S. patent application number 12/053728 was filed with the patent office on 2008-11-06 for hcv e1 comprising specific disulfide bridges.
This patent application is currently assigned to INNOGENETICS N.V. Invention is credited to Alfons Bosman, Erik Depla, Stany Depraetere, Gert Verheyden.
Application Number | 20080274144 12/053728 |
Document ID | / |
Family ID | 34814476 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274144 |
Kind Code |
A1 |
Depraetere; Stany ; et
al. |
November 6, 2008 |
HCV E1 COMPRISING SPECIFIC DISULFIDE BRIDGES
Abstract
The invention relates to recombinantly or synthetically produced
HCV E1 envelope proteins or parts thereof comprising disulfides
between specific cysteine residues. The invention further relates
to viral-like particles and compositions comprising said HCV E1
envelope proteins or parts thereof as well as to methods using said
HCV E1 envelope proteins or parts thereof, and to kits comprising
said HCV E1 envelope proteins or parts thereof.
Inventors: |
Depraetere; Stany;
(Oudenaarde, BE) ; Depla; Erik; (Destelbergen,
BE) ; Verheyden; Gert; (Gent, BE) ; Bosman;
Alfons; (Opwijk, BE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
INNOGENETICS N.V
Ghent
BE
|
Family ID: |
34814476 |
Appl. No.: |
12/053728 |
Filed: |
March 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073942 |
Mar 8, 2005 |
7413741 |
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12053728 |
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60550421 |
Mar 8, 2004 |
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Current U.S.
Class: |
424/228.1 ;
435/339; 435/7.1; 530/387.1; 530/388.1 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 37/00 20180101; C07K 2317/34 20130101; C07K 2317/56 20130101;
A61K 2039/5258 20130101; C07K 14/005 20130101; C12N 2770/24222
20130101; C07K 16/109 20130101; A61P 31/14 20180101 |
Class at
Publication: |
424/228.1 ;
530/387.1; 530/388.1; 435/339; 435/7.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 5/06 20060101
C12N005/06; A61P 37/00 20060101 A61P037/00; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 9, 2004 |
EP |
04447057.3 |
Claims
1. A recombinant or synthetic HCV E1 envelope protein or a part
thereof comprising at least one of the following disulfides: a
disulfide between the pair of cysteines at amino acid positions 207
and 226, a disulfide between the pair of cysteines at amino acid
positions 229 and 238, or a disulfide between the pair of cysteines
at amino acid positions 272 and 281, wherein said amino acid
positions are relative to the HCV polyprotein which is starting
with the methionine of the Core protein at amino acid position 1 of
said HCV polyprotein.
2. The HCV E1 envelope protein or a part thereof according to claim
1 further comprising at least one of the cysteines at amino acid
positions 304 or 306 wherein said at least one cysteine is carrying
a free thiol group or a thiol group which is blocked.
3. The HCV E1 envelope protein or a part thereof according to claim
2 wherein said blocked thiol group is reversibly or irreversibly
blocked.
4. The HCV E1 envelope protein or a part thereof according to claim
2 wherein said at least one cysteine at amino acid position 304 or
306 is mutated to a non-cysteine amino acid or is deleted.
5. A viral-like particle comprising the HCV E1 envelope protein or
part thereof according to any one of claims 1 to 4.
6. A composition comprising the HCV E1 envelope protein or part
thereof according to any one of claims 1 to 4 or the viral-like
particle according to claim 5 and at least one of a
pharmaceutically acceptable carrier, adjuvant or vehicle.
7. An HCV vaccine comprising the HCV E1 envelope protein or part
thereof according to any one of claims 1 to 4 or the viral-like
particle according to claim 5 and at least one of a
pharmaceutically acceptable carrier, adjuvant or vehicle.
8. The HCV vaccine according to claim 7 which is a prophylactic or
therapeutic HCV vaccine.
9. An antibody selectively binding to an HCV E1 envelope protein or
a part thereof comprising at least one of the following disulfides:
a disulfide between the pair of cysteines at amino acid positions
207 and 226, a disulfide between the pair of cysteines at amino
acid positions 229 and 238, or a disulfide between the pair of
cysteines at amino acid positions 272 and 281, wherein said amino
acid positions are relative to the HCV polyprotein which is
starting with the methionine of the Core protein at amino acid
position 1 of said HCV polyprotein; or selectively binding to a
viral-like particle comprising any of said HCV E1 envelope proteins
or parts thereof, an antibody capable of competing with said
antibody for selectively binding said HCV E1 envelope protein, or a
fragment of any of said antibodies.
10. The antibody according to claim 9 that is isolated from a
mammal immunized with the HCV E1 envelope protein or part thereof
according to any one of claims 1 to 4 or with the viral-like
particle according to claim 5.
11. The antibody according to claim 9 or 10 which is a monoclonal
antibody.
12. The antibody according to any of claims 9 to 11 which is
secreted by the hybridoma cell line of DSM deposit with accession
number ACC 2470.
13. The hybridoma cell line of DSM deposit with accession number
ACC 2470.
14. A method for determining the presence of HCV antibodies in a
sample, said method comprising the steps of: (i) contacting said
sample with the HCV E1 envelope protein or part thereof according
to any one of claims 1 to 4 or the viral-like particle according to
claim 5 under conditions allowing the formation of an immunological
antigen-antibody complex; (ii) determining the immunological
complex formed in (i); (iii) inferring from (ii) the presence of
HCV antibodies in said sample.
15. A method for determining HCV E1 antigens in a sample, said
method comprising the steps of: (i) contacting said sample with the
antibody according to any one of claims 9 to 12 under conditions
allowing the formation of an immunological antigen-antibody
complex; (ii) determining the immunological complex formed in (i);
(iii) inferring from (ii) the presence of HCV E1 antigens in said
sample.
16. The method according to claim 14 or 15 wherein in step (i) the
HCV E1 envelope protein or part thereof according to any one of
claims 1 to 4, the viral-like particle according to claim 5 or the
antibody according to any one of claims 9 to 12 is added as
competitor; and wherein said HCV E1 envelope protein or part
thereof, viral-like particle or antibody used in said step (i) or
said HCV E1 envelope protein or part thereof, viral-like particle
or antibody added as competitor are labeled.
17. A method for screening compounds capable of modulating the
binding between an HCV E1 envelope protein and an E1 ligand, said
method comprising: (i) contacting said E1 ligand with an HCV E1
envelope protein or part thereof according to any one of claims 1
to 4 or with the viral-like particle according to claim 5 under
conditions allowing the formation of an HCV E1-E1 ligand complex;
(ii) adding a compound suspected of modulating the binding between
an HCV E1 envelope protein and an E1 ligand to the HCV E1-E1 ligand
complex formed in (i); (iii) determining the change in amount of
HCV E1-E1 ligand complex formed in (i) and (ii); (iv) inferring
from (iii) whether the compound added in (ii) is a modulator of
binding between an HCV E1 envelope protein and an E1 ligand.
18. A method for screening compounds capable of modulating the
binding between an HCV E1 envelope protein and an E1 ligand, said
method comprising: (i) contacting, under conditions allowing the
formation of an HCV E1-E1 ligand complex, said E1 ligand with an
HCV E1 envelope protein or part thereof according to any one of
claims 1 to 4 or with the viral-like particle according to claim 5
in the presence and absence, respectively, of a compound suspected
of modulating the binding between an HCV E1 envelope protein and an
E1 ligand; (ii) determining the amount of HCV E1-E1 ligand complex
formed in (i) in the presence and absence of said compound; (iii)
inferring from (ii) whether said compound is a modulator of binding
between an HCV E1 envelope protein and an E1 ligand.
19. A method according to claim 17 or 18 wherein in step (i) the
HCV E1 envelope protein or part thereof according to any one of
claims 1 to 4 or the viral-like particle according to claim 5 is
added as competitor; and wherein said HCV E1 envelope protein or
part thereof or viral-like particle used in said step (i) or said
HCV E1 envelope protein or part thereof or viral-like particle
added as competitor are labeled.
20. A diagnostic kit for determining the presence of HCV antibodies
in a sample, said kit comprising at least one of an HCV E1 envelope
protein or part thereof according to any one of claims 1 to 4, the
viral-like particle according to claim 5 and the antibody according
to any of claims 9 to 12.
21. A diagnostic kit for determining the presence of HCV E1
envelope protein antigens in a sample comprising at least one of an
HCV E1 envelope protein or part thereof according to any one of
claims 1 to 4, the viral-like particle according to claim 5 and the
antibody according to any of claims 9 to 12.
22. A method of producing the recombinant HCV E1 envelope protein
or part thereof according to any one of claims 1 to 4, said method
comprising the steps of (i) expressing of an HCV E1 envelope
protein or part thereof in a eukaryotic host; (ii) isolating from
the pool of HCV E1 envelope protein or part thereof expressed in
(i) the non-aggregated monomeric fraction of HCV E1 envelope
protein or part thereof according to any one of claims 1 to 4.
23. A method of producing the synthetic HCV E1 envelope protein or
part thereof according to any one of claims 1 to 4 wherein said
method comprises: (i) chemical synthesis of an HCV E1 envelope
protein or part thereof; (ii) introducing during or after the
chemical synthesis of said HCV E1 envelope protein or part thereof
at least one disulfide according to claim 1.
24. Use of a HCV E1 envelope protein or part thereof according to
any of claims 1 to 4, the viral-like particle according to claim 5
and/or the antibody according to any of claims 9 to 12 for the
preparation of a medicament or a vaccine or for the preparation of
an immunoassay or immunoassay kit.
25. Use of a HCV E1 envelope protein or part thereof according to
any of claims 1 to 4 or the viral-like particle according to claim
5 as a carrier of another protein or of a non-proteinaceous
molecule.
26. An isolated protein comprising the HCV E1 envelope protein or
part thereof according to any of claims 1 to 4.
27. The isolated protein according to claim 26 further comprising
at least one of: an N-terminal flanking amino acid or amino acid
sequence of an HCV protein or part thereof not naturally contiguous
with said HCV E1 envelope protein or part thereof; a C-terminal
flanking amino acid or amino acid sequence of an HCV protein or
part thereof not naturally contiguous with said HCV E1 envelope
protein or part thereof; an N-terminal flanking non-HCV amino acid
or amino acid sequence; a C-terminal flanking non-HCV amino acid or
amino acid sequence.
28. The isolated protein according to claim 26 comprising the HCV
E1 envelope protein or part thereof as carrier protein.
Description
FIELD OF THE INVENTION
[0001] The invention relates to recombinantly or synthetically
produced HCV E1 envelope proteins or parts thereof comprising
disulfides between specific cysteine residues. The invention
further relates to viral-like particles and compositions comprising
said HCV E1 envelope proteins or parts thereof as well as to
methods using said HCV E1 envelope proteins or parts thereof, and
to kits comprising said HCV E1 envelope proteins or parts
thereof.
BACKGROUND OF THE INVENTION
[0002] The about 9.6 kb single-stranded RNA genome of the HCV virus
comprises a 5'- and 3'-non-coding region (NCRs) and, in between
these NCRs a single long open reading frame of about 9 kb encoding
an HCV polyprotein of about 3000 amino acids.
[0003] HCV polypeptides are produced by translation from the open
reading frame and cotranslational proteolytic processing.
Structural proteins are derived from the amino-terminal one-fourth
of the coding region and include the capsid or Core protein (about
21 kDa), the E1 envelope glycoprotein (about 35 kDa) and the E2
envelope glycoprotein (about 70 kDa, previously called NS1), and p7
(about 7 kDa). The E2 protein can occur with or without a
C-terminal fusion of the p7 protein (Shimotohno et al. 1995).
Recently, an alternative open reading frame in the Core-region was
found which is encoding and expressing a protein of about 17 kDa
called F (Frameshift) protein (Xu et al. 2001; Ou & Xu in US
Patent Application Publication No. US2002/0076415). In the same
region, ORFs for other 14-17 kDa ARFPs (Alternative Reading Frame
Proteins), A1 to A4, were discovered and antibodies to at least A1,
A2 and A3 were detected in sera of chronically infected patients
(Walewski et al. 2001). From the remainder of the HCV coding
region, the non-structural HCV proteins are derived which include
NS2 (about 23 kDa), NS3 (about 70 kDa), NS4A (about 8 kDa), NS4B
(about 27 kDa), NS5A (about 58 kDa) and NS5B (about 68 kDa)
(Grakoui et al. 1993).
[0004] HCV is the major cause of non-A, non-B hepatitis worldwide.
Acute infection with HCV (20% of all acute hepatitis infections)
frequently leads to chronic hepatitis (70% of all chronic hepatitis
cases) and end-stage cirrhosis. It is estimated that up to 20% of
HCV chronic carriers may develop cirrhosis over a time period of
about 20 years and that of those with cirrhosis between 1 to
4%/year is at risk to develop liver carcinoma (Lauer & Walker
2001, Shiffman 1999). An option to increase the life-span of
HCV-caused end-stage liver disease is liver transplantation (30% of
all liver transplantations world-wide are due to
HCV-infection).
[0005] It is generally accepted that the more a recombinantly
expressed HCV envelope protein is resembling a naturally produced
HCV envelope protein (naturally produced in the sense of being the
consequence of infection of a host by HCV), the better such an HCV
envelope protein is suited for diagnostic, prophylactic and/or
therapeutic uses or purposes, and for use in drug screening
methods. HCV envelope proteins are currently obtained via
recombinant expression systems such as mammalian cell cultures
infected with E1 or E2-recombinant vaccinia virus (see, e.g.,
WO96/04385), stably transformed mammalian cell lines, and
recombinant yeast cells (see, e.g., WO02/086101). These expression
systems suffer from the drawback that the expressed HCV envelope
proteins tend to form aggregates that comprise contaminating
proteins and which are in part stabilized by intermolecular
disulfide bridges. In order to obtain sufficient amounts of
recombinant HCV envelope proteins the bulk of intracellularly
accumulated HCV envelope proteins is reduced and/or cysteines are
blocked and/or a detergent is used during the purification process.
As such the obtained recombinant HCV envelope proteins are not
closely resembling naturally produced HCV envelope proteins.
