U.S. patent application number 11/445274 was filed with the patent office on 2006-12-07 for particles of hcv envelope proteins: use for vaccination.
This patent application is currently assigned to INNOGENETICS N.V.. Invention is credited to Alfons Bosman, Erik Depla, Geert Maertens, Frans Van Wijnendaele.
Application Number | 20060275323 11/445274 |
Document ID | / |
Family ID | 26152268 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275323 |
Kind Code |
A1 |
Depla; Erik ; et
al. |
December 7, 2006 |
Particles of HCV envelope proteins: use for vaccination
Abstract
The present invention is based on the finding that the envelope
proteins of HCV induce a beneficial immune response in chronically
HCV-infected chimpanzees. The immunization can preferentially be
carried out using HCV envelope proteins in the form of particles
which are produced in a detergent-assisted manner. The envelope
proteins when presented as such to chronic HCV carriers are highly
immunogenic and stimulate both the cellular and humoral immune
response.
Inventors: |
Depla; Erik; (Destelbergen,
BE) ; Maertens; Geert; (Brugge, BE) ; Bosman;
Alfons; (Opwijk, BE) ; Van Wijnendaele; Frans;
(Laarne, BE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
INNOGENETICS N.V.
Ghent
BE
|
Family ID: |
26152268 |
Appl. No.: |
11/445274 |
Filed: |
June 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10414219 |
Apr 16, 2003 |
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11445274 |
Jun 2, 2006 |
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09355040 |
Jul 23, 1999 |
6635257 |
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PCT/EP99/04342 |
Jun 23, 1999 |
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10414219 |
Apr 16, 2003 |
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Current U.S.
Class: |
424/204.1 ;
424/130.1 |
Current CPC
Class: |
A61K 2039/5258 20130101;
A61K 39/00 20130101; A61K 2039/55566 20130101; C12N 2770/24234
20130101; A61K 39/12 20130101; A61K 2039/545 20130101; C07K 14/005
20130101; A61K 39/29 20130101; A61K 39/29 20130101; A61K 2300/00
20130101; A61P 31/12 20180101; Y10S 530/826 20130101; C12N
2770/24222 20130101; A61P 31/14 20180101 |
Class at
Publication: |
424/204.1 ;
424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/12 20060101 A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 1998 |
EP |
98870142.1 |
Feb 22, 1999 |
EP |
99870033.0 |
Claims
1. A purified HCV envelope protein or a part thereof wherein at
least one cysteine residue is alkylated.
2. The purified HCV envelope protein according to claim 1, wherein
the hydrogen on the sulphur atom of said cysteine is replaced by
(CH.sub.2).sub.nR, in which n is 0, 1, 2, 3 or 4 and R.dbd.H, COOH,
NH.sub.2, CONHO.sub.2, phenyl, or any derivative thereof.
3. The purified HCV envelope protein according to claim 1, wherein
said envelope protein is E1 or E1s.
4. A composition containing the purified HCV envelope protein
according to claim 1.
5. A specific antibody generated against the purified HCV envelope
protein or parts thereof according to claim 1.
6. A composition containing the specific antibody according to
claim 5.
7. Kit for detecting HCV antigens comprising the specific antibody
according to claim 5 in a suitable container.
8. Kit for detecting HCV antibodies present in a biological sample
comprising the purified HCV envelope protein or parts thereof
according to claim 1, in a suitable container.
9. Kit for detecting HCV related T-cell response, comprising the
purified HCV envelope protein or parts thereof according to claim
1.
10. A method for detecting antibodies to HCV in a biological
sample, said method comprising the steps of: (i) providing the
purified HCV envelope protein according to claim 1, (ii) incubating
said biological sample with said HCV envelope protein of (i) under
conditions allowing formation of a complex between said HCV
envelope protein and said antibodies to HCV, and (iii) determining
from (ii) whether said complex has formed and, therefrom, the
presence of antibodies to HCV in said biological sample.
11. A method for detecting HCV antigens in a biological sample,
said method comprising the steps of: (i) providing the specific
antibody according to claim 5, (ii) incubating said biological
sample with said specific antibody of (i) under conditions allowing
formation of a complex between said antibodies and said antigens to
HCV, and (iii) determining from (ii) whether said complex has
formed and, therefrom, the presence of HCV antigens in said
biological sample.
Description
[0001] The present application is a divisional of U.S. application
Ser. No. 10/414,219, filed Apr. 16,2003 (pending), which is a
divisional of U.S. application Ser. No.09/355,040, filed Jul. 23,
1999 (issued as U.S. Pat. No. 6,635,257), which is a 371 of U.S.
National Phase of PCT/EP99/04342, filed Jun. 23, 1999, which claims
benefit of EP 98870142.1, filed Jun. 24, 1998 and EP 99870033.0,
filed Feb. 22, 1999, the entire contents of each of which is hereby
incorporated by reference in this application.
FIELD OF THE INVENTION
[0002] The present invention is based on the finding that the
envelope proteins of HCV induce a beneficial immune response in
chimpanzees which are chronically infected with a heterologous
subtype 1a or subtype 1b HCV strain. More specifically, the present
invention relates to the finding that envelope proteins are highly
immunogenic and result in the stimulation of both the cellular and
humoral immune response. Moreover, the present invention relates to
the finding that blocking of cysteines by alkylation results in
even more immunogenic proteins. In addition, the envelope proteins
of the present invention can be incorporated in particles which
display a high immunogenecity and immunoreactivity. It was further
demonstrated that such particles may incorporate other
proteins.
BACKGROUND OF THE INVENTION
[0003] Hepatitis C virus (HCV) infection is a major health problem
in both developed and developing countries. It is estimated that
about 1 to 5% of the world population is affected by the virus. HCV
infection appears to be the most important cause of
transfusion-associated hepatitis and frequently progresses to
chronic liver damage. Moreover, there is evidence implicating HCV
in induction of hepatocellular carcinoma. Consequently, the demand
for reliable diagnostic methods and effective therapeutic agents is
high. Also sensitive and specific screening methods of
HCV-contaminated blood-products and improved methods to culture HCV
are needed.
[0004] HCV is a positive stranded RNA virus of approximately 9,600
bases which encode at least three structural and six non-structural
proteins. Based on sequence homology, the structural proteins have
been functionally assigned as one single core protein and two
envelope proteins: E1 and E2. The E1 protein consists of 192 amino
acids and contains 5 to 6 N-glycosylation sites, depending on the
HCV genotype. The E2 protein consists of 363 to 370 amino acids and
contains 9-11 N-glycosylation sites, depending on the HCV genotype
(for reviews see: Major and Feinstone, 1997; Maertens and Stuyver,
1997). The E1 protein contains various variable domains (Maertens
and Stuyver, 1997), while the E2 protein contains three
hypervariable domains, of which the major domain is located at the
N-terminus of the protein (Maertens and Stuyver, 1997). The latter
envelope proteins have been produced by recombinant techniques in
Escherichia coli, insect cells, yeast cells and mammalian cells.
The usage of an expression system in higher eukaryotes and
especially in mammalian cell culture leads to envelope proteins
that are effectively recognized by antibodies in patient samples
(Maertens et al., 1994).
[0005] It has been suggested that the E1 envelope protein needs the
E2 envelope protein to reach a proper folding status (Deleersnyder
et al., 1997). In addition, it has been suggested that E1 and E2
form heterodimers which may form the basic unit of the viral
envelope (Yi et al., 1997). In WO 98/21338 to Liang et al. these
presumptions have been used to construct HCV particles, which
consist of E1 and E2, as well as Core and P7. In other words, the
usage of E1 or E2 separately for immunization and other purposes is
not suggested in the prior art. But, Houghton (1997) reported that
repeated immunizations with recombinant gpE1E2 (4.times.25 .mu.g)
of 3 chronically HCV-infected chimpanzees did not induce a
significant immune response. The inventors of the present
application reasoned that the induction of an anti-envelope immune
response in patients with chronic hepatitis C would indeed be
desirable and beneficial to the patient, since higher levels of
such antibodies seem to correlate with good response to interferon
therapy, and may therefore help the patient to clear the virus
(PCT/EP 95/03031 to Maertens et al.). The inventors of the present
invention further reasoned that, as the antibody levels against E1
in chronic HCV carriers are among the lowest of all HCV antibodies,
it may be beneficial to raise those antibody levels, and possibly
the cellular response, to induce control or even clearance of the
infection by the host. Also higher levels of cellular immunity
against E1 seem to correlate with good response towards interferon
therapy (Leroux-Roels et al., 1996).
[0006] Besides the importance of anti-E1 immunity in relation to
interferon therapy, other indications point-out that some other
parts of the HCV genome may be important to induce a specific
immune response which may allow control of the infection. Also
T-cell reactivity against the C-terminal region of the core protein
has been observed more frequently in patients responding to
interferon therapy (Leroux-Roels et al, 1996). Potentially
neutralizing antibodies against the NS4B protein were demonstrated
in patients clearing HCV after liver transplant (Villa et al.,
1998). Also within NS3 several T-cell epitopes have been mapped
which seem to correlate with clearing of HCV during the acute phase
(see: PCT/EP 94/03555 to Leroux-Roels et al.; Leroux-Roels et al.,
1996; Rehermann et al., 1996 and 1997; Diepolder et al., 1995 and
1997). Furthermore, antibodies to NS5A, like E1 antibodies, show
higher levels at baseline before interferon-alpha therapy in long
term responders (LTR) as compared to non-responders.
[0007] At present, therapeutic vaccination for HCV has not been
successful. Also prophylactic vaccination has only been shown to be
effective against a homologous strain of HCV (Choo et al., 1994).
The present invention relates to the surprising finding that
administration of an HCV envelope antigen can dramatically improve
the state of chronic active hepatitis in an individual infected
with a heterologous strain or isolate, both in a heterologous
subtype 1a or heterologous subtype 1b infection. Indeed,
chronically infected chimpanzees who were administered six doses of
50 .mu.g E1s (i.e. aa 192-326 of E1) surprisingly showed vigourous
humoral and cellular immune responses, which had not been mounted
over the entire period of chronic infection before the latter
vaccination. Moreover, viral antigen became undetectable in the
liver over a period of two to five months and remained undetectable
for at least 5 months post-vaccination. Although HCV-RNA titers in
the serum did not decrease, liver enzyme levels in the serum showed
a clear tendency to normalize. Most importantly, liver histology
improved dramatically in both vaccinees. The present invention
further relates to the surprising finding that the E1 protein used
for vaccination, which was expressed as a single HCV protein
without its hydrophobic anchor, forms stable particles. It should
also be noted that, to avoid induction of an immune response
against non-relevant epitopes, the E1 protein used for vaccination
was constructed as a consensus sequence of individual clones
derived from a single serum sample from one chronic carrier. In
addition, the present application relates to the finding that the
induction of such anti-E1 responses may be increased by using
antigens of a different genotype than the ones of the infection
present in the host. Moreover, the present application relates to
the finding that when cysteines of HCV envelope proteins are
alkylated, for instance by means of
N-(iodoethyl)-trifluoroacetamide, ethylenimine or active halogens,
such as iodoacetamide, the oligomeric particles as described above
display an even higher immunogenicity. Finally, the present
invention relates also to the finding that mutation of cysteines of
HCV envelope proteins to any other naturally occuring amino acid,
preferentially to methionine, glutamic acid, glutamine or lysine,
in the oligomeric particles as described above also results in
higher immunogenicity, compared to the original envelope
proteins.
AIMS OF THE INVENTION
[0008] It is clear from the literature that there is an urgent need
to develop reliable vaccines and effective therapeutic agents for
HCV. Therefore, the present invention aims at providing an antigen
preparation, which is able to induce specific humoral and cellular
immunity to HCV envelope proteins, even (but not solely) in chronic
HCV carriers. The same antigens can be used for diagnosis of the
immune response.
[0009] More specifically, the present invention aims at providing
an antigen preparation as defined above, which consists of stable
particles of single envelope proteins of HCV. It should be clear
that, at present, such particles or a method to prepare such
particles, are not known in the art. Moreover, there is no
indication in the art that any antigen preparation, including such
stable particles or such purified single HCV envelope proteins,
could successfully be used as (heterologous) prophylactic or
therapeutic vaccine against HCV. The present invention thus also
aims at providing a method to produce stable particles, which can
be successfully used as a prophylactic or therapeutic agent against
HCV, in addition to provide DNA vaccines encoding HCV antigens.
More specifically, the present invention aims at providing a method
to produce the latter particles based on detergent-assisted
particle formation (see further). Furthermore, the present
invention aims at providing methods to prepare particles consisting
of antigens obtained from different HCV genotypes.
[0010] Moreover, the present invention aims at providing an antigen
which is a consensus sequence from individual clones, which may
allow a more correct folding of the proteins. This in order to
avoid stimulation of immunity against non-relevant epitopes.
[0011] Furthermore, the present invention aims at providing an
antigen formulation, in particular for therapeutic vaccination,
based on the genotype of HCV by which the chronic carrier is
infected. In this regard, the present invention aims at providing
an envelope protein of either a different or a homologous genotype
or subtype compared to the genotype or subtype of the chronic
carrier.
[0012] A further aim of the invention is to provide a method for
treating or therapeutically vaccinating chronically infected
patients using the above-indicated antigens or DNA vaccines,
possibly in combination with other compounds. The present invention
also aims to provide a method to prophylactically vaccinate humans
against HCV.
[0013] Another aim of the invention is to provide oligomeric
particles which have a superior immunogenicity, due to the mutation
of at least one cysteine residue of HCV envelope protein into a
natural occuring amino acid, preferentially methionine, glutamic
acid, glutamine or lysine. Alternatively, alkylation of at least
one cysteine residue of HCV envelope protein may be performed. In
particular, the latter protein can be alkylated by means of
ethylenimine, N-(iodoethyl)trifluoroacetamide or active halogens.
In this regard, the instant invention aims to provide the
additional use of oligomeric particles as vehicles for presenting
non-HCV epitopes efficiently.
[0014] It is a further aim of the present invention to provide a
method to treat patients, acutely or chronically infected, with an
anti-envelope antibody, such as anti-E1 antibody, e.g. anti-E1 V2
region antibody, either alone or in combination with other
treatments.
[0015] Another aim of the invention is to provide a T cell
stimulating antigen such as Core, E1, E2, P7, NS2, NS3, NS4A, NS4B,
NS5A, or NS5B along with the envelope proteins of the
invention.
[0016] All the aims of the present invention are considered to have
been met by the embodiments as set out below.
BRIEF DESCRIPTION OF TABLES AND DRAWINGS
[0017] Table 1 provides sequences of E1 clones obtained from a
single chronic carrier, the E1 construct used for production of a
vaccine is the consensus of all these individual clones. V1-V5,
variable regions 1-5; C4, constant domain 4; HR, hydrophobic
region; HCV-B con, consensus sequence at positions that are
variable between clones and HCV-J.
[0018] Table 2 provides sequences of the E1 vaccine protein and the
E1 protein as found in the infected chimpanzees Phil and Ton. The
subtype 1b isolate of Phil differed by 5.92% from the vaccine
strain. The difference between the vaccine and the subtype 1a
isolate of Ton was 20.74%.
[0019] Table 3 provides a schematic overview of the changes induced
by therapeutic vaccination in two chronically infected chimpanzees
(Ton and Phil). Analysis was performed as explained for FIGS. 8 and
11. In addition, histology and inflammation were scored from the
liver biopsies.
[0020] Table 4 provides sequences of peptides used to map the
B-cell epitopes. Note that HR overlaps with V4V5.
[0021] Table 5 shows the rearrangement of NS3 in order to make a
shorter protein carrying all major epitopes correlating with viral
clearance.
[0022] Table 6 shows the reactivity in LIA of E1s-acetamide versus
E1s-maleimide with sera of chronic HCV carriers. Proteins were
immobilized on the LIA membranes. E1s-acetamide was sprayed as such
on the LIA strips while E1s-maleimide (also containing
biotin-maleimide) was complexed with streptavidin before spraying.
Antigens were bound to LIA-membranes, and strips were processed
essentially as described in Zrein et al. (1998). Human antibodies
directed against these antigens were visualized using a
human-anti-IgG conjugated with alkaline phosphatase. NBT and BCIP
were used for color development of the strip. Staining was scored
from 0.5 to 4, as explained in Zrein et al. (1998). Using a cut-off
for this assay of 0.5 the number of positive samples (#pos) and
percentage (% pos) is mentioned at the bottom of the table.
[0023] FIG. 1. Superimposed size exclusion chromatography profiles
in PBS/3% Empigen-BB of E1s and E2s proteins expressed and purified
according to Maertens et al. (PCT/EP95/0303 1)
[0024] FIG. 2. Superimposed size exclusion chromatography (SEC)
profiles of E1s and E2s proteins expressed and purified according
to Maertens et al. (PCT/EP95/03031), and submitted to another run
on the same SEC column in PBS/0.2% CHAPS, to obtain specific
oligomeric structures of an estimated apparent molecular weight of
250-300 kDa. Similar degrees of association can be obtained by
using 3% betaine.
[0025] FIG. 3. Superimposed size exclusion chromatography profiles
of E1s and E2s proteins expressed and purified according to
Maertens et al. (PCT/EP95/03031), submitted to a second run in 0.2%
CHAPS or 3% betaine to obtain specific oligomeric structures as
shown in FIG. 2, and submitted to a third run on the same SEC
column in 0.05% CHAPS, to obtain specific homo-oligomeric
structures with an estimated apparent molecular weight of 250-300
kDa (E2s) and >600 kDa (E1s). Similar degrees of association can
be obtained by using 0.1 or 0.5% betaine.
[0026] FIG. 4 Dynamic light scattering analysis, expressed as
percentage of the number of particles in relation to the observed
diameter in nm, of E1s in PBS/0.05% CHAPS.
[0027] FIG. 5 Dynamic light scattering analysis, expressed as
percentage of the number of particles in relation to the observed
diameter in nm, of E1s in PBS/0.1% betaine (top) or 0.5% betaine
(bottom).
[0028] FIG. 6 EM staining of (A) E1s in PBS/0.05% CHAPS and (B) E1s
in PBS/3% betaine.
[0029] FIG. 7 Size distribution of particles of E1s in PBS/0.05%
CHAPS.