[0006] Folding of the HCV E1 envelope protein is dependent on the
formation of disulfide bridges. At present not much is known about
the requirements needed for an HCV E1 envelope protein to assume
its folding. It has been suggested that at least some of the
cysteines of the HCV E1 envelope protein are involved in
intramolecular disulfide bridges. In an in vitro assay it was shown
that oxidation of HCV E1 (i.e., the formation of disulfides in HCV
E1) is requires the presence of both Core and E2 (Merola et al.
2001). Recently, the results of a computer prediction of the
disulfide bridges within HCV E1 was published. Disulfides between
the cysteine residues 207 and 306, 226 and 304, 229 and 281, and
238 and 272 were predicted (Garry and Dash 2003). Note that the HCV
E1 amino acid sequences in FIG. 1 and FIG. 5 of this reference are
not identical to each other and that the HCV E1 amino acid sequence
in FIG. 1 is missing amino acid 250; the above-indicated numbering
of the cysteine residues has been adapted relative to Garry and
Dash (2003) to correspond to the numbering of the cysteine residues
as used hereafter in the description of the invention.
SUMMARY OF THE INVENTION
[0007] The present invention relates in a first aspect to a
recombinant or synthetic HCV E1 envelope protein or a part thereof
comprising at least one of the following disulfides: [0008] a
disulfide between the pair of cysteines at amino acid positions 207
and 226, [0009] a disulfide between the pair of cysteines at amino
acid positions 229 and 238, or [0010] a disulfide between the pair
of cysteines at amino acid positions 272 and 281, wherein said
amino acid positions are relative to the HCV polyprotein which is
starting with the methionine of the Core protein at amino acid
position 1 of said HCV polyprotein. Furthermore, said recombinant
or synthetic HCV E1 envelope protein or a part thereof can further
comprise at least one of the cysteines at amino acid positions 304
or 306 wherein said at least one cysteine is carrying a free thiol
group or a thiol group which is blocked, i.e., reversibly or
irreversibly blocked. Alternatively, said at least one cysteine at
amino acid position 304 or 306 is mutated to a non-cysteine amino
acid or is deleted.
[0011] In a further aspect of the invention any of the above HCV E1
envelope proteins or parts thereof is comprised in a viral-like
particle.
[0012] The invention also relates to compositions comprising any of
the above HCV E1 envelope protein or part thereof or a viral-like
particle and at least one of a pharmaceutically acceptable carrier,
adjuvant or vehicle. In particular said composition is an HCV
vaccine, such as a prophylactic or therapeutic HCV vaccine
[0013] The invention further embodies antibodies or fragments
thereof selectively binding to an HCV E1 envelope protein or a part
thereof comprising at least one of the following disulfides: [0014]
a disulfide between the pair of cysteines at amino acid positions
207 and 226, [0015] a disulfide between the pair of cysteines at
amino acid positions 229 and 238, or [0016] a disulfide between the
pair of cysteines at amino acid positions 272 and 281, wherein said
amino acid positions are relative to the HCV polyprotein which is
starting with the methionine of the Core protein at amino acid
position 1 of said HCV polyprotein; or selectively binding to a
viral-like particle comprising any of said HCV E1 envelope proteins
or parts thereof, antibodies capable of competing with said
antibodies for selectively binding said HCV E1 envelope protein, or
a fragment of any of said antibodies.
[0017] Said antibodies can be isolated from a mammal immunized with
any of the above HCV E1 envelope proteins or parts thereof or with
the above viral-like particle. Fragments of said antibodies can
than be prepared. In particular said antibody or fragment thereof
is a monoclonal antibody or a fragment thereof. A specific antibody
is the antibody secreted by the hybridoma cell line of DSM deposit
with accession number ACC 2470. Again, fragments of this specific
antibody can than be prepared. Determining if two antibodies are
competing with each other for binding an epitope can easily be
performed.
[0018] The invention also relates to the hybridoma cell line of DSM
deposit with accession number ACC 2470.
[0019] Methods for determining the presence of HCV antibodies in a
sample are part of another embodiment of the invention, said
methods comprising the steps of: [0020] (i) contacting said sample
with any of the above HCV E1 envelope proteins or parts thereof or
with the above viral-like particle under conditions allowing the
formation of an immunological antigen-antibody complex; [0021] (ii)
determining the immunological complex formed in (i); [0022] (iii)
inferring from (ii) the presence of HCV antibodies in said
sample.
[0023] Methods for determining the presence of HCV E1 antigens in a
sample are part of another embodiment of the invention, said
methods comprising the steps of: [0024] (i) contacting said sample
with any of the above antibodies under conditions allowing the
formation of an immunological antigen-antibody complex; [0025] (ii)
determining the immunological complex formed in (i); [0026] (iii)
inferring from (ii) the presence of HCV E1 antigens in said
sample.
[0027] In particular, in step (i) of said methods any of the above
HCV E1 envelope proteins or parts thereof, the above viral-like
particle or any of the above antibodies is added as competitor; and
said HCV E1 envelope protein or part thereof, viral-like particle
or antibody used in said step (i) or said HCV E1 envelope protein
or part thereof, viral-like particle or antibody added as
competitor are labeled.
[0028] Another aspect of the invention relates to methods for
screening compounds capable of modulating the binding between an
HCV E1 envelope protein and an E1 ligand, said methods comprising:
[0029] (i) contacting said E1 ligand with any of the above HCV E1
envelope proteins or parts thereof with the above viral-like
particle under conditions allowing the formation of an HCV E1-E1
ligand complex; [0030] (ii) adding a compound suspected of
modulating the binding between an HCV E1 envelope protein and an E1
ligand to the HCV E1-E1 ligand complex formed in (i); [0031] (iii)
determining the change in amount of HCV E1-E1 ligand complex formed
in (i) and (ii); [0032] (iv) inferring from (iii) whether the
compound added in (ii) is a modulator of binding between an HCV E1
envelope protein and an E1 ligand.
[0033] Further methods for screening compounds capable of
modulating the binding between an HCV E1 envelope protein and an E1
ligand, comprise the steps of: [0034] (i) contacting, under
conditions allowing the formation of an HCV E1-E1 ligand complex,
said E1 ligand with any of the above HCV E1 envelope proteins or
parts thereof or with the above viral-like particle in the presence
and absence, respectively, of a compound suspected of modulating
the binding between an HCV E1 envelope protein and an E1 ligand;
[0035] (ii) determining the amount of HCV E1-E1 ligand complex
formed in (i) in the presence and absence of said compound; [0036]
(iii) inferring from (ii) whether said compound is a modulator of
binding between an HCV E1 envelope protein and an E1 ligand.
[0037] In particular, in steps (i) of said methods any of above HCV
E1 envelope proteins or parts thereof or the above viral-like
particle is added as competitor; and said HCV E1 envelope protein
or part thereof or viral-like particle used in said step (i) or
said HCV E1 envelope protein or part thereof or viral-like particle
added as competitor are labeled.
[0038] The invention additionally relates to diagnostic kits for
determining the presence of HCV antibodies or HCV E1 envelope
proteins in a sample, said kits comprising any of the above HCV E1
envelope proteins or parts, the above viral-like particle and/or
any of the above antibodies.
[0039] Another aspect of the invention envisages methods of
producing a recombinant HCV E1 envelope protein or part thereof
according to the invention, said methods comprising the steps of
[0040] (i) expressing of an HCV E1 envelope protein or part thereof
in a eukaryotic host; [0041] (ii) isolating from the pool of HCV E1
envelope protein or part thereof expressed in (i) the
non-aggregated monomeric fraction of HCV E1 envelope protein or
part thereof according to the invention.
[0042] Methods of producing a synthetic HCV E1 envelope protein or
part thereof according to the invention are part of a further
aspect wherein said methods comprise: [0043] (i) chemical synthesis
of an HCV E1 envelope protein or part thereof; [0044] (ii)
introducing during or after the chemical synthesis of an HCV E1
envelope protein or part thereof at least one disulfide as
determined in the current invention.
[0045] Yet another aspect of the invention relates to the use of
any of the above HCV E1 envelope proteins or parts thereof, the
above viral-like particle and/or any of the above antibodies for
the preparation of a medicament or a vaccine or for the preparation
of an immunoassay or a diagnostic kit.
[0046] The use of any of the above HCV E1 envelope proteins or
parts thereof or the above viral-like particle as a carrier of
another protein or of a non-proteinaceous molecule form a further
aspect of the invention.
[0047] In another aspect, the invention covers isolated proteins
comprising an HCV E1 envelope protein or part thereof according to
the invention. In particular said isolated protein is further
comprising at least one of: [0048] an N-terminal flanking amino
acid or amino acid sequence of an HCV protein or part thereof not
naturally contiguous with said HCV E1 envelope protein or part
thereof; [0049] a C-terminal flanking amino acid or amino acid
sequence of an HCV protein or part thereof not naturally contiguous
with said HCV E1 envelope protein or part thereof; [0050] an
N-terminal flanking non-HCV amino acid or amino acid sequence;
[0051] a C-terminal flanking non-HCV amino acid or amino acid
sequence.
[0052] Alternatively, said isolated protein is comprising the HCV
E1 envelope protein or part thereof as carrier protein.
FIGURE LEGENDS
[0053] FIG. 1. Schematic representation of a full-length HCV E1
envelope protein with indication of the relative position of the 8
cysteine residues numbered C1 to C8. The arabic numbers refer to
amino acid positions in the HCV polyprotein and indicate the
positions of the amino- and carboxy-terminal amino acids of a
full-length HCV E1 envelope protein and the E1s protein (192 and
383 for E1, and 192 and 326 for E1s, respectively; dotted lines)
and the positions of said 8 cysteine residues. The indicated
intramolecular disulfide bridges ("S--S") were determined as
outlined in the Examples. Further indicated is the relative
position of the epitope selectively recognized by the antibody
IGH388 (see also FIG. 5 and Legend thereto).
[0054] FIG. 2. Non-reducing SDS-PAGE followed by silver staining
(A) and western blotting (B) of the E1s viral-like particles
obtained as described in Example 1. To the left are indicated the
molecular weights of the molecular weight markers.
[0055] FIG. 3. Mid-range (3-10 kDa) MALDI-TOF-MS spectrum of
T2'-T7'-T9' peptide mixture left to oxidize for 31 h as described
in Example 5. T9' (Thr del) represents the side product of T9'
formed during peptide synthesis and which lacks one of the two
threonine-residues compared to T9.
[0056] FIG. 4. Amino acid sequences and nucleic acid sequences of
the VH and VKL chains of the monoclonal antibody IGH388 obtained
and characterized as described in Examples 7 and 8 hereafter.
[0057] FIG. 5. Binding of the monoclonal antibodies IGH 201 and IGH
388 with monomeric E1 or sulphonated E1 ("E1 SO3") expressed as
optical density measured in ELISA. Monomeric E1 or sulphonated E1
("E1 SO3") was coated on ELISA plates at a concentration of 2
.mu.g/mL. After blocking a serial dilution of the antibodies was
incubated, the starting concentration for IGH 201 was 1 .mu.g/mL,
for IGH 388 3 .mu.g/mL. The dilution factor applied is about 3.16
fold. Finally after washing bound antibodies are detected using an
anti-mouse or anti-human secondary antiserum conjugated with
peroxidase.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein. Likewise, the terms "disulfide", "disulfide
bond" and "disulfide bridge" are used interchangeably herein.
[0059] In work leading to the present invention a non-aggregated
monomeric HCV E1 envelope protein fraction was isolated from the
bulk of recombinantly expressed HCV E1 envelope protein in the
absence of a reducing agent (see Example 1; see Example 1 also for
a description of monomeric HCV E1 envelope protein). Due to the
omission of conditions disrupting intra- and intermolecular
disulfides during purification this monomeric recombinant HCV E1
envelope protein is believed to advantageously resemble naturally
produced HCV E1 envelope protein.
[0060] A first surprising observation was that the obtained
non-aggregated monomeric HCV E1 envelope protein fraction contained
intramolecular disulfide bonds. This is in contradiction with the
report by Merola et al. (2002) who showed that both the HCV Core
and E2 proteins assist the HCV E1 protein in its folding and
oxidation. The results of the present invention indicate that no
other HCV protein was required for formation of intramolecular
disulfide bonds in the HCV E1 envelope protein. It is thus possible
to obtain oxidized HCV E1 envelope protein closely resembling
naturally produced HCV E1 envelope protein in a production and
purification process not involving other HCV proteins such as Core
and/or E2.
[0061] A second surprising aspect emerged after determining the
position of the disulfide bonds in the obtained non-aggregated
monomeric HCV E1 envelope protein fraction, i.e., after determining
which cysteines of the HCV E1 envelope protein are involved in
which disulfide bonds. The experimentally determined position of
the disulfide bonds is completely different from the disulfide
pattern predicted by Garry and Dash (2003). Furthermore surprising
is that two of the eight cysteines in the non-aggregated monomeric
HCV E1 envelope protein fraction of the invention are not engaged
in intramolecular disulfide bonds.
[0062] In a first aspect the invention relates to a recombinant or
synthetic HCV E1 envelope protein or a part thereof comprising at
least one of the following disulfides: [0063] a disulfide between
the pair of cysteines at amino acid positions 207 and 226, [0064] a
disulfide between the pair of cysteines at amino acid positions 229
and 238, or [0065] a disulfide between the pair of cysteines at
amino acid positions 272 and 281, wherein said amino acid positions
are relative to the HCV polyprotein which is starting with the
methionine of the Core protein at amino acid position 1 of said HCV
polyprotein.
[0066] The amino acid positions indicated above are to be
considered as relative: a skilled person will recognize that the
numbering of the cysteines of the HCV E1 envelope protein in the
HCV polyprotein can be subject of changes. Such changes in amino
acid numbering can be the consequence of HCV genotype-, HCV
subtype-, or HCV isolate-specific amino acid insertions, deletions
or mutations in the Core and/or E1 portion of the HCV polyprotein.
It is to clear that said changes in amino acid numbering are
relative to the amino acid numbering of the HCV E1 envelope protein
as indicated in FIG. 1. In relation to the first aspect of the
invention it is furthermore clear that any fragment of said HCV E1
envelope protein has to comprise at least one pair of cysteines
that can engage in the formation of a disulfide bond.