[0030] FIG. 8 Evolution of anti-E1 antibodies induced by six
consecutive and 3 boost immunizations (indicated by small arrows)
in a 1b infected chimpanzee (Phil), and the evolution of ALT, HCV
RNA, and anti-E1 antibodies. Anti-E1 antibodies binding to solid
phase E1 were detected using an anti-human IgG specific secondary
antiserum conjugated with peroxidase. TMB was used as substrate for
colour development. The results are expressed as end-point titer.
ALT levels were determined with a clinical analyser, and are
expressed as U/l. HCV RNA in serum was determined using HCV Monitor
(Roche, Basel, Switzerland). Viral load in the liver was determined
by semi-quantitative determination of the amount of E2 antigen
stained in the liver biopsy using a specific monoclonal (ECACC
accession number 98031215 as described in EP application No
98870060.5).
[0031] FIG. 9 Epitope mapping of the antibody responses induced by
immunization with E1 in chimpanzee Phil. Antibodies reactivity
towards the various peptides was measured by an indirect ELISA in
which biotinylated peptides (see also Table 4) are adsorbed on the
microtiterplates via streptavidin. Specific antibodies are detected
using an anti-human IgG specific secondary antiserum conjugated
with peroxidase. TMB was used as substrate for colour
development.
[0032] FIG. 10 Results of the lymphocyte proliferation assay before
and after vaccination in chimpanzee Phil. Frozen PBMC were thawed
and stimulated in triplicate with different antigens. Negative
control was medium alone, while concanavalin A was used as positive
control at a concentration of 5 .mu.g/ml. PBMC at a concentration
of 2-4.times.10.sup.5 cells/well in a total volume of 150 .mu.l
were cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated FCS in U-shaped 96-well microtiterplates together
with the controls or 1 .mu.g/ml of E1 for 90 h at 37.degree. C. in
a humidified atmosphere containing 5% CO.sub.2. During the last 18
h the cells were pulsed with 0.5 .mu.Ci (.sup.3H) thymidine per
well. Subsequently, the cultures, were harvested on glass fibre
filters and label uptake was determined. Results are expressed as
Stimulation Indices (SI): mean cpm antigen/mean cpm medium alone of
triplicate determinations.
[0033] FIG. 11 Evolution of anti-E1 antibodies induced by six
consecutive and 3 boost immunizations (indicated by small arrows)
in HCV subtype 1a infected chimpanzee Ton. Evolution of ALT, HCV
RNA in serum and determination of HCV antigen in liver are shown.
Anti-E1 antibodies were determined by means of an indirect ELISA:
specific antibodies binding to solid phase-coated E1 are detected
using a anti-human IgG specific secondary antiserum conjugated with
peroxidase. TMB was used as substrate for color development. The
results are expressed as end-point titres. ALT levels were
determined with a clinical analyser, and are expressed as U/l. HCV
RNA was determined using HCV Monitor (Roche, Basel, Switzerland).
E2 antigen was stained in the liver biopsy using a specific
monoclonal (ECACC accession number 98031215 as described in EP
application N.sup.o 98870060.5). The semi-quantitative scoring is
indicated by black squares for clearly positive staining in the
majority of the cells, by grey squares for clear staining in the
minority of the cells and by white squares for biopsies showing no
detectable staining. HCV RNA is indicated by small black boxes.
Staining of E2 could be confirmed by Core and E1 staining (data not
shown).
[0034] FIG. 12 Epitope mapping of the antibody response induced by
immunization with E1 in Ton. Antibodies reactivity towards the
various peptides was measured by an indirect ELISA in which
biotinylated peptides (see also Table 4) are adsorbed on the
microtiterplates via streptavidin. Specific antibodies are detected
using an anti-human IgG specific secondary antiserum conjugated
with peroxidase. TMB was used as substrate for color
development.
[0035] FIG. 13 Analysis of E1 antibody responses to subtype 1a and
subtype 1b E1 proteins in chimpanzee Ton. For this purpose an E1
genotype 1a, derived from the HCV-H sequence, recombinant vaccinia
virus was generated expressing the same part of E1 as for genotype
1b (see infra). E1 was derived from crude lysates from vaccinia
virus infected RK13 cells (prepared as described in Maertens et al.
(PCT/EP95/03031)). Antibody reactivity was measured by an indirect
ELISA in which E1 was adsorbed on the microtiterplates via the
high-mannose binding Galanthus nivalis agglutinin (GNA). Specific
antibodies were detected using an anti-human IgG specific secondary
antiserum conjugated with peroxidase. TMB was used as substrate for
colour development. The results are expressed as differential OD
(OD of well with adsorbed E1 minus OD of well without adsorbed
E1).
[0036] FIG. 14 Results of the lymphocyte proliferation assay before
and after vaccination of chimpanzee Ton. Frozen PBMC were thawed
and stimulated in triplicate with different antigens. Negative
control was medium alone, while concanavalin A was used as positive
control at a concentration of 5 .mu.g/ml. PBMC at a concentration
of 2-4 10.sup.5 cells/well in a total volume of 150 .mu.l were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated
FCS in U-shaped 96-well microtiterplates together with the controls
or 1 .mu.g/ml of E1 for 90 h at 37.degree. C. in a humidified
atmosphere containing 5% CO.sub.2. During the last 18 h the cells
are pulsed with 0.5 .mu.Ci (.sup.3H) thymidine per well.
Subsequently, the cultures, are harvested on glass fibre filters
and label uptake is determined. Results are expressed as
Stimulation Indices (SI): mean cpm antigen/mean cpm medium alone of
triplicate determinations.
[0037] FIG. 15 Maps of the constructs used to obtain expression of
an E2 protein with its N-terminal hypervariable region deleted.
Constructs pvHCV-92 and pvHCV-99 are intermediate constructs used
for the construction of the deletion mutants pvHCV-100 and
pvHCV-101.
[0038] FIG. 16 Sequence (nucleotides: A (SEQ ID NO:28);
translation: B (SEQ ID NO:29)) corresponding with the constructs
depicted in FIG. 15 (see above).
[0039] FIG. 17 Antibody titers obtained in mice upon immunization
with different E1 preparations as described in example 9. Titers
were determined by means of ELISA: murine sera were diluted 1/20
and further on (0.5 log.sub.10) and incubated on E1s (either
acetamide or maleimide modified) coated on microtiterplates. After
washing binding antibodies are detected using an anti-mouse IgG
specific secondary antiserum conjugated with peroxidase. TMB was
used as substrate for colour development. The results are expressed
as end-point titer and standard deviations are shown (n=6).
[0040] FIG. 18 Epitope mapping of the antibody response induced by
immunization with different E1s preparations in mice. Antibody
reactivity towards the various peptides was measured by an indirect
ELISA, in which biotinylated peptides (listed in Table 4) are
adsorbed on the microtiterplates via streptavidin. Murine sera were
diluted 1/20 and specific antibodies are detected using an
anti-mouse-IgG specific secondary antiserum conjugated with
peroxidase. TMB was used as substrate for colour development.
[0041] FIG. 19 Immunoglobulin isotyping profile of mice immunized
with different E1s preparations. Specific Ig class and subclass
antibodies were adsorbed at the microtiterplate. After capturing of
the murine Ig out of immune sera diluted 1/500, E1s was incubated
at 1 .mu.g/ml. The formed immunecomplexes were further incubated
with a polyclonal rabbit antiserum directed against E1. Finally,
the rabbit antibodies were detected using a goat-anti-rabbit Ig
secondary antiserum conjugated with peroxidase. TMB was used as
substrate for color development. The results were normalized for
IgG.sub.1 (ie the IgG.sub.1 signal was for each animal separately
considered to be 1 and all the results for the other isotypes were
expressed relative to this IgG.sub.1 result).
[0042] FIG. 20 Antibody titers induced by two immunizations around
day 1000 with E1s-acetamide in chimp Phil. Anti-E1 antibodies were
determined by means of an indirect ELISA: specific antibodies
binding to solid phase E1 are detected using anti-human IgG
specific secondary antiserum conjugated with peroxidase. The titer
is expressed in units/ml, these units refer to an in house standard
which is based on human sera.
[0043] FIG. 21 Antibody titers induced by two immunizations around
day 900 with E1s-acetamide in chimp Ton. Anti-E1 antibodies were
determined by means of an indirect ELISA: specific antibodies
binding to solid phase E1 are detected using anti-human IgG
specific secondary antiserum conjugated with peroxidase. The titer
is expressed in units/ml, these units refer to an in house standard
which is based on human sera.
[0044] FIG. 22 SEC profile of the final detergent reduction step
(0.2 to 0.05% CHAPS): E1 alone particle (A), E2 alone particle (B)
or an equimolar mixture of E1 and E2; mixed particle (C). The
figure also shows an overlay of the OD values of an ELISA
specifically detecting E1 only (top), E2 only (middle) and an ELISA
detecting only mixed particles (bottom).
[0045] FIG. 23 SEC profile of the final detergent reduction step
(0.2 to 0.05% CHAPS): E1 genotype 1b alone particle (top), E1
genotype 4 alone particle (middle) or an equimolar mixture of E1
genotype 1b and 4, mixed particle (bottom). The figure also shows
an overlay of the OD values of an ELISA specifically detecting only
mixed particles (see also FIG. 22).
DETAILED DESCRIPTION OF THE INVENTION
[0046] The invention described herein draws on previously published
work and pending patent applications. By way of example, such work
consists of scientific papers, patents or pending patent
applications. All these publications and applications, cited
previously or below are hereby incorporated by reference.
[0047] The present invention relates to HCV vaccination. For the
first time successful immunotherapy of chimpanzees with severe
chronic active hepatitis C could be achieved by vaccination with an
HCV antigen. The vaccine not only induced high immune responses,
but also induced clearance of viral antigen from the liver, and
considerable improvement of the histological activity and of liver
disease. The present invention further relates to purified single
HCV envelope proteins and in particular to oligomeric particles.
The oligomeric particles consist essentially of HCV envelope
proteins and have a diameter of 1 to 100 nm as measured by dynamic
light scattering or possibly electron microscopy. In this regard it
should be stressed that the particles can be formed by E1 and/or E2
proteins only, or parts thereof (see infra). Therefore, the
oligomeric particles of the present invention differ fundamentally
with the HCV-like particles described in WO 98/21338, which
necessarily consist of E1 and E2 and Core and P7. The terms
"oligomeric particles consisting essentially of HCV envelope
proteins" are herein defined as structures of a specific nature and
shape containing several basic units of the HCV E1 and/or E2
envelope proteins, which on their own are thought to consist of one
or two E1 and/or E2 monomers, respectively. It should be clear that
the particles of the present invention are defined to be devoid of
infectious HCV RNA genomes. The particles of the present invention
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 "viral-like
particles" or VLPs. Alternatively, the higher-order particles can
be solid spherical structures, in which the complete sphere
consists of HCV E1 or E2 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 (see further)-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 which consist of a lower number of HCV
envelope proteins, usually between 4 and 16. A specific example of
the latter includes the smaller particles obtained with E1s in 0.2%
CHAPS as exemplified herein which apparently contain 8-10 monomers
of E1s. 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 E1 or E2 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 the well-known-in-the-art dynamic light
scattering techniques (see further in examples section), is usually
between 1 to 100 nm, more preferentially between 2 to 70 nm, even
more preferentially between 2 and 40 nm, between 3 to 20 nm,
between 5 to 16 nm, between 7 to 14 nm or between 8 to 12 nm.
[0048] The invention further relates to an oligomeric particle as
defined above, wherein said envelope proteins are selected from the
group consisting of HCV E1, HCV E1s, HCV E2 proteins, SEQ ID No 13
or SEQ ID No 14, or parts thereof. The proteins HCV E1 and HCV E2,
and a detailed description of how to purify the latter proteins,
are well-described and characterized in PCT/EP 95/03031 to Maertens
et al. HCV E1s, SEQ ID No 13 or SEQ ID No 14, or parts thereof, can
be purified similarly as described for HCV E1 or HCV E1s in PCT/EP
95/03031 to Maertens et al. It should be stressed that the whole
content, including all the definitions, of the latter document is
incorporated by reference in the present application. The protein
HCV E1s refers to amino acids 192 to 326 of E1, and represents the
E1 protein without its C-terminal hydrophobic anchor. The term "or
parts thereof" refers to any part of the herein-indicated proteins
which are immunogenic, once they are part of a particle of the
present invention.
[0049] The invention further pertains to oligomeric particles as
described herein, wherein at least one cysteine residue of the HCV
envelope protein as described above is alkylated, preferably
alkylated by means of alkylating agents, such as, for example,
active halogens, ethylenimine or N-(iodoethyl)trifluoroacetamide.
In this respect, it is to be understood that alkylation of
cysteines refers to cysteines on which the hydrogen on the sulphur
atom is replaced 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. Other methods of alkylation
include the use of ethylenimine or N-(iodoethyl)trifluoroacetamide
both resulting in substitution of H by
--CH.sub.2--CH.sub.2--NH.sub.2 (Hermanson, 1996). The term
"alkylating agents" as used herein refers to compounds which are
able to perform alkylation as described herein. Such alkylations
finally result in a modified cysteine, which can mimic other
aminoacids. Alkylation by an ethylenimine results in a structure
resembling lysine, in such a way that new cleavage sites for
trypsine are introduced (Hermanson 1996). Similarly, the usage of
methyliodide results in an amino acid resembling methionine, while
the usage of iodoacetate and iodoacetamide results in amino acids
resembling glutamic acid and glutamine, respectively. In analogy,
these amino acids are preferably used in direct mutation of
cysteine. Therefore, the present invention pertains to oligomeric
particles as described herein, wherein at least one cysteine
residue of the HCV envelope protein as described herein is mutated
to a natural amino acid, preferentially to methionine, glutamic
acid, glutamine or lysine. The term "mutated" refers to
site-directed mutagenesis of nucleic acids encoding these amino
acids, ie to the well kown methods in the art, such as, for
example, site-directed mutagenesis by means of PCR or via
oligonucleotide-mediated mutagenesis as described in Sambrook et
al. (1989).
[0050] The term "purified" as applied herein refers to a
composition wherein the desired components, such as, for example,
HCV envelope proteins or oligomeric particles, comprises at least
35% of the total components in the composition. The desired
components preferably comprises at least about 40%, more preferably
at least about 50%, still more preferably at least about 60%, still
more preferably at least about 70%, even more preferably at least
about 80%, even more preferably at least about 90%, even more
preferably at least about 95%, and most preferably at least about
98% of the total component fraction of the composition. The
composition may contain other compounds, such as, for example,
carbohydrates, salts, lipids, solvents, and the like, without
affecting the determination of the percentage purity as used
herein. An "isolated" HCV oligomeric particle intends an HCV
oligomeric particle composition that is at least 35% pure. In this
regard it should be clear that the term "a purified single HCV
envelope protein" as used herein, refers to isolated HCV envelope
proteins in essentially pure form. The terms "essentially purified
oligomeric particles" and "single HCV envelope proteins" as used
herein refer to HCV oligomeric particles or single HCV envelope
proteins such that they can be used for in vitro diagnostic methods
and therapeutics. These HCV oligomeric particles are substanially
free from cellular proteins, vector-derived proteins or other HCV
viral components. Usually, these particles or proteins are purified
to homogeneity (at least 80% pure, preferably 85%, more preferably
90%, more preferably 95%, more preferably 97%, more preferably 98%,
more preferably 99%, even more preferably 99.5%, and most
preferably the contaminating proteins should be undetectable by
conventional methods such as SDS-PAGE and silver staining).
[0051] The present invention also relates to an oligomeric particle
as defined above wherein said envelope proteins are any possible
mixture of HCV E1, HCV E1s, HCV E2, SEQ ID No 13 and/or SEQ ID No
14, or parts thereof, such as, for example, a particle of the
present invention can substantially consist of HCV E1- and HCV E2
proteins, HCV E1- and HCV E1s proteins, HCV E1s- and HCV E2
proteins, and HCV E1-, HCV E1s- and HCV E2 proteins. Furthermore,
the present invention also relates to an oligomeric particle as
defined above wherein said proteins are derived from different HCV
strains, subtypes or genotypes, such as, for example, said proteins
are derived from genotype 1b and genotype 4, or are a mixture
consisting of HCV envelope proteins from one strain or genotype of
HCV and at least one other strain or genotype of HCV. The different
HCV strains or genotypes are well-defined and characterized in
PCT/EP 95/04155 to Maertens et al. It is stressed again that the
whole content, including all the definitions, of the latter
document is incorporated by reference in the present application.
Thus, the present invention relates to oligomeric particles
comprising envelope proteins derived from any HCV strain or
genotype known in the art or to particles comprising a mixture of
proteins derived from any HCV strain or genotype known in the art.
In this regard the present invention also relates to a consensus
sequences derived from individual clones as exemplified below and
in the examples section (see further).
[0052] The present invention further relates to an oligomeric
particle as described herein obtainable by a method, as well as to
said method to produce said oligomeric particle. Said method is
characterized by the following steps:
[0053] (I) Purifying HCV envelope proteins, possibly including the
use of an optionally first detergent. In essence, the purification
procedure of step (I) has been described extensively in PCT EP
95/03031 to Maertens et al. Importantly, according to the present
invention, the blocking step in the purification procedure as
described in PCT EP 95/03031, eg with NEM-biotin, is carried out
with an alkylation step as described in the present application,
preferentially by using iodoacetamide. Moreover, the purification
procedure of step (I) can possibly include the use of a disulphide
bond cleavage agent, and possibly include the use of an alkylating
agent. Finally, the procedure of step (I) results in purified HCV
envelope proteins in a solution.
[0054] (II) Replacing the solution of said purified HCV envelope
proteins with a detergent or salt, resulting in the formation of
oligomeric particles.
[0055] (III) Recovering or purifying said oligomeric particles,
possibly including further reducing the concentration of the
detergent or salt of step (II), which further assists the formation
and stabilization of said oligomeric particles, formed after said
replacing.