[0067] To further clarify the scope of the HCV E1 envelope protein
variants envisaged by the current invention it is clear that any of
the above HCV E1 envelope proteins of the invention can further
comprise at least one of the cysteines at positions 304 and 306,
i.e., the cysteines of the HCV E1 envelope protein not involved in
an intramolecular disulfide bond. This at least one cysteine at
position 304 or 306 can be the naturally occurring cysteine which
is carrying a free thiol group or which is carrying a thiol group
that is blocked. Alternatively, this at least one cysteine at
position 304 or 306 is mutated to a non-cysteine amino acid or is
deleted from the HCV E1 envelope protein.
[0068] The mutation of a cysteine to a non-cysteine amino acid can
be conservative (e.g., to alanine or to serine) or
non-conservative. A conservative substitution will in general
affect the overall functioning of the protein wherein the
substitution is introduced less seriously than a non-conservative
substitution.
HCV E1 Envelope Protein
[0069] The HCV E1 envelope protein or part or variant thereof
according to the invention may be of synthetic origin, i.e.
synthesized by applying organic chemistry, or of recombinant
origin. As "HCV E1 envelope protein" is herein understood any
isolated HCV E1 envelope protein or any part thereof comprising at
least a cysteine pair as outlined above.
[0070] An HCV E1 envelope protein may be produced by expression in,
e.g., mammalian or insect cells infected with recombinant viruses,
yeast cells or bacterial cells.
[0071] More particularly, said mammalian cells include HeLa cells,
Vero cells, RK13 cells, MRC-5 cells, Chinese hamster ovary (CHO)
cells, Baby hamster kidney (BHK) cells and PK15 cells.
[0072] More particularly, said insect cells include cells of
Spodoptera frugiperda, such as Sf9 cells.
[0073] More particularly, said recombinant viruses include
recombinant vaccinia viruses, recombinant adenoviruses, recombinant
baculoviruses, recombinant canary pox viruses, recombinant Semlike
Forest viruses, recombinant alphaviruses, recombinant Ankara
Modified viruses and recombinant avipox viruses.
[0074] More particularly, said yeast cells include cells of
Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces
kluyveri, or Saccharomyces uvarum, Schizosaccharomyces, such as
Schizosaccharomyces pombe, Kluyveromyces, such as Kluyveromyces
lactis, Yarrowia, such as Yarrowia lipolytica, Hansenula, such as
Hansenula polymorpha, Pichia, such as Pichia pastoris, Aspergillus
species, Neurospora, such as Neurospora crassa, or Schwanniomyces,
such as Schwanniomyces occidentalis, or mutant cells derived from
any thereof. More specifically, the HCV peptide or part thereof
according to the invention is the product of expression in a
Hansenula cell.
[0075] More particularly, said bacterial cells include cells of
Escherichia coli or Streptomyces species.
Blocking of Cysteines
[0076] An "irreversibly blocked cysteine" is a cysteine of which
the cysteine thiol-group is irreversibly protected by chemical
means. In particular, "irreversible protection" or "irreversible
blocking" by chemical means refers to alkylation, preferably
alkylation of a cysteine in a protein by means of alkylating
agents, such as, for example, active halogens, ethylenimine or
N-(iodoethyl)trifluoro-acetamide. In this respect, it is to be
understood that alkylation of cysteine thiol-groups refers to the
replacement of the thiol-hydrogen by (CH.sub.2).sub.nR, in which n
is 0, 1, 2, 3 or 4 and R.dbd.H, COOH, NH.sub.2, CONH.sub.2, phenyl,
or any derivative thereof. Alkylation can be performed by any
method known in the art, such as, for example, active halogens
X(CH.sub.2).sub.nR in which X is a halogen such as I, Br, Cl or F.
Examples of active halogens are methyliodide, iodoacetic acid,
iodoacetamide, and 2-bromoethylamine.
[0077] A "reversibly blocked cysteine" is a cysteine of which the
cysteine thiol-groups is reversibly protected. In particular, the
term "reversible protection" or "reversible blocking" as used
herein contemplates covalently binding of modification agents to
the cysteine thiol-groups, as well as manipulating the environment
of the protein such, that the redox state of the cysteine
thiol-groups remains (shielding). Reversible protection of the
cysteine thiol-groups can be carried out chemically or
enzymatically.
[0078] The term "reversible protection by enzymatical means" as
used herein contemplates reversible protection mediated by enzymes,
such as for example acyl-transferases, e.g. acyl-transferases that
are involved in catalysing thio-esterification, such as palmitoyl
acyltransferase. The term "reversible protection by chemical means"
as used herein contemplates reversible protection: [0079] 1. by
modification agents that reversibly modify cysteinyls such as for
example by sulphonation and thio-esterification; [0080] 2. by
modification agents that reversibly modify the cysteinyls of the
present invention such as, for example, by heavy metals, in
particular Zn.sup.2+, Cd.sup.2+, mono-, dithio- and
disulfide-compounds (e.g. aryl- and alkylmethanethiosulfonate,
dithiopyridine, dithiomorpholine, dihydrolipoamide, Ellmann
reagent, Aldrothiol.TM. (Aldrich) (Rein et al. 1996),
dithiocarbamates), or thiolation agents (e.g. gluthathion, N-Acetyl
cysteine, cysteineamine). Dithiocarbamate comprise a broad class of
molecules possessing an R.sub.1R.sub.2NC(S)SR.sub.3 functional
group, which gives them the ability to react with sulphydryl
groups. Thiol containing compounds are preferentially used in a
concentration of 0.1-50 mM, more preferentially in a concentration
of 1-50 mM, and even more preferentially in a concentration of
10-50 mM; [0081] 3. by the presence of modification agents that
preserve the thiol status (stabilise), in particular antioxidantia,
such as for example DTT, dihydroascorbate, vitamins and derivates,
mannitol, amino acids, peptides and derivates (e.g. histidine,
ergothioneine, carnosine, methionine), gallates, hydroxyanisole,
hydroxytoluene, hydroquinon, hydroxymethylphenol and their
derivates in concentration range of 10 .mu.M-10 mM, more
preferentially in a concentration of 1-10 mM; [0082] 4. by thiol
stabilising conditions such as, for example, (i) cofactors as metal
ions (Zn.sup.2+, Mg.sup.2+), ATP, (ii) pH control (e.g. for
proteins in most cases pH .about.5 or pH is preferentially thiol
pK.sub.a -2; e.g. for peptides purified by Reversed Phase
Chromatography at pH .about.2). Combinations of reversible
protection as described in (1), (2), (3) and (4) may be
applied.
[0083] The reversible protection and thiol stabilizing compounds
may be presented under a monomeric, polymeric or liposomic
form.
[0084] The removal of the reversible protection state of the
cysteine residues can be accomplished chemically or enzymatically
by, e.g.: [0085] a reductant, in particular DTT, DTE,
2-mercaptoethanol, dithionite, SnCl.sub.2, sodium borohydride,
hydroxylamine, TCEP, in particular in a concentration of 1-200 mM,
more preferentially in a concentration of 50-200 mM; [0086] removal
of the thiol stabilising conditions or agents by e.g. pH increase;
[0087] enzymes, in particular thioesterases, glutaredoxine,
thioredoxine, in particular in a concentration of 0.01-5 .mu.M,
even more particular in a concentration range of 0.1-5 .mu.M; or
[0088] combinations of the above described chemical and/or
enzymatical conditions.
[0089] The removal of the reversible protection state of the
cysteine residues can be carried out in vitro or in vivo, e.g., in
a cell or in an individual.
Viral-Like Particles
[0090] In a further aspect of the invention any of the above HCV E1
envelope proteins or parts thereof is comprised in a viral-like
particle.
[0091] The terms "oligomeric particle", "virus-like particle",
"viral-like particle", or "VLP" are used interchangeably herein and
are defined as structures of a specific nature and shape containing
several HCV E1 envelope proteins. In particular these VLPs are not
formed in cells but are reconstituted in vitro starting from
purified HCV envelope proteins (see Example 1 of WO99/67285). It
should be clear that the particles are defined to be devoid of
infectious HCV RNA genomes. The particles can be higher-order
particles of spherical nature which can be empty, consisting of a
shell of envelope proteins in which lipids, detergents, the HCV
core protein, or adjuvant molecules can be incorporated. The latter
particles can also be encapsulated by liposomes or apolipoproteins,
such as, for example, apolipoprotein B or low density lipoproteins,
or by any other means of targeting said particles to a specific
organ or tissue. In this case, such empty spherical particles are
often referred to as "virus-like particles" or VLPs. Alternatively,
the higher-order particles can be solid spherical structures, in
which the complete sphere consists of HCV envelope protein
oligomers, in which lipids, detergents, the HCV core protein, or
adjuvant molecules can be additionally incorporated, or which in
turn may be themselves encapsulated by liposomes or
apolipoproteins, such as, for example, apolipoprotein B, low
density lipoproteins, or by any other means of targeting said
particles to a specific organ or tissue, e.g. asialoglycoproteins.
The particles can also consist of smaller structures (compared to
the empty or solid spherical structures indicated above) which are
usually round-shaped and which usually do not contain more than a
single layer of HCV envelope proteins. A typical example of such
smaller particles are rosette-like structures. Such rosette-like
structures are usually organized in a plane and are round-shaped,
e.g. in the form of a wheel. Again lipids, detergents, the HCV core
protein, or adjuvant molecules can be additionally incorporated, or
the smaller particles may be encapsulated by liposomes or
apolipoproteins, such as, for example, apolipoprotein B or low
density lipoproteins, or by any other means of targeting said
particles to a specific organ or tissue. Smaller particles may also
form small spherical or globular structures consisting of a similar
smaller number of HCV envelope proteins in which lipids,
detergents, the HCV core protein, or adjuvant molecules could be
additionally incorporated, or which in turn may be encapsulated by
liposomes or apolipoproteins, such as, for example, apolipoprotein
B or low density lipoproteins, or by any other means of targeting
said particles to a specific organ or tissue. The size (i.e. the
diameter) of the above-defined particles, as measured by dynamic
light scattering (DLS) or electron microscope (EM) techniques, is
usually between 1 to 100 nm, or between 2 to 70 nm. Virus-like
particles of HCV envelope proteins have been described in
International Patent Application Publication Nos. WO99/67285,
WO02/055548 and in International Patent Publication No.
WO02/086101. The HCV viral-like particles as described above can
comprise HCV E1 envelope proteins or parts thereof according to the
invention and can furthermore comprise other HCV E1 envelope
proteins (e.g., from different HCV genotypes, -subtypes, or
-isolates) and/or HCV E2 envelope proteins or parts thereof (see
Examples 11 and 12 of WO99/67285).
Vaccines and Vaccine Compositions
[0092] The invention also relates to compositions comprising any of
the above HCV E1 envelope protein or part thereof or the above-said
viral-like particle and at least one of a pharmaceutically
acceptable carrier, adjuvant or vehicle. In particular said
composition is an HCV vaccine, such as a prophylactic or
therapeutic HCV vaccine
[0093] A vaccine or vaccine composition is an immunogenic
composition capable of eliciting an immune response sufficiently
broad and vigorous to provoke one or both of: [0094] a stabilizing
effect on the multiplication of a pathogen already present in a
host and against which the vaccine composition is targeted; and
[0095] an increase of the rate at which a pathogen newly introduced
in a host, after immunization with a vaccine composition targeted
against said pathogen, is resolved from said host.
[0096] A vaccine composition may also provoke an immune response
broad and strong enough to exert a negative effect on the survival
of a pathogen already present in a host or broad and strong enough
to prevent an immunized host from developing disease symptoms
caused by a newly introduced pathogen. A vaccine composition may
also induce an immune response in a host already infected with the
pathogen against which the immune response leading to a halting or
reversion of disease progression in the absence of eradication of
the pathogen. In particular the vaccine composition of the
invention is a HCV vaccine composition. The HCV vaccine composition
is comprising as active substance at least one HCV E1 envelope
protein or part thereof according to the invention, or a VLP
comprising said protein. In particular the HCV vaccine or HCV
vaccine composition is comprising an effective amount of said HCV
E1 envelope protein and/or of said VLP. Said HCV vaccine
composition may additionally comprise one or more further active
substances and/or at least one of a pharmaceutically acceptable
carrier, adjuvant or vehicle.
[0097] An effective amount of an antigen (either "free" or in the
form of a VLP) in a vaccine or vaccine composition is referred to
as an amount of antigen required and sufficient to elicit an immune
response. It will be clear to the skilled artisan that the immune
response sufficiently broad and vigorous to provoke the effects
envisaged by the vaccine composition may require successive (in
time) immunizations with the vaccine composition as part of a
vaccination scheme or vaccination schedule. The "effective amount"
may vary depending on the health and physical condition of the
individual to be treated, the age of the individual to be treated
(e.g. dosing for infants may be lower than for adults) the
taxonomic group of the individual to be treated (e.g. human,
non-human primate, primate, etc.), the capacity of the individual's
immune system to mount an effective immune response, the degree of
protection desired, the formulation of the vaccine, the treating
doctor's assessment, the strain of the infecting pathogen and other
relevant factors. It is expected that the effective antigen amount
will fall in a relatively broad range that can be determined
through routine trials. Usually, the antigen amount will vary from
0.01 to 1000 .mu.g/dose, more particularly from 0.1 to 100
.mu.g/dose. Dosage treatment may be a single dose schedule or a
multiple dose schedule. The vaccine may be administered in
conjunction with other immunoregulatory agents.
[0098] A vaccine composition may comprise more than one antigen,
i.e., a plurality of antigens, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more, e.g., up to 15, 20, 25, 30, 40 or 50 or more distinct
antigens. In particular, the HCV vaccine composition comprises as
antigen(s) the HCV E1 envelope protein(s) or part(s) thereof
according to the invention, or comprises VLPs comprising said HCV
E1 envelope proteins. The vaccine can be "monotypic" wherein the
HCV E1 antigens all are derived from the same HCV genotype, HCV
subtype or HCV isolate. The vaccine can also be "polytypic" by
comprising HCV E1 antigens derived from different (at least 2) HCV
genotypes, HCV subtypes or HCV isolates.