[0056] More preferably, the present invention relates to an
oligomeric particle as defined herein, as well as the method to
produce said particle, wherein said optionally first detergent is
Empigen-BB. More preferably, the present invention relates to an
oligomeric particle as defined herein, as well as the method to
produce said particle, wherein the detergent of step (II) is CHAPS,
octylglucaside or Tween, more preferably Tween-20 or Tween-80, or
any other detergent. More preferably, the present invention relates
to an oligomeric particle as defined herein, as well as the method
to produce said particle, wherein said salt is betaine. Even more
preferably, the present invention relates to an oligomeric particle
as defined above, as well as the method to produce said particle,
wherein said Empigen-BB is used at a concentration of 1% to 10% and
wherein said CHAPS or Tween is used at a concentration of 0.01% to
10%, or said betaine is used at a concentration of 0.01% to 10%.
Even more preferably, the present invention relates to an
oligomeric particle as defined above, as well as the method to
produce said particle, wherein said Empigen-BB is used at a
concentration of 3% and wherein said CHAPS or betaine are used at
concentrations of 0.2% or 0.3%, respectively, after which buffer is
switched and said CHAPS or betaine are used at concentrations of
0.05% or 0.1-0.5%, respectively. It is to be understood that all
percentages used in the method described above are given as
weight/volume. It should be clear that the method described above
(see also PCT/EP 95/03031 and the examples section of the present
application) is an example of how to produce the particles of the
present invention. In this regard, the present invention also
concerns any other method known in the art which can be used to
produce the oligomeric particles of the present invention, such as,
for example, omitting the reducing agent as described in PCT/EP
95/03031 and the examples section (infra), and using instead host
cells, which have an optimised redox state in the Endoplasmic
Reticulum for reducing cysteine bridges. In addition, it should be
clear that a whole range of alkylbetaines can be used, such as, for
example, with a C.sub.n tail, in which n= a positive integer
ranging from 1 to 20, as well as betaine derivatives, such as, for
example, sulfobetaines.
[0057] Since for the first time successful immunotherapy of
chimpanzees with severe chronic active hepatitis C was achieved by
vaccination with a purified HCV antigen, the present invention also
relates to purified single HCV envelope proteins, in particular E1
or E1s. Moreover, the present invention pertains to a composition
comprising said single HCV envelope proteins, and the use thereof
as an HCV vaccine, or for the manufacture of an HCV vaccine.
[0058] In order to avoid induction of an immune response against
irrelevant epitopes, the HCV envelope protein used for vaccination
is preferably constructed as a consensus sequence of individual
subtypes, strains, or clones. Therefore, the present invention also
pertains to the use of an HCV antigen (either in the form of
peptide, protein, or a polynucleotide) for vaccination or
diagnosis. Furthermore, the present invention also pertains to an
oligomeric particle, as defined herein, and the use thereof, in
which the HCV envelope protein is encoded by a consensus sequences
based on quasispecies variability within an isolate (isolate
consensus sequence) or based on the consensus sequence of different
isolates within a subtype (subtype consensus sequence), type or
species (type or species consensus sequence), or the complete HCV
genus (genus consensus sequence). Consequently, the amino acid
sequence of this consensus HCV envelope protein is a consensus
sequence derived from an isolate-, subtype-, strain-, or genus
consensus sequence. For the connotation of the term "consensus" is
particularly referred to Maertens and Stuyver (1997), and
references used therein.
[0059] The oligomeric particle of the present invention displays
epitopes extremely efficiently (see infra). Hence, the oligomeric
particle is a means to present epitopes in such a way that they can
elicit a proficient immune response. In this context, it is
comprehended that the HCV envelope proteins as defined herein do
not need to contain HCV epitopes exclusively. The HCV envelope
proteins, which form the oligomeric particles, may contain epitopes
that are derived from HCV solely, and possibly contain epitopes
that are derived from other exogenous agents, such as, for example,
HBV or HIV. In other words, the oligomeric particle with an HCV
envelope protein backbone, can be used as a vehicle to present
non-HCV epitopes, possibly in addition to HCV epitopes. Therefore,
the present invention also encompasses an oligomeric particle, as
defined herein but possibly without HCV epitopes, and its
applications and its manufacture, possibly containing non-HCV
epitopes. The term "exogenous agent" as used herein, refers to any
agent, whether living or not, able to elicit an immune response,
and which is not endogenous to the host, and which is not HCV.
Specifically, the latter term refers to the group consisting of
pathogenic agents, allergens and haptens. Pathogenic agents
comprise prions, virus, prokaryotes and eukaryotes. More
specifically, virus comprise in particular HBV, HIV, or
Herpesvirus, but not HCV. Allergens comprise substances or
molecules able to provoke an immune response in an host on their
own when a host is exposed to said allergens. Haptens behave
similarly to allergens with respect to the ability of provoking an
immune response, but in contrast to allergens, haptens need a
carrier molecule.
[0060] The present invention also relates to a composition
comprising an oligomeric particle as defined above. More
particularly the present invention relates to a vaccine
composition. The term "vaccine composition" relates to an
immunogenic composition capable of eliciting protection against
HCV, whether partial or complete. It therefore includes HCV
peptides, proteins, or polynucleotides. Protection against HCV
refers in particular to humans, but refers also to non-human
primates, trimera mouse (Zauberman et al., 1999), or other
mammals.
[0061] The particles of the present invention can be used as such,
in a biotinylated form (as explained in WO 93/18054) and/or
complexed to Neutralite Avidin (Molecular Probes Inc., Eugene,
Oreg., USA). It should also be noted that "a vaccine composition"
comprises, in addition to an active substance, a suitable
excipient, diluent, carrier and/or adjuvant which, by themselves,
do not induce the production of antibodies harmful to the
individual receiving the composition nor do they elicit protection.
Suitable carriers are typically large slowly metabolized
macromolecules such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric aa's, aa copolymers and inactive
virus particles. Such carriers are well known to those skilled in
the art. Preferred adjuvants to enhance effectiveness of the
composition include, but are not limited to: aluminium hydroxide,
aluminium in combination with 3-0-deacylated monophosphoryl lipid A
as described in WO 93/19780, aluminium phosphate as described in WO
93/24148, N-acetyl-muramyl-L-threonyl-D-isoglutamine as described
in U.S. Pat. No. 4,606,918,
N-acetyl-normuramyl-L-alanyl-D-isoglutamine,
N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine2-(1'2'dipalmitoyl-sn-gly-
cero-3-hydroxyphosphoryloxy)ethylamine and RIBI (ImmunoChem
Research Inc., Hamilton, Mont., USA) which contains monophosphoryl
lipid A, detoxified endotoxin, trehalose-6,6-dimycolate, and cell
wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any
of the three components MPL, TDM or CWS may also be used alone or
combined 2 by 2. Additionally, adjuvants such as Stimulon
(Cambridge Bioscience, Worcester, Mass., USA) or SAF-1 (Syntex) may
be used, as well as adjuvants such as combinations between QS21 and
3-de-O-acetylated monophosphoryl lipid A (WO94/00153), or MF-59
(Chiron), or poly[di(carboxylatophenoxy)phosphazene] based
adjuvants (Virus Research Institute), or blockcopolymer based
adjuvants such as Optivax (Vaxcel, Cythx) or inulin-based
adjuvants, such as Algammulin and GammaInulin (Anutech), Incomplete
Freund's Adjuvant (IFA) or Gerbu preparations (Gerbu Biotechnik).
It is to be understood that Complete Freund's Adjuvant (CFA) may be
used for non-human applications and research purposes as well. "A
vaccine composition" will further contain excipients and diluents,
which are inherently non-toxic and non-therapeutic, such as water,
saline, glycerol, ethanol, wetting or emulsifying agents, pH
buffering substances, preservatives, and the like. Typically, a
vaccine composition is prepared as an injectable, either as a
liquid solution or suspension. Solid forms, suitable for solution
on, or suspension in, liquid vehicles prior to injection may also
be prepared. The preparation may also be emulsified or encapsulated
in liposomes for enhancing adjuvant effect. The polypeptides may
also be incorporated into Immune Stimulating Complexes together
with saponins, for example Quil A (ISCOMS). Vaccine compositions
comprise an immunologically effective amount of the polypeptides of
the present invention, as well as any other of the above-mentioned
components. "Immunologically effective amount" means that the
administration of that amount to an individual, either in a single
dose or as part of a series, is effective for prevention or
treatment. This amount varies depending upon the health and
physical condition of the individual to be treated, the taxonomic
group of the individual to be treated (e.g. human, non-human
primate, primate, etc.), the capacity of the individual's immune
system to mount an effective immune response, the degree of
protection desired, the formulation of the vaccine, the treating
doctor's assessment, the strain of the infecting HCV and other
relevant factors. It is expected that the amount will fall in a
relatively broad range that can be determined through routine
trials. Usually, the amount will vary from 0.01 to 1000 .mu.g/dose,
more particularly from 0.1 to 100 .mu.g/dose. The vaccine
compositions are conventionally administered parenterally,
typically by injection, for example, subcutaneously or
intramuscularly. Additional formulations suitable for other methods
of administration include oral formulations and suppositories.
Dosage treatment may be a single dose schedule or a multiple dose
schedule. The vaccine may be administered in conjunction with other
immunoregulatory agents. Therefore, the instant invention pertains
to the use of an oligomeric particle as defined herein for
prophylactically inducing immunity against HCV. It should be noted
that a vaccine may also be useful for treatment of an individual as
pointed-out above, in which case it is called a "therapeutic
vaccine".