Carriers, Adjuvants and Vehicles
[0099] A "pharmaceutically acceptable carrier" or "pharmaceutically
acceptable adjuvant" is any suitable excipient, diluent, carrier
and/or adjuvant which, by themselves, do not induce the production
of antibodies harmful to the individual receiving the composition
nor do they elicit protection. Preferably, a pharmaceutically
acceptable carrier or adjuvant enhances the immune response
elicited by an antigen. Suitable carriers or adjuvantia typically
comprise one or more of the compounds included in the following
non-exhaustive list: [0100] large slowly metabolized macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers and inactive
virus particles; [0101] aluminium hydroxide, aluminium phosphate
(see International Patent Application Publication No. WO93/24148),
alum (KAl(SO.sub.4).sub.2.12H.sub.2O), or one of these in
combination with 3-0-deacylated monophosphoryl lipid A (see
International Patent Application Publication No. WO93/19780);
[0102] N-acetyl-muramyl-L-threonyl-D-isoglutamine (see U.S. Pat.
No. 4,606,918), N-acetyl-normuramyl-L-alanyl-D-isoglutamine,
N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1',2'-dipalmitoyl-sn--
glycero-3-hydroxyphosphoryloxy)ethylamine; [0103] RIBI (ImmunoChem
Research Inc., Hamilton, Mont., USA) which contains monophosphoryl
lipid A (i.e., a detoxified endotoxin), trehalose-6,6-dimycolate,
and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion. Any of the three components MPL, TDM or CWS may also be
used alone or combined 2 by 2; [0104] adjuvants such as Stimulon
(Cambridge Bioscience, Worcester, Mass., USA), SAF-1 (Syntex);
[0105] adjuvants such as combinations between QS21 and
3-de-O-acetylated monophosphoryl lipid A (see International Patent
Application Publication No. WO94/00153) which may be further
supplemented with an oil-in-water emulsion (see, e.g.,
International Patent Application Publication Nos. WO95/17210,
WO97/01640 and WO9856414) in which the oil-in-water emulsion
comprises a metabolisable oil and a saponin, or a metabolisable
oil, a saponin, and a sterol, or which may be further supplemented
with a cytokine (see International Patent Application Publication
No. WO98/57659); [0106] adjuvants such as MF-59 (Chiron), or
poly[di(carboxylatophenoxy)phosphazene] based adjuvants (Virus
Research Institute); [0107] blockcopolymer based adjuvants such as
Optivax (Vaxcel, Cytrx) or inulin-based adjuvants, such as
Algammulin and GammaInulin (Anutech); [0108] Complete or Incomplete
Freund's Adjuvant (CFA or IFA, respectively) or Gerbu preparations
(Gerbu Biotechnik). It is to be understood that Complete Freund's
Adjuvant (CFA) may be used for non-human applications and research
purposes as well; [0109] a saponin such as QuilA, a purified
saponin such as QS21, QS7 or QS17, .beta.-escin or digitonin;
[0110] immunostimulatory oligonucleotides comprising unmethylated
CpG dinucleotides such as [purine-purine-CG-pyrimidine-pyrimidine]
oligonucleotides. These immunostimulatory oligonucleotides include
CpG class A, B, and C molecules (Coley Pharmaceuticals), ISS
(Dynavax), Immunomers (Hybridon). Immunostimulatory
oligonucleotides may also be combined with cationic peptides as
described, e.g., by Riedl et al. (2002); [0111] Immune Stimulating
Complexes comprising saponins, for example Quil A (ISCOMS); [0112]
excipients and diluents, which are inherently non-toxic and
non-therapeutic, such as water, saline, glycerol, ethanol, wetting
or emulsifying agents, pH buffering substances, preservatives, and
the like; [0113] a biodegradable and/or biocompatible oil such as
squalane, squalene, eicosane, tetratetracontane, glycerol, peanut
oil, vegetable oil, in a concentration of, e.g., 1 to 10% or 2.5 to
5%; [0114] vitamins such as vitamin C (ascorbic acid or its salts
or esters), vitamin E (tocopherol), or vitamin A; [0115]
carotenoids, or natural or synthetic flavanoids; [0116] trace
elements, such as selenium; [0117] any Toll-like receptor ligand as
reviewed in Barton and Medzhitov (2002).
[0118] Any of the afore-mentioned adjuvants comprising
3-de-O-acetylated monophosphoryl lipid A, said 3-de-O-acetylated
monophosphoryl lipid A may be forming a small particle (see
International Patent Application Publication No. WO94/21292).
[0119] In any of the aforementioned adjuvants MPL or
3-de-O-acetylated monophosphoryl lipid A can be replaced by a
synthetic analogue referred to as RC-529 or by any other
amino-alkyl glucosaminide 4-phosphate (Johnson et al. 1999, Persing
et al. 2002). Alternatively it can be replaced by other lipid A
analogues such as OM-197 (Byl et al. 2003).
[0120] A "pharmaceutically acceptable vehicle" includes vehicles
such as water, saline, physiological salt solutions, glycerol,
ethanol, etc. Auxiliary substances such as wetting or emulsifying
agents, pH buffering substances, preservatives may be included in
such vehicles.
[0121] Typically, a vaccine or vaccine composition is prepared as
an injectable, either as a liquid solution or suspension. Injection
may be subcutaneous, intramuscular, intravenous, intraperitoneal,
intrathecal, intradermal, intraepidermal. Other types of
administration comprise implantation, suppositories, oral
ingestion, enteric application, inhalation, aerosolization or nasal
spray or drops. Solid forms, suitable for dissolving in, or
suspension in, liquid vehicles prior to injection may also be
prepared. The preparation may also be emulsified or encapsulated in
liposomes for enhancing adjuvant effect.
Antibodies
[0122] The invention further embodies antibodies selectively
binding to an HCV E1 envelope protein or a part thereof comprising
at least one of the following disulfides: [0123] a disulfide
between the pair of cysteines at amino acid positions 207 and 226,
[0124] a disulfide between the pair of cysteines at amino acid
positions 229 and 238, or [0125] a disulfide between the pair of
cysteines at amino acid positions 272 and 281, wherein said amino
acid positions are relative to the HCV polyprotein which is
starting with the methionine of the Core protein at amino acid
position 1 of said HCV polyprotein; or selectively binding to a
viral-like particle comprising any of said HCV E1 envelope proteins
or parts thereof, antibodies capable of competing with said
antibodies for selectively binding said HCV E1 envelope protein, or
a fragment of any of said antibodies.
[0126] Said antibodies can be isolated from a mammal immunized with
any of the above HCV E1 envelope proteins or parts thereof or with
the above viral-like particle. Fragments of said antibodies can
than be prepared. In particular said antibody or fragment thereof
is a monoclonal antibody or a fragment thereof. A specific antibody
is the antibody secreted by the hybridoma cell line of DSM deposit
with accession number ACC 2470. Again, fragments of this specific
antibody can than be prepared. Determining if two antibodies are
competing with each other for binding an epitope can easily be
performed. Such experiments usually involve labeling of one of the
two of said antibodies. Competition between antibodies can for
instance be determined by comparing the amount of labeled antibody
bound to the epitope of interest in the presence and absence,
respectively, of the non-labeled antibody.
[0127] The present invention thus relates to an antibody to the HCV
E1 envelope protein or part thereof according to the invention,
and/or to a viral-like particle comprising said HCV E1 envelope
protein. In particular, said antibody is raised upon immunization
of a mammal with at least one protein as defined herein, or with a
VLP comprising said protein. In a specific embodiment, said
antibody is specifically reactive with a protein of the present
invention, or with a viral-like particle comprising said protein.
In particular the binding of said antibody to an HCV E1 envelope
protein is dependent on the presence of at least one of the
intramolecular disulfides present in the HCV E1 envelope protein as
determined in the current invention. The selective binding of such
antibodies to an HCV E1 envelope protein with at least one
intramolecular disulfide as determined in the current invention is
thus higher than the binding to an HCV E1 envelope protein without
at least one of the intramolecular disulfides as determined in the
current invention. Such antibodies are not hypothetical as the
antibody IGH388 as disclosed in Example 7 hereafter binds with much
higher affinity to an HCV E1 envelope protein with the
intramolecular disulfides as determined in the current invention
than to an HCV E1 envelope protein without disulfides. In a further
specific embodiment, any of above-said antibodies is a monoclonal
antibody or a humanized (monoclonal) antibody or a single-chain
antibody. Fragments of any of above-said antibodies, e.g., Fab, are
also included in the term "antibody". In particular, said fragments
retain the binding specificity of the complete antibody. The
immunization process normally requires administration of said
protein or part thereof, or of said viral-like particle comprising
said protein, to said mammal.
[0128] The 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,
immunized with an HCV protein according to the invention, or with a
viral-like particles comprising said protein, on the one hand, and
of cells of a myeloma cell line on the other hand. Hybridomas are
subsequently selected which produce the monoclonal antibodies
recognizing the protein or viral-like particle comprising said
protein which has been initially used for the immunization of the
animals. The invention also relates to the hybridoma cell line of
DSM deposit with accession number ACC 2470 as outlined in Examples
7.
[0129] The antibodies involved in the invention can be labeled by
an appropriate label of the enzymatic, colorimetric,
chemiluminescent, fluorescent, or radioactive type.
[0130] Non-human mammalian antibodies or animal antibodies can be
humanized (see for instance Winter and Harris 1993). The antibodies
or monoclonal antibodies according to the invention may be
humanized versions of for instance rodent antibodies or rodent
monoclonal antibodies. Humanisation of antibodies entails
recombinant DNA technology, and is departing from parts of rodent
and/or human genomic DNA sequences coding for H and L chains or
from cDNA clones coding for H and L chains.
[0131] Alternatively, the monoclonal antibodies according to 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
immunized with a protein of the invention or with a VLP comprising
said protein. 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) or by screening Epstein
Barr-virus-transformed lymphocytes of immunized individuals for the
presence of reactive B-cells by means of the antigens of the
present invention.
[0132] The invention also relates to the use of the proteins or
parts thereof or the use of VLPs comprising said proteins for the
selection of recombinant antibodies by the process of repertoire
cloning (Persson et al., 1991).
[0133] The invention further relates to the use of an antibody
according to the invention for the manufacture of an immunogenic
composition or a vaccine composition. In particular the immunogenic
composition is an HCV immunogenic composition and the vaccine
composition is an HCV vaccine composition, a therapeutic HCV
vaccine composition or a prophylactic HCV vaccine composition. Any
of these compositions can be used for immunizing a mammal against
HCV infection or for treating a mammal infected with HCV.
Immunoassays and Diagnostic Kits
[0134] Methods for determining the presence of HCV antibodies in a
sample are part of another embodiment of the invention, said
methods comprising the steps of: [0135] (i) contacting said sample
with any of the above HCV E1 envelope proteins or parts thereof or
with the above viral-like particle under conditions allowing the
formation of an immunological antigen-antibody complex; [0136] (ii)
determining the immunological complex formed in (i); [0137] (iii)
inferring from (ii) the presence of HCV antibodies in said
sample.
[0138] Methods for determining HCV E1 antigens in a sample are part
of another embodiment of the invention, said methods comprising the
steps of: [0139] (i) contacting said sample with any of the above
antibodies under conditions allowing the formation of an
immunological antigen-antibody complex; [0140] (ii) determining the
immunological complex formed in (i); [0141] (iii) inferring from
(ii) the presence of HCV E1 antigens in said sample.
[0142] In particular, in step (i) of said methods any of the above
HCV E1 envelope proteins or parts thereof, the above viral-like
particle or any of the above antibodies is added as competitor; and
[0143] said HCV E1 envelope protein or part thereof, viral-like
particle or antibody used in said step (i), or [0144] said HCV E1
envelope protein or part thereof, viral-like particle or antibody
added as competitor are labeled.
[0145] The invention additionally relates to diagnostic kits for
determining the presence of HCV antibodies or HCV E1 envelope
proteins in a sample, said kits comprising at least one of the
above HCV E1 envelope proteins or parts, the above viral-like
particle and/or any of the above antibodies.
[0146] The HCV E1 envelope proteins or parts thereof according to
the present invention, or VLPs comprising said proteins, may be
employed in virtually any immunoassay format that employs a known
antigen to detect antibodies or a known antibody to detect
antigens. A common feature of all of these assays is that the
antigen is contacted with the body component containing or
suspected of containing HCV antibodies or HCV antigens under
conditions that permit binding between an antigen and an antibody,
i.e. under conditions allowing the formation of an immunological
complex. Such conditions will typically be physiologic temperature,
pH and ionic strength using an excess of antigen (in the case of
antibody detection) or antibody (in the case of antigen detection).
The incubation of the antigen or antibody with the specimen is
followed by detection of immune complexes.
[0147] The design of 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 and/or labeled
polypeptide, e.g. a labeled peptide or polypeptide according to the
present invention; 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 ELISA and RIA assays. Other immunoassay designs comprise line
immunoassays, sandwich immunoassays, antigen down immunoassays. An
immunoassay may be set up in a competitive format.
[0148] An 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 protein is
typically bound to a solid matrix, solid support or solid phase to
facilitate separation of the sample from the protein after
incubation. Examples of solid supports, matrices or phases are
listed above. The solid support containing the antigenic proteins
is typically washed after separating it from the test sample, and
prior to detection of bound antibodies. Both standard and
competitive formats are known in the art.
[0149] In a homogeneous format, the test sample is incubated with
the combination of antigens in solution. For example, it may be
under conditions that will precipitate any antigen-antibody
complexes that are formed. Both standard and competitive formats
for these assays are known in the art.
[0150] In a standard format, the amount of antibodies, such as
anti-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 said antibodies, such as said anti-HCV antibodies, will
bind due to complex formation. In a competitive format, the amount
of said antibodies, such as said anti-HCV antibodies, in a sample
is deduced by monitoring the competitive effect on the binding of a
known amount of (labeled) antibody (or other competing ligand) or
antigen in the complex.
[0151] Antigen-antibody complexes can be detected by any of a
number of known techniques, depending on the format. For example,
unlabeled antibodies such as anti-HCV antibodies in the complex may
be detected using a conjugate of anti-xenogeneic Ig complexed with
a label (e.g. an enzyme label).