[0062] The present invention also relates to a composition as
defined above which also comprises HCV core, E1, E2, P7, NS2, NS3,
NS4A, NS4B, NS5A and/or NS5B protein, or parts thereof. E1, E2,
and/or E1E2 particles may, for example, be combined with T cell
stimulating antigens, such as, for example, core, P7, NS3, NS4A,
NS4B, NS5A and/or NS5B. In particular, the present invention
relates to a composition as defined above wherein said NS3 protein,
or parts thereof, have an amino acid sequence given by SEQ ID 1 or
SEQ ID 2 (see further in examples section).
[0063] The purification of these NS3 proteins will preferentially
include a reversible modification of the cysteine residues, and
even more preferentially sulfonation of cysteines. Methods to
obtain such a reversible modification, including sulfonation have
been described for NS3 proteins in Maertens et al.
(PCT/EP99/02547). It should be stressed that the whole content,
including all the definitions, of the latter document is
incorporated by reference in the present application.
[0064] It is clear from the above that the present invention also
relates to the usage of an oligomeric particle as defined above or
a composition as defined above for the manufacture of an HCV
vaccine composition. In particular, the present invention relates
to the usage of an oligomeric particle as defined herein for
inducing immunity against HCV in chronic HCV carriers. More in
particular, the present invention relates to the usage of an
oligomeric particle as defined herein for inducing immunity against
HCV in chronic HCV carriers prior to, simultaneously to or after
any other therapy, such as, for example, the well-known interferon
therapy either or not in combination with the administration of
small drugs treating HCV, such as, for example, ribavirin. Such
composition may also be employed before or after liver
transplantation, or after presumed infection, such as, for example,
needle-stick injury. In addition, the present invention relates to
a kit containing the oligomeric particles or the single HCV
envelope proteins of the present invention to detect HCV antibodies
present in a biological sample. The term "biological sample" as
used herein, refers to a sample of tissue or fluid isolated from an
individual, including but not limited to, for example, serum,
plasma, lymph fluid, the external sections of the skin, respiratory
intestinal, and genitourinary tracts, oocytes, tears, saliva, milk,
blood cells, tumors, organs, gastric secretions, mucus, spinal cord
fluid, external secretions such as, for example, excrement, urine,
sperm, and the like. Since the oligomeric particles and the single
HCV envelope proteins of the present invention are highly
immunogenic, and stimulate both the humoral and cellular immune
response, the present invention relates also to a kit for detecting
HCV related T cell response, comprising the oligomeric particle or
the purified single HCV envelope protein of the instant invention.
HCV T cell response can for example be measured as described in the
examples section, or as described in PCT/EP 94/03555 to
Leroux-Roels et al. It should be stressed that the whole content,
including all the definitions, of this document is incorporated by
reference in the present application.
[0065] It should be clear that the present invention also pertains
to the use of specific HCV immunoglobulins for treatment and
prevention of HCV infection. It is here for the first time
demonstrated that sufficient levels of HCV antibodies, especially
HCV envelope antibodies, induce amelioration of Hepatitis C
disease. It is also demonstrated for the first time that sufficient
levels of antibodies can bind circulating virus, and that the
presence of Ab-complexed virus coincides with disappearance of HCV
antigen from the liver, and with amelioration of liver disease. HCV
envelope antibodies may be induced by vaccination or may be
passively transferred by injection after the antibodies have been
purified from pools of HCV-infected blood or from blood obtained
from HCV vaccinees. Therefore, the present invention pertains
further to specific antibodies, generated against an oligomeric
particle as described above or against a composition as described
above, or a single HCV envelope protein. In particular, the present
invention relates to a kit comprising said antibodies for detecting
HCV antigens. The term "specific antibodies" as used herein, refers
to antibodies, which are raised against epitopes which are specific
to the oligomeric particle as disclosed in the present invention.
In other words, specific antibodies are raised against epitopes
which result from the formation of, and are only present on
oligomeric particles. Moreover, there are various procedures known
to produce HCV peptides. These procedures might result in HCV
peptides capable of presenting epitopes. It is conceivable that the
HCV peptides, obtained by these various and different procedures,
are capable of presenting similar epitopes. Similar epitopes are
epitopes resulting from different production or purifying
procedures but recognizable by one and the same antibody. However,
the oligomeric particles of the instant invention present epitopes
extremely efficient. Consequently, the epitopes on the oligomeric
particles are highly immunogenic. Therefore, the present invention
also pertains to epitopes on oligomeric particles, said epitopes
are at least 10 times, preferentially at least 20 times,
preferentially at least 50, preferentially at least 100 times,
preferentially at least 500 times, and most preferentially at least
1000 times more immunogenic than epitopes on HCV-peptides, which
are not produced according to the present invention, ie not
produced by detergent-assisted particle formation. It will be
appreciated by the skilled that said immunogenecity can, for
example, be detected and therefore compared by immunising mammals
by means of administering comparable quantities of peptides,
produced by either method. Moreover, the term "specific antibody"
refers also to antibodies which are raised against a purified
single HCV envelope protein. As used herein, the term "antibody"
refers to polyclonal or monoclonal antibodies. The term "monoclonal
antibody" refers to an antibody composition having a homogeneous
antibody population. The term "antibody" is not limiting regarding
the species or source of the antibody, nor is it intended to be
limited by the manner in which it is made. In addition, the term
"antibody" also refers to humanized antibodies in which at least a
portion of the framework regions of an immunoglobulin are derived
from human immunoglobulin sequences and single chain antibodies,
such as, for example, as described in U.S. Pat. No. 4,946,778, to
fragments of antibodies such as F.sub.ab, F.sub.'(ab)2, F.sub.v,
and other fragments which retain the antigen binding function and
specificity of the parental antibody.
[0066] Moreover, the present invention also features the use of an
oligomeric particle as described above, or a composition as
described above to detect antibodies against HCV envelope proteins.
As used herein, the term "to detect" refers to any assay known in
the art suitable for detection. In particular, the term refers to
any immunoassay as described in WO 96/13590.
[0067] The terms "peptide", "polypeptide" and "protein" are used
interchangeably in the present invention. "Polypeptide" refers to a
polymer of amino acids (amino acid sequence) and does not refer to
a specific length of the molecule. Thus, oligopeptides are included
within the definition of polypeptide. It is to be understood that
peptidomimics are inherent in the terms "polypeptide", "peptide"
and "protein"
[0068] Also, the present invention relates to the use of an
oligomeric particle as described herein for inducing immunity
against HCV, characterized in that said oligomeric particle is used
as part of a series of time and compounds. In this regard, it is to
be understood that the term "a series of time and compounds" refers
to administering with time intervals to an individual the compounds
used for eliciting an immune response. The latter compounds may
comprise any of the following components: oligomeric particles, HCV
DNA vaccine composition, HCV polypeptides.
[0069] In this respect, a series comprises administering, either:
[0070] (I) an HCV antigen, such as, for example, an oligomeric
particle, with time intervals, or [0071] (II) an HCV antigen, such
as, for example, an oligomeric particle in combination with a HCV
DNA vaccine composition, in which said oligomeric particles and
said HCV DNA vaccine composition, can be administered
simultaneously, or at different time intervals, including at
alternating time intervals, or [0072] (III) either (I) or (II),
possibly in combination with other HCV peptides, with time
intervals.
[0073] In this regard, it should be clear that a HCV DNA vaccine
composition comprises nucleic acids encoding HCV envelope peptide,
including E1-, E2-, E1/E2-peptides, E1s peptide, SEQ ID No 13, SEQ
ID No 14, NS3 peptide, other HCV peptides, or parts of said
peptides. Moreover, it is to be understood that said HCV peptides
comprises HCV envelope peptides, including E1-, E2-,
E1/E2-peptides, E1s peptide, SEQ ID No 13, SEQ ID No 14, NS3
peptide, other HCV peptides, or parts thereof. The term "other HCV
peptides" refers to any HCV peptide or fragment thereof with the
proviso that said HCV peptide is not E1, E2, E1s, SEQ ID No 13, SEQ
ID No 14, or NS3. In item II of the above scheme, the HCV DNA
vaccine composition comprises preferentially nucleic acids encoding
HCV envelope peptides. In item II of the above scheme, the HCV DNA
vaccine composition consists even more preferentially of nucleic
acids encoding HCV envelope peptides, possibly in combination with
a HCV-NS3 DNA vaccine composition. In this regard, it should be
clear that an HCV DNA vaccine composition comprises a plasmid
vector comprising a polynucleotide sequence encoding an HCV peptide
as described above, operably linked to transcription regulatory
elements. As used herein, a "plasmid vector" refers to a nucleic
acid molecule capable of transporting another nucleic acid to which
it has been linked. Preferred vectors are those capable of
autonomous replication and/or expression of nucleic acids to which
they have been linked. In general, but not limited to those,
plasmid vectors are circular double stranded DNA loops which, in
their vector form, are not bound to the chromosome. As used herein,
a "polynucleotide sequence" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and single (sense or antisense) and double-stranded
polynucleotides. As used herein, the term "transcription regulatory
elements" refers to a nucleotide sequence which contains essential
regulatory elements, such that upon introduction into a living
vertebrate cell it is able to direct the cellular machinery to
produce translation products encoded by the polynucleotide. The
term "operably linked" refers to a juxtaposition wherein the
components are configured so as to perform their usual function.