[0152] In an immunoprecipitation or agglutination assay format the
reaction between an antigen and an antibody forms a protein cluster
that precipitates from the solution or suspension and forms a
visible layer or film of precipitate. If no antibody is present in
the test specimen or sample, no such precipitate is formed.
[0153] A diagnostic kit usually comprises a molecule for detecting
the presence of a sample reactant capable of interacting with said
molecule, of a sample reactant modifying said molecule (e.g., in a
chemical reaction), and/or of a sample reactant modifiable by said
molecule (e.g., in a chemical reaction). In a diagnostic kit for
detection of an antigen or antibody in a sample, one or more
antibodies or antigens, respectively, are part of said kit. In a
diagnostic kit for detecting antigens or antibodies, antibodies or
antigens, respectively, are often present on a solid phase, matrix
or support.
[0154] The proteins or parts thereof according to the present
invention, or the VLPs comprising said proteins, can be packaged
and be part of a diagnostic kit. The kit will normally contain in
separate containers or vials the peptides or polypeptides according
to the present invention (labelled or unlabelled), control antibody
formulations (positive and/or negative), labelled 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 proteins according to the present invention may be already
bound to a solid matrix or may be present in the kit in a separate
vial together 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. 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. 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.
[0155] Diagnostic kits for detecting antibodies to an HCV virus or
for typing of an HCV virus wherein said kits comprise at least one
protein according to the invention, or VLP comprising said protein,
are part of the invention. In said diagnostic kit said protein or
said VLP can be bound to a solid support.
[0156] Solid phases, solid matrices or solid supports on which the
proteins or VLPs of the present invention, may be bound (or
captured, absorbed, adsorbed, linked, coated, immobilized;
covalently or non-covalently) comprise beads or the wells or cups
of microtiter plates, or may be in other forms, such as solid or
hollow rods or pipettes, particles, e.g., from 0.1 .mu.m to 5 mm in
diameter (e.g. "latex" particles, protein particles, or any other
synthetic or natural particulate material), microspheres or beads
(e.g. protein A beads, magnetic beads). A solid phase may be of a
plastic or polymeric material such as nitrocellulose, polyvinyl
chloride, polystyrene, polyamide, polyvinylidine fluoride or other
synthetic polymers. Other solid phases include membranes, sheets,
strips, films and coatings of any porous, fibrous or bibulous
material such as nylon, polyvinyl chloride or another synthetic
polymer, a natural polymer (or a derivative thereof) such as
cellulose (or a derivative thereof such as cellulose acetate or
nitrocellulose). Fibers or slides of glass, fused silica or quartz
are other examples of solid supports. Paper, e.g., diazotized paper
may also be applied as solid phase. Clearly, proteins of the
present invention may be bound, captured, absorbed, adsorbed,
linked or coated to any solid phase suitable for use in
immunoassays. Said proteins can be present on a solid phase in
defined zones such as spots or lines.
[0157] Any of the solid phases described above can be developed,
e.g. automatically developed in an assay device.
[0158] With "developed" or "development" is meant that a sample or
samples, suspected of comprising a binding partner to a molecule
present on a solid phase, is or are applied to said solid phase and
that the necessary steps are performed in order to detect binding
of the binding partner to a molecule on a solid phase. This can,
e.g., be the detection of binding of an antibody suspected to be
present in a biological sample to or with an antigen, in casu a
protein or peptide of the present invention, present on a solid
phase. Automatic development hence refers to a development process,
or any one or more steps thereof, in an automated or robotized
fashion. A development automate or robot (or, generally, an assay
device) generally is connected to or comprises one, more or all of
the development or assay reagents and may in addition comprise a
means to "read" the developed assay. Said "reading" will logically
depend on the assay and may, e.g., confer to determining color
intensities, to determining optical density or absorption at a
given wavelength, to determining fluoresence, fosforescence or
(chemi)luminescence, to determining turbidity, to determining the
decay of a radio-active element or to determining other physical or
physico-chemical characteristics that are related to the binding of
a binding partner in a sample to a molecule present on a solid
phase.
[0159] Another aspect of the invention relates to methods for
screening compounds capable of modulating the binding between an
HCV E1 envelope protein and an E1 ligand, said methods comprising:
[0160] (i) contacting said E1 ligand with any of the above HCV E1
envelope proteins or parts thereof with the above viral-like
particle under conditions allowing the formation of an HCV E1-E1
ligand complex; [0161] (ii) adding a compound suspected of
modulating the binding between an HCV E1 envelope protein and an E1
ligand to the HCV E1-E1 ligand complex formed in (i); [0162] (iii)
determining the change in amount of HCV E1-E1 ligand complex formed
in (i) and (ii); [0163] (iv) inferring from (iii) whether the
compound added in (ii) is a modulator of binding between an HCV E1
envelope protein and an E1 ligand.
[0164] Further methods for screening compounds capable of
modulating the binding between an HCV E1 envelope protein and an E1
ligand, comprise the steps of: [0165] (i) contacting, under
conditions allowing the formation of an HCV E1-E1 ligand complex,
said E1 ligand with any of the above HCV E1 envelope proteins or
parts thereof or with the above viral-like particle in the presence
and absence, respectively, of a compound suspected of modulating
the binding between an HCV E1 envelope protein and an E1 ligand;
[0166] (ii) determining the amount of HCV E1-E1 ligand complex
formed in (i) in the presence and absence of said compound; [0167]
(iii) inferring from (ii) whether said compound is a modulator of
binding between an HCV E1 envelope protein and an E1 ligand.
[0168] In particular, in step (i) of said methods any of above HCV
E1 envelope proteins or parts thereof or the above viral-like
particle is added as competitor; and [0169] said HCV E1 envelope
protein or part thereof or viral-like particle used in said step
(i), or [0170] said HCV E1 envelope protein or part thereof or
viral-like particle added as competitor are labeled.
E1 Ligands
[0171] Ligands of the HCV E1 envelope protein (i.e., E1 ligands)
include peptides, antibodies or other molecules binding with E1,
such as E1 ligands inhibiting the viral fusion or receptor domains,
which are expected to be located on the HCV E1 envelope proteins.
E1 ligands/(putative) HCV receptors known to date include L-SIGN
and DC-SIGNR (Pohlmann et al. 2003), and apolipoprotein B, annexin
V and tubulin (WO 99/24054) and heparin or derivatives thereof. A
particular E1 ligand is an antibody of the current invention, such
as an antibody specifically binding part of an E1 structure
comprising an intramolecular disulfide of the invention, e.g. the
monoclonal antibody IGH388 described herein.
Compounds
[0172] A compound capable of modulating the binding between an HCV
E1 envelope protein and an E1 ligand can be a compound of any kind
or chemical nature. Said compound can be of proteinaceous or
non-proteinaceous nature. In particular said compound is targeting
part of an E1 structure comprising an intramolecular disulfide
bridge according to the invention. "Modulating" in this respect
includes modification of the binding between an HCV E1 envelope
protein and an E1 ligand in either way. Modulating thus includes
increasing or decreasing the strength of the binding between an HCV
E1 envelope protein and an E1 ligand.
[0173] Another aspect of the invention envisages methods of
producing a recombinant HCV E1 envelope protein or part thereof
according to the invention, said methods comprising the steps of
[0174] (i) expressing of an HCV E1 envelope protein or part thereof
in a eukaryotic host; [0175] (ii) isolating from the pool of HCV E1
envelope protein or part thereof expressed in (i) the
non-aggregated monomeric fraction of HCV E1 envelope protein or
part thereof according to the invention.
[0176] Methods of producing a synthetic HCV E1 envelope protein or
part thereof according to the invention are part of a further
aspect wherein said methods comprise: [0177] (i) chemical synthesis
of an HCV E1 envelope protein or part thereof; [0178] (ii)
introducing during or after the chemical synthesis of an HCV E1
envelope protein or part thereof at least one disulfide as
determined in the current invention.
[0179] Yet another aspect of the invention relates to the use of
any of the above HCV E1 envelope proteins or parts thereof, the
above viral-like particle and/or any of the above antibodies for
the preparation of a medicament or a vaccine or for the preparation
of an immunoassay or a diagnostic kit.
[0180] The use of any of the above HCV E1 envelope proteins or
parts thereof or the above viral-like particle as a carrier of
another protein or of a non-proteinaceous molecule form a further
aspect of the invention.
Fusion Proteins and the HCV E1 Envelope Protein as Carrier
Protein
[0181] The HCV E1 envelope protein or part thereof according to the
present invention can further be flanked by at least one amino acid
or amino acid sequence that is a non-HCV amino acid or amino acid
sequence, or that is a HCV amino acid or amino acid sequence that
is not naturally contiguous to said HCV E1 envelope protein (or
part or variant thereof). Said flanking amino acid or amino acid
sequence is contiguous with the N- and/or C-terminus of the HCV E1
envelope protein (or part or variant thereof). Any fusion protein
comprising an said HCV E1 envelope protein or part thereof is
included in the present invention.
[0182] In particular the non HCV E1-part of any of such fusion
proteins can be another HCV antigen (non-contiguous core,
non-contiguous E1, E2, p7, NS2, NS3, NS4, NS5, or a part of any
thereof; or a combination of any thereof) or an antigen of another
pathogen such as, but not limited to, HBV (e.g., HBsAg or part
thereof), HIV (e.g., p53 or part thereof), HTLV, influenza virus,
pathogenic Clostridia species, pathogenic Salmonella species,
pathogenic Neisseria species etc. Obviously the non HCV E1-part of
any of such fusion proteins can be any other protein involved in
provoking any disease symptom. In general the non HCV E1-part(s) of
any of such fusion proteins comprises or consists of any epitope
(B-cell epitope, T helper cell epitope, cytotoxic T cell epitope)
of any given protein, or a combination of such epitopes (e.g., a
polyepitope).
[0183] In the use of an HCV E1 envelope protein or part thereof, or
a viral-like particle of any thereof, according to the present
invention as a carrier of other proteins or of non-proteinaceous
molecules said other proteins or non-proteinaceous molecules are
linked to, or coupled to, or carried by said E1 or E1-part or
E1-particle in a covalent or non-covalent fashion. In particular,
any amino acid of said HCV E1 envelope protein or part thereof can
be mutated to a lysine in order to facilitate directed (covalent)
coupling of any other protein or of a non-proteinaceous molecule
wherein said coupling is via the .epsilon.-NH.sub.2 group of the
lysine. Even more particular, an arginine residue in the HCV E1
envelope protein or part thereof is mutated to a lysine. The
carried other protein can be another HCV antigen (E1, E2, p7, NS2,
NS3, NS4, NS5, or a part of any thereof; or a combination of any
thereof) or an antigen of another pathogen such as, but not limited
to, HBV (e.g., HBsAg or part thereof), HIV (e.g., p53 or part
thereof), HTLV, influenza virus, pathogenic Clostridia species,
pathogenic Salmonella species, pathogenic Neisseria species etc. In
general the carried protein comprises or consists of any epitope
(B-cell epitope, T helper cell epitope, cytotoxic T cell epitope)
of any given protein, or a combination of such epitopes (e.g., a
polyepitope). Obviously the carried other protein can be any other
protein involved in provoking any disease symptom. Carried
non-proteinaceous molecules include any molecule with prophylactic
or therapeutic action.
[0184] Thus the invention covers isolated proteins comprising an
HCV E1 envelope protein or part thereof according to the invention.
In particular said isolated protein is further comprising at least
one of: [0185] an N-terminal flanking amino acid or amino acid
sequence of an HCV protein or part thereof not naturally contiguous
with said HCV E1 envelope protein or part thereof; [0186] a
C-terminal flanking amino acid or amino acid sequence of an HCV
protein or part thereof not naturally contiguous with said HCV E1
envelope protein or part thereof; [0187] an N-terminal flanking
non-HCV amino acid or amino acid sequence; [0188] a C-terminal
flanking non-HCV amino acid or amino acid sequence.
[0189] Alternatively, said isolated protein is comprising the HCV
E1 envelope protein or part thereof as carrier protein.
[0190] The invention further relates to methods for inducing
immunity or an immune response in healthy or HCV-infected mammals
wherein said methods comprise administering the HCV E1 envelope
protein or part thereof according to the invention, or the
viral-like particle comprising it, to said mammals. The invention
further relates to methods for passively immunizing healthy or
HCV-infected mammals wherein said methods comprise administering an
antibody to an HCV E1 envelope protein or part thereof according to
the invention to said mammals. In particular said mammal is a
human.
EXAMPLES
Example 1
Purification of Monomeric HCV E1 Envelope Protein Population
[0191] The HCV E1s protein (amino acids 192-326:
YEVRNVSGMYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRENNSSRCWVALTPTLA
ARNASVPTTTIRRHVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPRRHETVQDC
NCSIYPGHITGHRMAWDMMMNR; SEQ ID NO:1) was expressed in Hansenula
polymorpha cells as described in, e.g., Example 16 of International
Patent Publication WO 02/086101. The HCV E1s protein was purified
without disruption of intra- and intermolecular disulfide bridges
as outlined below.
[0192] Since the HCV E1s (aa 192-326) was expressed as a C-terminal
(His).sub.6-tagged protein [(CL)-E1s-(His).sub.6], a first capture
and purification step of the Gu.HClu-solubilized product could be
performed on Ni-IDA after cell disruption and clarification.
[0193] In brief, cell pellets were resuspended in 6 M Gu.HCl, 10 mM
iodoacetamide, 100 mM HEPES, pH 8.0 [2 mL buffer/g cells (wet
weight)]. Iodoacetamide was added to block present free thiol
groups. After homogenisation of the cell suspension, cells were
disrupted by high-pressure homogenisation (3 passages at 1.8 kbar
and 10.degree. C. on a high-pressure homogenizer, Constant
Systems). The lysate was clarified by centrifugation
(13.000.times.g for 1 hour at 4.degree. C.). The obtained
supernatant was diluted 4 times with 6 M Gu.HCl, 50 mM phosphate,
pH 7.2 and n-dodecyl-N,N-dimethylglycine (also known as
lauryldimethylbetaine or Empigen BB.RTM., Albright & Wilson)
and imidazole were added to a final concentration of 1% (w/v) and
20 mM respectively.
[0194] All further chromatographic steps were executed on an Akta
FPLC (or Explorer) workstation (Pharmacia). The sample was
filtrated through a 0.22 .mu.m pore size membrane (cellulose
acetate) and loaded on a Ni.sup.2+-IDA column (Chelating Sepharose
FF, Pharmacia), that was equilibrated with 50 mM phosphate, 6 M
Gu.HCl, 1% Empigen BB, pH 7.2 (IMAC-A buffer) supplemented with 20
mM imidazole. The column was washed sequentially with IMAC-A buffer
containing 20 mM and 50 mM imidazole respectively till the
absorbance at 280 nm reached the baseline level. A further washing
and elution step of the his-tagged products was performed by the
sequential application of IMAC-B buffer (PBS, 1% Empigen BB, pH
7.2) supplemented with 50 mM imidazole and 200 mM imidazole,
respectively. The fractions were analysed by SDS-PAGE (and silver
staining) and western-blot using a specific monoclonal antibody
directed against E1s (IGH201). The elution fractions containing
mainly E1s were pooled (`IMAC pool`) and concentrated 20 times by
ultrafiltration (MWCO 10 kDa, centriplus, Amicon, Millipore).
[0195] In order to separate the monomeric E1s from the oligomeric
fraction, the concentrated sample was loaded on a Superdex.RTM. 200
HR 10/30 column (Pharmacia) equilibrated with PBS, 3% (w/v)
n-dodecyl-N,N-dimethylglycine. Fractions were screened for the
presence of monomeric E1s by SDS-PAGE under reducing and
non-reducing conditions (and silver staining) and western-blot
analysis using a specific monoclonal antibody directed against E1s
(IGH201). This analysis showed that fractions containing monomeric
E1s (Mr between .about.10 kDa and .about.35 kDa based on the
migration on non-reducing SDS-PAGE) with a purity of more than 90%
could be separated. These fractions were pooled (`SEC pool`) for
further virus like particle (VLP) formation. Western blotting and
peak integration of the IMAC- and SEC-runs indicated that the
monomeric E1s fraction constitutes less than 5% of the total
intracellular E1s protein population. Removal of the E1s in
intermolecularly disulfide-linked material was thus necessary for
obtaining a monomeric E1s population allowing the accurate analysis
and localization of intramolecular disulfide bridges. After PNAGase
F-treatment of the monomeric E1s fraction, all proteins migrate
between Mr .about.10 kDa and .about.25 kDa in a non-reducing
SDS-PAGE gel.
[0196] VLP formation of the purified monomeric E1s was enforced by
exchanging n-dodecyl-N,N-dimethylglycine for 3% betain. For
(His).sub.6-tagged E1s, this buffer switch was realized on a
Ni.sup.2+-IDA column. Therefore, the `SEC pool` was 3 times diluted
in PBS buffer to a final concentration of PBS, 1% (w/v)
n-dodecyl-N,N-dimethylglycine, pH 7.5 and was applied to a
Ni.sup.2+-IDA column (Chelating Sepharose FF, Pharmacia), that was
equilibrated with PBS, 1% (w/v) n-dodecyl-N,N-dimethylglycine, pH
7.5. Further VLP formation was accomplished by application of 7
column volumes washing buffer (PBS, 3% (w/v) betaine, pH 7.5).
Elution of the obtained VLPs was accomplished by addition of 500 mM
imidazole to this buffer. After pooling of the elution fractions,
Dynamic Light Scattering Analysis (using a particle-size analyser
Model Zetasizer 1000 HS, Malvern Instruments Ltd, controlled by
photon correlation spectroscopy (PCS) software) on the final
product showed the presence of E1s-particles with an average size
of 29 nm. Furthermore, SDS-PAGE (and silver staining) and
western-blot analysis using a specific monoclonal antibody directed
against E1s (IGH201) indicated that the final VLP product--obtained
by an initial purification of monomeric E1s without disruption of
disulfide bridges--is obtained with more than 90% purity (see FIG.
2A for silver-stained gel, and FIG. 2B for western blot).
[0197] The hybridoma cell line producing the monoclonal antibody
directed against E1 (IGH201) was deposited on Mar. 12, 1998 under
the conditions of the Budapest Treaty at the European Collection of
Cell Cultures, Centre for Applied Microbiology & Research,
Salisbury, Wiltshire SP4 0JG, UK, and has the accession number
ECACC 98031216.
Example 2
Localization of Disulfide Bridges in Monomeric E1s Population Using
Tryptic Digestion
[0198] In order to find disulfide bridges in the monomeric E1s
fraction purified as in Example 1, this product was compared to a
fully reduced and sulphonated E1s-(His)6 prepared as described in,
e.g., Example 15 of International Patent Publication WO 02/086101,
further referred to as sulphonated E1s. This sulphonated E1s sample
was used as control sample and treated in the same way as the
monomeric E1s material from Example 1.
[0199] In order to generate a tryptic digest the following steps
were applied to both samples: [0200] Alkylation with iodoacetamide,
to block cysteines that are free: the material was incubated in 20
mM iodoacetamide during 20 minutes, in the dark at room
temperature. [0201] Deglycosylation with PNGase F: PNGase F was
added to reach a concentration of 0.2 U/.mu.g E1s. The PNGase F
digest was incubated overnight at 37.degree. C., in the dark
(because of the presence of iodoacetamide). [0202] Purification of
deglycosylated E1s on RPC: purification was performed using a Vydac
C4, 2.1 mm.times.250 mm column. The following gradient was used 0
min-20% B, 5 min-20% B, 65 min-80% B, 66 min-20% B, 86 min-20% B
(solvent A: 0.1% TFA in water, solvent B: 0.1% trifluoroacetic acid
(TFA) in acetonitrile, flow: 150 .mu.L/min). [0203] Trypsine
digestion in solution was performed at a 1/20 enzyme/substrate
ration (buffer 50 mM NH.sub.4HCO.sub.3 pH 7.8, 1 M urea, 10%
acetonitrile; incubation: overnight at 37.degree. C.). [0204]
Purification of 5 .mu.g of tryptic peptides was performed by ZipTip
C18 (elution in 3 .mu.l 80% acetonitrile/0.1% TFA), followed by
mass spectrometry (MALDI-TOF-MS) in linear and reflector mode
(dried droplet method).
[0205] A theoretical exercise based on the E1s protein sequence
learns that the peptides as presented in Table 1 can theoretically
be generated by a tryptic digest.
[0206] Analysis of the MALDI-TOF-MS spectrum of the tryptic digest,
allowed to assign a number of peaks to theoretical peptides as
presented in Table 1. An overview of the assigned MALDI-TOF-MS
peaks is given in Table 2. Peptides recovered only from the
monomeric E1s protein but not from the control sulphonated E1s are
highlighted.
[0207] From Table 2 the following conclusions are formulated:
[0208] Surprisingly only trace amounts of the peptides expected on
the basis of Garry and Dash (2003) were recovered; [0209] One
disulfide bridge was present in the T2 peptide; [0210] A disulfide
bridge was linking T2 to T4; [0211] Some masses could be assigned
to T2/T4, containing two S--S-bridges; [0212] One disulfide bridge
was present in the T7 peptide, and the cysteines of T7 were
apparently not involved in bridging T7 with other peptides. The
cysteine bridge within T7 was formed with high efficiency as
MS-spectra do not support the presence of alkylated T7 forms;
[0213] The two cysteines, which are in the T9-peptide, were only
measured in the alkylated form, so the T9 peptide does not contain
a disulfide bridge and the cysteines are apparently also not
involved in bridging T9 with other peptides; [0214] Some peaks of
the sulphonated E1s were assigned to peptides having a disulfide
bridge within T2 or linking T2/T4, these peptides could have been
present in the starting material or may have formed during the
treatment of the samples as sulphonation can be reversed; [0215]
General remarks: [0216] (i) the material was overalkylated,
probably because PNGase F digestion has been performed in the
presence of iodoacetamide. This explains the modification of
methionine to dehydroamino-2-butyric acid (Dhb) and to alkylated
methionine (+42.99 Da) (Lapko et al. 2000). The overalkylation of
the His.sub.6 tail is probably the reason why T10 was not measured.
[0217] (ii) The indications "W.fwdarw.D" in Tables 1 and 2 (as well
as in Table 3) refer to the phenomenon in which a tryptophan
(186.21 Da) degrades to a mass of 115.1 Da, i.e. putatively
asparagine. This phenomenon has also been observed in other
proteins analysed by mass spectrometry after liquid chromatography,
e.g., for tryptophan at position 61 in the human transthyretin.
TABLE-US-00001 [0217] TABLE 1 Theoretical tryptic digest of
E1s-(His).sub.6. Where appropriate, peptides expected based on Gary
and Dash (2003) have also been indicated. The numbering of the
amino acids presented for E1 starts at 1 and can be compared with
the HCV poly- protein in which the first amino acid of E1 is 192,
by adding 191. A schematic representation of the cysteines in the
E1 and E1s protein is depicted in FIG. 1. Fragment Amino acid SEQ
ID # start-end Sequence NO Reduced S-S-bridges (free cysteines --SH
HS--) T1 1-4 YEVR 2 T2 5-40 DVSGMYHVTNDCSDSSIVYEAADMIMHTPGCVPCVR 3
T3 41-46 ENDSSR 4 T4 47-58 CWVALTPTLAAR 5 T5 59-68 DASVPTTTIR 6 T6
69-69 R T7 70-105 HVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPR 7 T8 106-106
R T9 107-126 HETVQDCDCSIYPGHITGHR 8 T10 127-141 MAWDMMMNWHHHHHH 9
Theoretical S-S-bridges potentially present in follow- Expected by
Garry and ing peptides or peptide combinations Dash 2003 T2 5-40
DVSGMYHVTNDCSDSSIVYEAADMIMHTPGCVPCVR 3 T7 70-105
HVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPR 7 T9 107-126
HETVQDCDCSIYPGHITGHR 8 T2/T4 5-40
DVSGMYHVTNDCSDSSIVYEAADMIMHTPGCVPCVR 3 47-58 CWVALTPTLAAR 5 T2/T7
5-40 DVSGMYHVTNDCSDSSIVYEAADMIMHTPGCVPCVR 3 linked by one bridge
70-105 HVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPR 7 T2/T9 5-40
DVSGMYHVTNDCSDSSIVYEAADMIMHTPGCVPCVR 3 linked by two bridges
107-126 HETVQDCDCSIYPGHITGHR 8 T4/T7 47-58 CWVALTPTLAAR 5 linked by
one bridge 70-105 HVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPR 7 T4/T9
47-58 CWVALTPTLAAR 5 107-126 HETVQDCDCSIYPGHITGHR 8 Combinations of
three or more peptides may also be possible
TABLE-US-00002 TABLE 2 Assigned MALDI-TOF-MS peaks for tryptic
digests of monomeric and sulphonated E1s. The numbering of the
amino acids presented for E1 starts at 1 and can be compared with
the HCV polyprotein in which the first amino acid of E1 is 192, by
adding 191. Sulphonated cysteines are generally measured as free
cysteines (desulphonation during MALDI-TOF-MS). Cys-CAM =
acetamide-derivative of cysteine; IAM on M = acetamide-derivative
of methionin; W .fwdarw. D = artefactual degradation of tryptophan
to aspartate (see General remark (ii) in Example 2). ##STR00001##
##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006##
Example 3
Further Support for Localization of Disulfide Bridges in the
Monomeric E1s by Tryptic Digestion, Using Additional Reduction and
Alkylation
[0218] Five microgram of the tryptic digest sample of the monomeric
E1s from Example 2 was reduced with DTT followed by alkylation with
iodoacetamide. The material was purified by ZipTip C18, eluted in 3
.mu.L of 80% acetonitrile/0.1% TFA and analyzed by
MALDI-TOF-MS.
[0219] The MALDI-TOF-MS measurement of the tryptic digest of the
monomeric sample, before and after reduction/alkylation, was
compared and is summarized in Table 3.
[0220] From Table 3 the following conclusions are formulated:
[0221] The peaks coming from T7 with an S--S-bridge (3738.96,
3979.31 and 3844.17 Da), disappeared after reduction and alkylation
with iodoacetamide. One peak at mass 3846.80 Da can be explained as
T7 with a reduced S--S-bridge (1 C=cys-CAM). This provides further
evidence for the existence of a disulfide bridge in T7; [0222] The
masses between 3854.91 Da and 4115.29 Da all increased with 2 Da
after reduction but it seems that alkylation of the thiols did not
take place. This is indicative for an S--S-bridge in the
T2-peptide, which was reduced after reduction and alkylation;
[0223] The masses around 5100.29 Da disappeared after reduction and
alkylation, which provided evidence for the S--S-bridge between T2
and T4.
[0224] From the combined results of Examples 2 and 3 it is
concluded that: [0225] the cysteines in T9 were not involved in a
disulfide bridge; [0226] the cysteine in T7 were involved in a
intra-peptide disulfide bridge, thus allowing the localization of a
first disulfide bridge between amino acids 272 and 281; [0227] a
second disulfide bridge was present between T2 and T4 thus linking
amino acid 238 with either amino acid 207 or 226 or 229; [0228] a
third disulfide bridge was present within the T2 peptide and thus
links either amino acid 207 with 226 or 207 with 229 or 226 with
229.