Thus, transcription regulatory elements operably linked to a
nucleotide sequence are capable of effecting the expression of said
nucleotide sequence. Those skilled in the art can appreciate that
different transcriptional promoters, terminators, carrier vectors
or specific gene sequences may be used succesfully.
[0074] Finally, the present invention relates to an immunoassay for
detecting HCV antibody, which immunoassay comprises: (1) providing
the oligomeric particle or the purified single HCV envelope protein
as defined herein, or a functional equivalent thereof, (2)
incubating a biological sample with said oligomeric particle, or
said HCV envelope protein under conditions that allow the formation
of antibody-antigen complex, (3) determining whether said
antibody-antigen complex comprising said oligomeric particle or
said HCV envelope protein is formed.
[0075] The present invention will now be illustrated by reference
to the following examples which set forth particularly advantageous
embodiments. However, it should be noted that these embodiments are
merely illustrative and can not be construed as to restrict the
invention in any way.
EXAMPLES
Example 1
Expression, Purification, and Detergent-Assisted
Homo-Oligomerization of the HCV E1 Protein
[0076] The HCV E1s protein (amino acids 192-326) was expressed and
purified from RK13 cells using recombinant vaccinia virus pvHCV-11A
according to the protocol described in Maertens et al. (PCT/EP
95/03031). In addition, the purified E1 protein in 3% Empigen-BB
which displays an apparent molecular weight corresponding to an E1
homo-dimer (approximately about 60 kDa; FIG. 1), was pooled and the
pooled fractions were again applied to a size exclusion
chromatography column (according to PCT/EP 95/03031) and run in the
presence of 0.2% CHAPS or 3% betaine. Surprisingly, although the
E1s protein is devoid of its membrane anchor region, a homogeneous
population of specifically associated E1 homo-oligomers with an
apparent molecular weight of 260-280 kDa could be obtained with
both detergents (FIG. 2). Such a homo-oligomeric structure could
contain an approximate number of 9 E1s monomers. It should be clear
that the latter is a rough estimate as the shape of the oligomer
may drastically influence its apparent molecular weight as measured
by size exclusion chromatography. By switching from 0.2% CHAPS to
0.05% CHAPS and repeating the same procedure, the apparent
molecular weight further shifted beyond the resolution of the
column (void of the column, >600 kDa, FIG. 3), suggesting the
formation of particles. Switching from 3% betaine to 0.1% betaine
yielded a population of E1s oligomers with a similar behaviour
(data not shown). Other detergents could be chosen by means of
which similar detergent-assisted oligomerization could be achieved.
The oligomerization leading to the particle formation is not unique
for CHAPS or betaine, since similar results were obtained by using
Tween-20 or Tween-80, or octylglucaside. Moreover, further removal
of the detergent may be possible which may allow to generate even
larger particles. The presence of detergent may, therefore, not
longer be needed to obtain particles. The particles may be obtained
by e.g. SCC, without any detergent. Notably, an E1 monomer is
approximately 31 kDa, while an E2 monomer is approximately 70 kDa.
These values, however, may differ depending on the glycosylation
status of the protein.
Example 2
Analysis of the Higher order Oligomeric Structures of E1s by means
of Dynamic Light Scattering
[0077] In order to confirm the unexpected result that particles
have been created, E1s preparations in 0.05% CHAPS and 0.1%
betaine, prepared according to example 1, or in 0.1% betaine,
prepared by dilution of preparations in 0.5 % betaine, were
subjected to analysis by means of dynamic light scattering
(DLS).
[0078] The dynamic light scattering technique measures Brownian
motion and relates this to the size of particles. The larger the
particle, the slower the Brownian motion will be. The velocity of
the Brownian motion is defined by a property known as the diffusion
coefficient (usually given by the symbol D). The size of the
particle is calculated from the diffusion coefficient by using the
Stokes-Einstein equation: d(H)=kT/3B0D, in which d(H) is the
hydrodynamic diameter, k is the Boltzmann constant, T is the
absolute temperature, 0 is the viscosity. Notebly, the measured
diameter is a value which refers to how a particle diffuses within
a fluid. So, it is referred to as hydrodynamic diameter. The
diffusion coefficient is derived from an autocorrelation function
(variation of intensity fluctuation of light with time). The
instrument uses a computer-controlled correlator to calculate the
intensity of the autocorrelation function automatically.
[0079] For measuring size distributions, the above autocorrelation
function is corrected to obtain linear curves and the instrument is
equipped with a computer program for analysis of the size
distribution. However, the technique has restrictive assumptions
similar to those of the technique called multi angle laser light
scattering (MALLS) and neither method can be considered to yield
absolute data. The results of size distributions from DLS have to
be interpreted as semi-quantitative indicators of polydispersity,
rather than as a true representation of the distribution.
[0080] Samples containing E1s particles (80-400 .mu.g E1s/ml
PBS-0.05% CHAPS, 0.1% or 0.5% betaine) were pipetted in the
measuring cell of an LSP 3.53 DLS instrument equipped with a 10 mW
HeNe Laser (PolymerLabs). A readout of the analysis is provided in
FIGS. 4 (E1s in 0.05% CHAPS) and 5 (E1s in 0.1% or 0.5%
betaine).
[0081] These analyses indeed confirmed the unexpected result that
the obtained E1s structures were spherical, monodisperse particles.
The E1s particles in PBS/0.1% betaine showed an average size
distribution of 21.3.+-.4 nm, in PBS/0.5% betaine: 27.9.+-.5 nm,
whereas a diameter of 12.5 was obtained for E1s in PBS/0.05%
CHAPS.
Example 3
Size and Shape Analysis by means of Electron Microscopy
[0082] Ten .mu.l of E1s (226 .mu.g/ml in PBS/0.05% CHAPS; and 143
.mu.g/ml in PBS 3% betaine) was visualized with a standard negative
staining with 1% uranyl acetate on carbon stabilized formvar grids.
The sample was applied for 30 seconds and then rinsed with
dH.sub.2O before staining for 5 seconds and photography (FIG.
6).
[0083] Statistical analysis yielded the following results: the E1s
particle in CHAPS had a mean diameter of 8.7.+-.0.27 nm (range
4.3-29.0; 95% CI 5.4) and that the E1s particle in betaine was less
homogeneous with a mean diameter of 9.7.+-.0.55 nm (range 4.3-40.5;
95% CI 11.0). Surprisingly, the 3% betaine preparation, which
initially showed a MW of 250-300 kDa as analysed by SEC even shows
larger particles than the 0.05% CHAPS preparation, which initially
showed a MW of >600 kDa. We therefore hypothesized that
intermediate homo-oligomeric forms of E1s obtained by 3% betaine
may have formed higher order particles over time. This surprising
effect points to other possibilities for obtaining higher-order
particles. A size distribution of the particles (FIG. 7) shows that
the CHAPS preparation is monodisperse, although a tailing to larger
size particles is observed (up to 29 nm for 0.05% CHAPS). Since
larger structures are overestimated in DLS analyses, the presence
of these larger particles, although less in number, may explain the
larger diameter obtained by DLS analysis (example 2). The
difference in diameter may also be explained by the fact that DLS
measures a particle in motion, while electron microscopy measures
static particles. It should be clear that the immunogenicity of
these preparations as shown in the examples below is due to the
entirety of the preparation, and may be due to the average,
smaller, or larger particles, or to the mixture thereof.
Example 4
Immunization of a Chimpanzee Chronically Infected with HCV Subtype
1b
[0084] A chimpanzee (Phil) already infected for over 13 years (5015
days before immunization) with an HCV subtype 1b strain was
vaccinated with E1 (aa 192-326) which was derived from a different
strain of genotype 1b, with a 95.1% identity on the amino acid
level (see also Table 2), and which was prepared as described in
examples 1-3. The chimpanzee received in total 6 intramuscular
immunizations of each 50 .mu.g E1 in PBS/0.05% CHAPS mixed with
RIBI R-730 (MPLA+TDM+CWS) according to the manufacturer's protocol
(Ribi Inc. Hamilton, Mont.). The 6 immunizations were given in two
series of three shots with a three week interval and with a lag
period of 6 weeks between the two series. Starting 150 days prior
to immunization, during the immunization period and until 1 year
post immunization (but see below) the chimpanzee was continuously
monitored for various parameters indicative for the activity of the
HCV induced disease. These parameters included blood chemistry,
ALT, AST, gammaGT, blood chemistry, viral load in the serum, viral
load in the liver and liver histology. In addition, the immune
answer to the immunization was monitored both on the humoral and
cellular level. During this period the animal was also monitored
for any adverse effects of the immunization, such as change in
behaviour, clinical symptoms, body weight, temperature and local
reactions (redness, swelling, indurations). Such effects were not
detected.
[0085] Clearly, ALT (and especially gammaGT, data not shown) levels
decreased as soon as the antibody level against E1 reached its
maximum (FIG. 8). ALT rebounded rather rapidly as soon as the
antibody levels started to decline, but gammaGT remained at a lower
level as long as anti-E1 remained detectable.