TABLE-US-00003 [0228] TABLE 3 Assigned MALDI-TOF-MS peaks for
tryptic digests of monomeric E1s with or without additional
DTT/iodoacetamide treatment. The numbering of the amino acids
presented for E1 starts at 1 and can be compared with the HCV
polyprotein in which the first amino acid of E1 is 192, by adding
191. Measured mass (M + H).sup.+ Corresponding Theoretical for
monomeric E1s Table Amino acid mass +DTT 1 number start-end
Interpretation (average) +Iodoacetamide T7 70-105 S-S-bridge/1 M =
Dhb 3738.34 3739.04 -- T7 70-105 S-S-bridge/1 M = Dhb/+57.02 Da
3795.36 3797.26 -- T7 70-105 S-S-bridge/+57.02 Da 3843.47 3844.94
-- T7 70-105 S-S bridge reduced/1 C = cys-CAM 3845.48 -- 3846.80 T2
5-40 S-S-bridge/free C/1 M = Dhb 3854.35 3854.78 -- T2 5-40
S-S-bridge reduced/free C/1 M = Dhb 3856.36 -- 3858.76 T2 5-40
S-S-bridge/free C 3902.46 3902.54 -- T2 5-40 S-S bridge
reduced/free C 3904.47 -- 3905.33 T2 5-40 S-S-bridge/1 C =
cys-CAM/1 M = Dhb 3911.37 3911.21 -- T2 5-40 S-S-bridge/1 C =
cys-CAM 3959.48 3959.44 -- T2 5-40 S-S-bridge reduced/1 C = cys-CAM
3961.49 -- 3962.45 T2 5-40 S-S-bridge/1 C = cys-CAM/1 M =
Dhb/+57.02 Da/ 4011.38 4012.00 -- +42.99 Da (IAM on M) T2 5-40
S-S-bridge/1 C = cys-CAM/+57.02 Da/+42.99 Da 4059.49 4059.10 --
(IAM on M) T2 5-40 S-S-bridge reduced/2 C = cys-CAM/1 free cys/
4061.50 -- 4062.15 +42.99 Da (IAM on M) T2 5-40 S-S-bridge
reduced/3 C = cys-CAM 4075.53 -- 4076.30 T2 5-40 S-S-bridge/1 C =
cys-CAM/+2 * 57.02 Da/+42.99 4116.51 4115.95 -- Da (IAM on M) T2
5-40 S-S-bridge reduced/3 C = cys-CAM/+42.99 Da 4118.52 -- 4118.57
(IAM on M) T2/T4 5-40/47-58 2 S-S-bridges/W .fwdarw. D/2 M = Dhb/1
* O 5050.65 5052.95 -- T2/T4 5-40/47-58 2 S-S-bridges/W .fwdarw.
D/1 M = Dhb/1 * O 5098.75 5100.28 -- T2/T4 5-40/47-58 2
S-S-bridges/W .fwdarw. D/1 M = Dhb/1 * O/+42.99 Da 5141.74 5142.71
-- (IAM on M) T2/T4 5-40/47-58 2 S-S-bridges/W .fwdarw. D/1 M =
Dhb/1 * O/+57.02 Da 5155.77 5157.76 -- T2/T4 5-40/47-58 2
S-S-bridges/W .fwdarw. D/1 M = Dhb/1 * O/+42.99 Da 5255.78 5256.84
-- (IAM on M)/+2 * 57.02 Da Cys-CAM = acetamide-derivative of
cysteins; IAM on M = acetamide-derivative of methionine; W .fwdarw.
D = artefactual degradation of tryptophan to aspartate (see General
remark (ii) in Example 2).
Example 4
Unraveling the Disulfide Bridges within T2 and Linking T2 with
T4
[0229] In order to unravel the localization of the disulfide
bridges within T2 end T2/T4, a number of peptides were analyzed. A
first experiment was designed to find out whether the two first
cysteines in the sequence of T2, i.e. cysteines 207 and 226 are
forming a disulfide bridge. As additional controls, a peptide was
analyzed containing the cysteines of T9 which apparently are unable
to bridge and a peptide containing the cysteines of T7 which based
on the evidence of examples 2 and 3 should be forming an
intrapeptide bridge.
[0230] The peptides are: [0231] IGP 1634:
Ac-SQLFTISPRRHETVQDCNCS-NH.sub.2 (SEQ ID NO:10); contains the
cysteines of T9 and is further referred to as T9'; [0232] IGP 2133:
Ac-AFCSAMYVGDLCGS-NH.sub.2 (SEQ ID NO:11); contains the cysteines
of T7 and is further referred to as T7'; and [0233] IGP 2134:
Ac-NDCSNSSIVYEAADMIMHTPGCVP-NH.sub.2 (SEQ ID NO:12); contains the
first 2 cysteines of T2 and is further referred to as T2' wherein
Ac is acetyl.
[0234] These peptides were dissolved at 450 .mu.M in 0.1% TFA
except T7'. Due to its lower solubility T7' was dissolved at 300
.mu.M in dichloromethane (DCM). Peptide stock solutions or mixtures
thereof as shown below were dispensed in 4 mL glass vials (Wheaton)
and dried using speed-vacuum drying. To study the interaction of
each individual peptide, a complete set of different peptide
combinations was prepared: [0235] homogeneous peptide solutions:
T2', T7', and T9' [0236] heterogeneous di-peptide mixtures: T2' and
T7', T2' and T9', and T7' and T9' [0237] heterogeneous tri-peptide
mixtures: T2' and T7' and T9'
[0238] MALDI-TOF-MS analysis confirmed that the individual peptides
were reduced, with free thiols, at the start of the experiment (see
Table 4). Samples were obtained from the 10-fold acidic stock
solutions prior to speed-vacuum drying.
TABLE-US-00004 TABLE 4 Low range MALDI-TOF-MS measurements
(500-3000 Da) of 10-fold stock solutions. Reduced peptide
Theoretical Measured mass mass M + H.sup.+ Peptide stock
(monoisotopic) (monoisotopic) Interpretation T7' (IGP 2133) 1463.6
1486.5 (Na adduct) free Cys T2' (IGP 2134) 2594.1 2595.0 free Cys
T9' (IGP 1634) 2361.1 2362.4 free Cys
[0239] Oxidation of the peptide or peptide mixtures was initialised
by adding 1 mL of 0.1 M NH.sub.4HCO.sub.3 pH 8.0 and stirring in
close contact with air. Estimated net peptide concentration of this
final oxidation mixture was 45 .mu.M or 65.8, 116.7 and 108.6
.mu.g/mL for T7', T2' and T9' respectively. The oxidation was
stopped after 31 hours by adding 1% TFA and 10% acetonitrile
(pH.about.=2) and vials were stored at -20.degree. C.
Simultaneously, a 1 .mu.L sample was spotted for MALDI-TOF-MS
analysis and combined with 1 .mu.L matrix mixture (dried droplet).
After drying, the spot was re-dissolved in situ using 1 .mu.L 1%
TFA/70% acetonitrile. By "in situ" acidification, peptide
precipitation was avoided. The details of the MALDI matrix and
instrument setting can be found in Table 5, and the results in
Tables 6 (low range: 500-3000 Da) and 7 (mid range: 3000-10000
Da).
TABLE-US-00005 TABLE 5 MALDI matrix and instrument settings used as
a function of the targeted mass range Mass range Structure (Da)
Matrix Settings monomer 500-3000 20 mg/ml .alpha.-cyano Reflector
mode peptide in 70% acetonitrile/ (Monoisotopic 0.1% TFA mass)
di-and trimer 3000-10000 10 mg/ml sinapinic acid/ Linear mode
peptide 10 mg/ml fucose (Average mass) in 50% acetonitrile/ 0.1%
TFA
TABLE-US-00006 TABLE 6 Low range MALDI-TOF-MS measurements
(500-3000 Da) after 31 h oxidation. Oxidised peptides Theoretical
Measured mass Peptide mass M + H.sup.+(1.sup.o solution
(monoisotopic) isotope) Interpretation T7' 1461.6 1484.6 (Na
intra-molecular S--S bridged adduct) T2' 2592.1 2593.4
intra-molecular S--S bridged T9' 2359.1 2360.1 intra-molecular S--S
bridged T7'/T2' 1461.6/2592.1 1484.5 (Na.sup.+)/ intra-molecular
S--S bridged 2593.0 T2'/T9' 2592.1/2359.1 2593.0/2359.9
intra-molecular S--S bridged T7'/T9' 1461.6/2359.1 1484.4
(Na.sup.+)/ intra-molecular S--S bridged 2359.8 T7'/T2'/
1461.6/2592.1/ 1484.4 (Na.sup.+)/ intra-molecular S--S bridged T9'
2359.1 2592.6/2359.6
[0240] To illustrate the clear signals of T9'T9' dimers versus the
signals that corresponds with traces of intermolecular disulfide
bridging involving T7' and T2' (T7'T9', T7'T2', T2'T9', T2'T2',
T7'T7') the mass spectrum of the T7'T2'T9' solution after 31 h of
oxidation is shown in FIG. 3. The T9' peptide preparation contained
a peptide synthesis side product consisting of the T9' sequence
with one out of the two threonine residues lacking (T9' Thr del).
This side product was also recovered in part in dimers, explaining
the additional peak in FIG. 3.
[0241] Data obtained for the mid range showed that T9' multimers
(mainly dimers) are formed independent from the presence of one or
both of the other peptides. From this it is obvious that, even
though inter-molecular interaction in this set-up is possible, as
shown by the T9' dimerisation, intra-molecular disulfide bounds are
favourable for T7' and T2'. This selectivity for intra-molecular
interaction of T7' and T2', possibly induced through energetically
favourable folding of the individual peptide, is indicative for the
presence of these disulfide bounds in the E1s protein localized in
T2' and T7' and not in T9'. None or only minor traces (FIG. 3) of
intermolecular disulfide bridging involving T7' and T2' (T7'T9',
T7'T2', T2'T9') were detected.
[0242] From these oxidation experiments it is concluded that:
[0243] The lack of a disulfide bridge in T9 between amino acids 304
and 306 as evident from Example 2 was further supported by the
finding that intramolecular disulfide bridging which is almost
exclusively found for T2' and T7', is for T9' accompanied with a
significant amount of intermolecularly disulfide bridged peptides.
Consequently the driving force to form an intramolecular bridge
between T2' and T7' seems to be higher than for T9'. In the context
of E1s this leads to the absence of detectable amounts of disulfide
bridges between amino acids 304 and 306; [0244] The disulfide
bridge within T2 was localized between aa 207 and 226 as in T2';
[0245] Consequently the disulfide bridge between T2 and T4 should
be localized between 229 and aa 238;
[0246] No evidence was found for other disulfide bridge
interactions.
TABLE-US-00007 TABLE 7 Mid range MALDI-TOF-MS measurements
(3000-10000 Da) after 31 h oxidation. Oxidised peptides Peptide
Theoretical mass dimer Measured mass solution peptide (average) M +
H.sup.+ Interpretation T7' 2925.4 (T7' T7' dimer) -- T2' 5187.6
(T2' T2' dimer) -- T9' 4721.2 (T9' T9' dimer) 4723.54 2 .times.
S--S inter-molecular linked T9' T9' dimer 4620.1 (T9' Thr deletion
4622.41 2 .times. S--S inter-molecular linked dimer) T9' T9' (Thr
del) dimer 7083.9 (T9' T9' T9' 7083.7 3 .times. S--S
inter-molecular linked trimer) T9' T9' T9' trimer 9446.5 9446.81 4
.times. S--S inter-molecular linked (T9' T9' T9' T9' T9' T9' T9'
T9' tetramer tetramer) T7'/T2' 2925.4 (T7' T7' dimer) -- 5187.6
(T2' T2' dimer) 5187.2 2 .times. S--S inter-molecular linked T2'
T2' dimer (trace) 4056.6 (T7' T2' dimer) -- T2'/T9' 5187.6 (T2' T2'
dimer) -- 4721.2 (T9' T9' dimer) 4723.7 2 .times. S--S
inter-molecular linked T9' T9' dimer 4954.5 (T2' T9' dimer) --
T7'/T9' 2925.4 (T7' T7' dimer) -- 4721.2 (T9' T9' dimer) 4722.8 2
.times. S--S inter-molecular linked T9' T9' dimer 3823.3 (T7' T9'
dimer) -- -- T7'/T2'/ 2925.4 (T7' T7' dimer) -- T9' 5187.6 (T2' T2'
dimer) -- 2 .times. S--S inter-molecular linked T2' T2' dimer
(trace) 4721.2 (T9' T9' dimer) 4721.9 2 .times. S--S
inter-molecular linked T9' T9' dimer 6652.6 (T7' T2' T9' --
trimer)
Example 5
Confirming the Localization of the Disulfide Bridge Between T2 and
T4
[0247] As the results of Example 4 strongly hint for a disulfide
bridge between the aa 229 and 238 another peptide was analyzed.
This peptide, IGP 1629 (Ac-PCVRENNSSRCWVALTPTLA-NH.sub.2; SEQ ID
NO:13; Ac=acetyl) represents part of the sequence of the monomeric
E1 as produced in example 1 and contains the two cysteines
potentially linking T2 and T4. The MALDI-TOF-MS spectrum of the
acidic stock solution (10% acetic acid, 10% DMSO and 20%
acetonitrile/0.1% TFA) of this peptide was analyzed. Surprisingly
this stock solution contained mainly a peptide with a disulfide
bridge. The oxidizing ability of sulfoxides, e.g., DMSO is often
used as an oxidant aid for peptide cyclisation by intramolecular
disulfide bridge formation. Sulfoxides are mostly used in
combination with buffers at neutral or slightly higher pH; in close
contact with air; at peptide concentrations.ltoreq.0.25 mg/mL;
overnight incubation and often with addition of stronger
oxidants.
[0248] Based on this experience, it is concluded that the peptide
IGP 1629 has the intrinsic ability to form intramolecular disulfide
bridges. The peptide contains probably one or two cysteine residues
with a low pKa value, making it possible to create preferentially
the cyclic peptide (intramolecular disulfide bridge) at this low pH
and even at high peptide concentrations of the stock solution
(.about.=1 mg/mL). In itself, this high peptide concentration,
without significant dimer- or oligomerisation, is a clear
indication that the sequences (and peptide conformation) are
favorable to the formation of the cyclic monomer structures, and
confirmed the presence of a disulfide bridge already in the acidic
stock solution. This finding further adds to the data generated in
the previous Examples linking peptide T2 with T4 with a disulfide
bridge between amino acids 229 and 238. The intramolecular
disulfide bridges in the E1s (and thus E1 as E1s comprises all
eight cysteines) protein as determined in this and previous
Examples are schematically summarized in FIG. 1.
Example 6
Mutation of Cysteine residues 304 and 306 of E1
[0249] The cysteine residues at relative amino acid positions 304
and 306 of E1s are mutated by point mutagenesis to serine or
alanine and this E1s protein is expressed as C-terminal
(His).sub.6-tagged protein [E1s-C304>S--C306>S-(His).sub.6,
or E1s-C304>A-C306>A-(His).sub.6] in Hansenula polymorpha as
described in Example 1. Cell lysates of these cultures are compared
to cell lysates of E1s-(His).sub.6-expressing cultures by
western-blot using a monoclonal antibody directed against E1s
(IGH201). The increased content of monomeric E1 in the cultures
containing E1 with mutation allows to purify large quantities of E1
comprising specific disulfide bridges.
Example 7
Antibodies with Higher Affinity for Monomeric than Sulphonated
E1
[0250] The monoclonal antibodies directed against E1 and used in
this example were generated in two different experiments. [0251] 1.
The monoclonal antibody IGH 201 is derived from a Balb/c mouse
immunized with irreversibly blocked E1 as described in Examples 1
and 2 of WO99/50301. The hybridoma cell line secreting this
antibody has been deposited as described in Example 1 herein.
[0252] 2. The monoclonal antibody 1C4 and its subclone, IGH 388,
have been derived from an HCV infected individual testing positive
for E1 antibodies. The antibody was generated based on the method
as described in Example 6 of WO99/60846, with some minor
modifications. [0253] Immune-deficient
NOD/ltSz-Prkdc.sup.scid/Prkdc.sup.scid (NOD/SCID) mice were bred
under sterile conditions and fed ad libitum with autoclaved food
and water without addition of prophylactic antibiotics and used
between 8 and 12 weeks of age. Mice were pretreated by sublethal
total body irradiation (3 Gy), administered using a linear
accelerator, and by intraperitoneal injection of 1 mg purified
TMbetal in 500 .mu.l phosphate buffered saline (PBS). TMbetal is a
rat monoclonal antibody (Ab) directed against the murine IL2
receptor beta chain used for in vivo depletion of mouse natural
killer cell activity. [0254] Heparinised venous blood was drawn
from a patient with chronic hepatitis C virus (HCV) infection. The
patient was serologically negative for hepatitis B virus or human
immunodeficiency virus infection. The patient was infected with HCV
genotype 1b as determined by INNO-LiPA HCV II (INNOGENETICS, Ghent
Belgium) and its serum showed positive reactivity in the INNO-TEST
HCV Ab III assay (Abs present directed towards Core, NS3, NS4 and
NS5) and an HCV E1 ELISA test. HCV RNA was detectable in the serum
using the Amplicor assay (Roche Diagnostics). Values of serum
alanine transaminase (ALT) were elevated for 6 month at least two
times above the normal values. The patient has not been treated yet
with Interferon, Ribavirin or other anti-viral agents. [0255] Human
peripheral blood lymphocytes (Hu-PBL) were isolated from the
heparinised venous blood by Ficoll-Hypaque (Nycomed, Oslo, Norway)
centrifugation. For intrasplenic engraftment in NOD/SCID mice,
animals were anesthetized and a subcostal incision of the skin was
made followed by incisions of the abdominal wall and the
peritoneum. The spleen was carefully exposed and injected with 50
.mu.l of cell suspension in PBS containing 2.times.10.sup.7 Hu-PBL.
After injection, the spleen was repositioned in the abdominal
cavity, and the abdominal wall and skin were sutured separately.
[0256] Recombinant hepatitis C envelope protein E1, produced via
Vaccinia-infected mammalian cell culture system as described in
Examples 1-3 and 5 of WO96/04385 and adjuvanted with Complete
Freunds Adjuvant (CFA), was injected subcutaneously in the hind leg
of NOD/SCID mice a few hours after Hu-PBL transfer. [0257] Seven
days after intrasplenic engraftment, a Hu-PBL-NOD/SCID spleen cell
suspension was prepared by gently squeezing the tissue with angled
forceps followed by filtration on a sterile gauze to remove larger
fragments. Spleen cell suspension consisted for more than 75% of
human B lymphocytes. For cell fusion, Hu-PBL-NOD/SCID spleen cells
and K6H6/B5 heteromyeloma cells, washed in PBS, were mixed at 5:1
ratio. Polyethylene glycol 1500 (50%; Boehringer Mannheim,
Mannheim, Germany) was added for 2 minutes and washed away. Fused
cells (10.sup.5 per microculture well) were cultured in 200 .mu.l
of RPMI 1640 culture medium supplemented with sodium pyruvate (1
mM), L-glutamine (2 mM), 2-ME (5.times.10-5 M), penicillin (100
U/ml), streptomycin (100 .mu.g/ml), non-essential amino acids,
hypoxantine-aminopterin-thymidine (all from Life technologies,
Paisley, UK), 10% Fetal Clone I serum (Hyclone, Logan, Utah), human
recombinant insulin (10 .mu.g/ml; Boehringher Mannheim), ouabain (1
.mu.M, Sigma, St. Louis, Mo.) and 10% BM condimed H1 (Boehringher
Mannheim). [0258] The in vitro anti-HCV envelope 1 Ab production by
the hybridoma cells was evaluated 10 to 14 days after initiation of
culture using an HCV E1 ELISA. One mothercolony 1C4 showed strong
reactivity and was subcloned until a monoclonal hybridoma cell line
was obtained that showed high and stable production of anti-E1 Abs:
1C4/3F3/1A3/6B12. The hybridoma cell line, also referred to as IGH
388, has been deposited in accordance with the Budapest Treaty on
Sep. 13, 2000 at the DSMZ (Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig,
Germany) under accession number DSM ACC2470. [0259] The monoclonal
Ab produced by the hybridoma IGH 388, is of the IgG1 isotype and
contains a kappa light chain. The VH and VL chain were sequenced
and the nucleic acid and amino acid sequences of the variable
domains is shown in FIG. 4.
[0260] Both these monoclonal antibodies were tested in a dilution
series for their reactivity with the sulphonated or monomeric E1 in
ELISA. Both the monomeric and sulphonated E1 were presented as a
VLP, prepared as described in Example 1 and in WO 02/085932. The
results are shown in FIG. 5. Remarkably, a large difference in
reactivity was noted with the IGH 388 monoclonal if tested on
monomeric versus sulphonated E1 while no such difference was noted
for the antibody IGH 201. This clearly indicates that IGH 388 which
has been generated as a consequence of natural infection and thus
been induced by natural E1, does preferentially recognize an E1 in
which disulfide bridges over an E1 without disulfide bridges.
Alternatively the antibody IGH 201 which has been generated in mice
using an E1 in which at least part of the cysteines were
irreversibly blocked does recognize both types of E1 with very
similar affinity.
Example 8
Epitope Mapping of IGH 388
[0261] The epitope of IGH201 was already known from WO 99/50301
(Example 4). This antibody reacts with the peptides V1V2 (IGP 888,
NH.sub.2--YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGC-GGK(biotin)-CONH.sub.2;
SEQ ID NO:14) and V2V3 (IGP 1036
acetyl-IVYEAADMIMHTPGCVPCVRENNSSRCWV-GK(biotin)GG; SEQ ID NO:15) of
E1 which have the amino acid region 212-226 in common. This region
(IYEAADMIMHTPGC; SEQ ID NO:16) contains only one cysteine, which is
located at the C-terminal end and is thus not expected to be
crucial for the binding of the antibody.
[0262] The antibody IGH 388 was similarly tested on a series of E1
peptides and was found to react both with V2V3 (IGP 1036) and V3V4'
(IGP 1087,
acetyl-PCVRENNSSRCWVALTPTLAARNASVPTTTIRRHVD-K(biotin)-CONH.sub.2;
SEQ ID NO:17) of E1 which have the amino acid region 228-240
(PCVRENNSSRCWV; SEQ ID NO:18) in common. This region contains two
cysteines which were found to form a disulfide bridge as described
in the examples 2-4.
[0263] Peptide equivalents of this common region were generated
from a various number of HCV genotypes and tested in ELISA for
reactivity with IGH 388. Good recognition was seen for at least 70%
of the HCV genotypes tested, confirming that the epitope recognized
by IGH 388 is located in the region 228-240.
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Sequence CWU 1
1
221135PRThepatitis C virus 1Tyr Glu Val Arg Asn Val Ser Gly Met Tyr
His Val Thr Asn Asp Cys1 5 10 15Ser Asn Ser Ser Ile Val Tyr Glu Ala
Ala Asp Met Ile Met His Thr 20 25 30Pro Gly Cys Val Pro Cys Val Arg
Glu Asn Asn Ser Ser Arg Cys Trp 35 40 45Val Ala Leu Thr Pro Thr Leu
Ala Ala Arg Asn Ala Ser Val Pro Thr 50 55 60Thr Thr Ile Arg Arg His
Val Asp Leu Leu Val Gly Ala Ala Ala Phe65 70 75 80Cys Ser Ala Met
Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val 85 90 95Ser Gln Leu
Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp 100 105 110Cys
Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala 115 120
125Trp Asp Met Met Met Asn Trp 130 13524PRThepatitis C virus 2Tyr
Glu Val Arg1336PRThepatitis C virus 3Asp Val Ser Gly Met Tyr His
Val Thr Asn Asp Cys Ser Asp Ser Ser1 5 10 15Ile Val Tyr Glu Ala Ala
Asp Met Ile Met His Thr Pro Gly Cys Val 20 25 30Pro Cys Val Arg
3546PRThepatitis C virus 4Glu Asn Asp Ser Ser Arg1 5512PRThepatitis
C virus 5Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg1 5
10610PRThepatitis C virus 6Asp Ala Ser Val Pro Thr Thr Thr Ile Arg1
5 10736PRThepatitis C virus 7His Val Asp Leu Leu Val Gly Ala Ala
Ala Phe Cys Ser Ala Met Tyr1 5 10 15Val Gly Asp Leu Cys Gly Ser Val
Phe Leu Val Ser Gln Leu Phe Thr 20 25 30Ile Ser Pro Arg
35820PRThepatitis C virus 8His Glu Thr Val Gln Asp Cys Asp Cys Ser
Ile Tyr Pro Gly His Ile1 5 10 15Thr Gly His Arg 20915PRThepatitis C
virus 9Met Ala Trp Asp Met Met Met Asn Trp His His His His His His1
5 10 151020PRThepatitis C virusMISC_FEATURE(1)..(1)Xaa 1 is an
N-terminally acetylated serine 10Xaa Gln Leu Phe Thr Ile Ser Pro
Arg Arg His Glu Thr Val Gln Asp1 5 10 15Cys Asn Cys Xaa
201114PRThepatitis C virusMISC_FEATURE(1)..(1)Xaa is an
N-terminally acetylated alanine 11Xaa Phe Cys Ser Ala Met Tyr Val
Gly Asp Leu Cys Gly Xaa1 5 101224PRThepatitis C
virusMISC_FEATURE(1)..(1)Xaa is an N-terminally acetylated
asparagine 12Xaa Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala
Asp Met Ile1 5 10 15Met His Thr Pro Gly Cys Val Xaa
201320PRThepatitis C virusMISC_FEATURE(1)..(1)Xaa is an
N-terminally acetylated proline 13Xaa Cys Val Arg Glu Asn Asn Ser
Ser Arg Cys Trp Val Ala Leu Thr1 5 10 15Pro Thr Leu Xaa
201438PRThepatitis C virusMISC_FEATURE(38)..(38)Xaa is a
biotinylated and C-terminally amidated lysine 14Tyr Glu Val Arg Asn
Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys1 5 10 15Ser Asn Ser Ser
Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr 20 25 30Pro Gly Cys
Gly Gly Xaa 351533PRThepatitis C virusMISC_FEATURE(1)..(1)Xaa is an
N-terminally acetylated isoleucine 15Xaa Val Tyr Glu Ala Ala Asp
Met Ile Met His Thr Pro Gly Cys Val1 5 10 15Pro Cys Val Arg Glu Asn
Asn Ser Ser Arg Cys Trp Val Gly Xaa Gly 20 25 30Gly1614PRThepatitis
C virus 16Ile Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys1
5 101737PRThepatitis C virusMISC_FEATURE(1)..(1)Xaa is an
N-terminally acetylated proline 17Xaa Cys Val Arg Glu Asn Asn Ser
Ser Arg Cys Trp Val Ala Leu Thr1 5 10 15Pro Thr Leu Ala Ala Arg Asn
Ala Ser Val Pro Thr Thr Thr Ile Arg 20 25 30Arg His Val Asp Xaa
351813PRThepatitis C virus 18Pro Cys Val Arg Glu Asn Asn Ser Ser
Arg Cys Trp Val1 5 1019127PRTHomo sapiens 19Glu Glu Gln Leu Val Glu
Ser Gly Gly Gly Pro Val Lys Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30Ala Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45Ser Ser Ile
Ser Ser Ser Gly Ser Tyr Val Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Val Phe65 70 75
80Leu Gln Leu Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Arg Cys
85 90 95Thr Arg Asp Val Asn Tyr Tyr Asp Thr Ser Glu Asp Tyr Tyr Gly
Glu 100 105 110Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val
Ser Ser 115 120 12520108PRTHomo sapiens 20Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Tyr Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Val Ser Arg Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala
Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Thr Tyr Ser Asn
85 90 95Thr Phe Ala Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10521381DNAHomo sapiens 21gaggagcagt tggtagagtc tgggggaggc
ccggtcaagc ctggaaggtc cctgagactc 60tcctgtgcag cctctggatt caccctcagt
agttatgcca tcaattgggt ccgccaggct 120ccagggcagg ggctggaatg
ggtctcatct atcagtagta gtgggagtta tgtgtcctac 180gcagactcgg
tgaagggccg cttcaccatc tccagagaca acgccaagaa cttagtgttt
240ctgcaattga acagcctgag agccggcgac acggctgttt atagatgtac
aagagatgta 300aattattatg atactagtga agattattac ggtgaggctt
ttgatatctg gggccaaggg 360acaatggtca ccgtctcttc a 38122324DNAHomo
sapiens 22gacatccaga tgacccagtc tccttccacc ctgtctgcat atgtaggaga
cagagtcacc 60atcacttgcc gggccagtca gagtgttagt cgctggttgg cctggtatca
gcaaagacca 120gggaaagccc ccaaactcct gatctataag gcgtctaatt
tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta
ttgccaacaa tataaaactt attctaacac ttttgcccag 300gggaccaagc
tggagatcaa gcga 324
* * * * *