[0086] E2 antigen in the liver decreased to almost undetectable
levels during the period in which anti-E1 was detectable and the E2
antigen rebounded shortly after the disappearance of these
antibodies. Together with the Core and E2 antigen becoming
undetectable in the liver, the inflammation of the liver markedly
decreased (see also Table 3). This is a major proof that the
vaccine induces a reduction of the liver damage, probably by
clearing, at least partially, the viral antigens from its major
target organ, the liver.
[0087] The viraemia level, as measured by Amplicor HCV Monitor
(Roche, Basel, Switzerland), remained approximately unchanged in
the serum during the whole study period.
[0088] More detailed analyses of the humoral response revealed that
the maximum end-point titer reached 14.5.times.10.sup.3 (after the
sixth immunization) and that this titer dropped to undetectable 1
year post immunization (FIG. 8). FIG. 9 shows that the main
epitopes, which can be mimicked by peptides, recognized by the
B-cells are located at the N-terminal region of E2 (peptides V1V2
and V2V3, for details on the peptides used see Table 4). Since the
reactivity against the recombinant E1 is higher and longer lasting,
it can also be deduced from this figure, that the antibodies
recognizing these peptides represent only part of the total
antibody population against E1. The remaining part is directed
against epitopes which cannot be mimicked by peptides, i.e
discontinuous epitopes. Such epitopes are only present on the
complete E1 molecule or even only on the particle-like structure.
Such an immune response against E1 is unique, at least compared to
what is normally observed in human chronic HCV carriers (WO
96/13590 to Maertens et al.) and in chimpanzees (van Doorn et al.,
1996), who raise anti-E1 antibodies in their natural course of
infection. In those patients, anti-E1 is in part also directed to
discontinuous epitopes but a large proportion is directed against
the C4 epitope (.+-.50% of the patient sera), a minor proportion
against V1V2 (ranging from 2-70% depending on the genotype), and
reactivity against V2V3 was only exceptionally recorded (Maertens
et al., 1997).
[0089] Analysis of the T-cell reactivity indicated that also this
compartment of the immune system is stimulated by the vaccine in a
specific way, as the stimulation index of these T-cells rises from
1 to 2.5, and remains somewhat elevated during the follow up period
(FIG. 10). It is this T cell reactivity that is only seen in Long
term responders to interferon therapy (see: PCT/EP 94/03555 to
Leroux-Roels et al.; Leroux-Roels et al., 1996).
Example 5
Immunization of a Chronic HCV carrier with Different Subtype
[0090] A chimpanzee (Ton) already infected for over 10 years (3809
days before immunization) with HCV from genotype 1a was vaccinated
with E1 from genotype 1b, with only a 79.3 % identity on the amino
acid level (see also Table 2), and prepared as described in the
previous examples. The chimpanzee received a total of 6
intramuscular immunizations of 50 .mu.g E1 in PBS/0.05% CHAPS each
mixed with RIBI R-730 according to the manufacturer's protocol
(Ribi Inc. Hamilton, Mont.). The 6 immunizations were given in two
series of three shots with a three week interval and with a lag
period of 4 weeks between the two series. Starting 250 days prior
to immunization, during the immunization period and until 9 months
(but see below) post immunization the chimpanzee was continuously
monitored for various parameters indicative for the activity of the
HCV induced disease. These parameters included blood chemistry,
ALT, AST, gammaGT, viral load in the serum, viral load in the liver
and liver histology. In addition, the immune answer to the
immunization was monitored both on the humoral and cellular level.
During this period the animal was also monitored for any adverse
effects of the immunization, such as change in behaviour, clinical
symptoms, body weight, temperature and local reactions (redness,
swelling, indurations). Such effects were not detected.
[0091] Clearly, ALT levels (and gammaGT levels, data not shown)
decreased as soon as the antibody level against E1 reached its
maximum (FIG. 11). ALT and gammaGT rebounded as soon as the
antibody levels started to decline, but ALT and gammaGT remained at
a lower level during the complete follow up period. ALT levels were
even significantly reduced after vaccination (62.+-.6 U/l) as
compared to the period before vaccination (85.+-.11 U/l). Since
less markers of tissue damage were recovered in the serum, these
findings were a first indication that the vaccination induced an
improvement of the liver disease.
[0092] E2 antigen levels became undetectable in the period in which
anti-E1 remained above a titer of 1.0.times.10.sup.3, but became
detectable again at the time of lower E1 antibody levels. Together
with the disappearance of HCV antigens, the inflammation of the
liver markedly decreased from moderate chronic active hepatitis to
minimal forms of chronic persistent hepatitis (Table 3). This is
another major proof that the vaccine induces a reduction of the
liver damage, probably by clearing, at least partially, the virus
from its major target organ, the liver.
[0093] The viraemia level, as measured by Amplicor HCV Monitor
(Roche, Basel, Switzerland), in the serum remained at approximately
similar levels during the whole study period. More detailed
analysis of the humoral response revealed that the maximum
end-point titer reached was 30.times.103 (after the sixth
immunization) and that this titer dropped to 0.5.times.10.sup.3
nine months after immunization (FIG. 11). FIG. 12 shows that the
main epitopes, which can be mimicked by peptides and are recognized
by the B-cells, are located at the N-terminal region (peptides V1V2
and V2V3, for details on the peptides used see Table 4). Since the
reactivity against the recombinant E1 is higher and longer lasting,
it can also be deduced from this figure, that the antibodies
recognizing these peptides represent only part of the total
antibody population against E1. The remaining part is most likely
directed against epitopes which cannot be mimicked by peptides,
i.e. discontinuous epitopes. Such epitopes are probably only
present on the complete E1 molecule or even only on the
particle-like structure. Such an immune response against E1 is
unique, at least compared to what is normally observed in human
chronic HCV carriers, which have detectable anti-E1. In those
patients, anti-E1 is in part also discontinuous, but a large
proportion is directed against he C4 epitope (50% of the patient
sera), a minor proportion against V1V2 (ranging from 2-70%
depending on the genotype) and exceptionally reactivity against
V2V3 was recorded (Maertens et al., 1997). As this chimpanzee is
infected with an 1a isolate the antibody response was also
evaluated for cross-reactivity towards a E1-1a antigen. As can be
seen in FIG. 13, such cross-reactive antibodies are indeed
generated, although, they form only part of the total antibody
population. Remarkable is the correlation between the reappearance
of viral antigen in the liver and the disappearance of detectable
anti-1a E1 antibodies in the serum.
[0094] Analysis of the T-cell reactivity indicated that also this
compartment of the immune system is stimulated by the vaccine in a
specific way, as the stimulation index of these T-cells rises from
0.5 to 5, and remains elevated during the follow up period (FIG.
14).
Example 6
Reboosting of HCV Chronic carriers with E1
[0095] As the E1 antibody titers as observed in examples 4 and 5
were not stable and declined over time, even to undetectable levels
for the 1b infected chimp, it was investigated if this antibody
response could be increased again by additional boosting. Both
chimpanzees were immunized again with three consecutive
intramuscular immunization with a three week interval (50 .mu.g E1
mixed with RIBI adjuvant). As can be judged from FIGS. 8 and 11,
the anti-E1 response could indeed be boosted, once again the viral
antigen in the liver decreased below detection limit. The viral
load in the serum remained constant although in Ton (FIG. 11). A
viremia level of <10.sup.5 genome equivalents per ml was
measured for the first time during the follow up period.
[0096] Notable is the finding that, as was already the case for the
first series of immunizations, the chimpanzee infected with the
subtype 1b HCV strain (Phil) responds with lower anti-E1 titers,
than the chimpanzee infected with subtype 1a HCV strain (maximum
titer in the first round 14.5.times.10.sup.3 versus
30.times.10.sup.3 for Ton and after additional boosting only
1.2.times.10.sup.3 for Phil versus 40.times.10.sup.3 for Ton).
Although for both animals the beneficial effect seems to be
similar, it could be concluded from this experiment that
immunization of a chronic carrier with an E1 protein derived from
another subtype or genotype may be especially beneficial to reach
higher titers, maybe circumventing a preexisting and specific
immune suppression existing in the host and induced by the
infecting subtype or genotype. Alternatively, the lower titers
observed in the homologous setting (1b vaccine+1b infection) may
indicate binding of the bulk of the antibodies to virus. Therefore,
the induced antibodies may possess neutralizing capacity.
Example 7a
Construction of a NS3 Protein Combining the Major Epitopes known to
Correlate with Control of Infection
[0097] Also other epitopes besides the ones in E1 may be linked
with clearing of HCV during acute phase or by interferon therapy.
Several of these epitopes are localized within NS3 (Leroux-Roels et
al., 1996; Rehermann et al., 1996 and 1997; Diepolder et al., 1995
and 1997). Two of the major epitopes are the CTL epitope mapped by
Rehermann and coworkers (aa 1073-1081), and the T-cell (CD4)
epitope mapped by Diepolder and coworkers (aa 1248-1261).
Unfortunately, these epitopes are scattered all over the NS3
protein. In order to have at least those epitopes available, a
relatively large protein would be needed (aa 1073-1454). Producing
such a large protein usually results in low yields, and may result
upon vaccination in a response which is only for a small part
targeted to the important epitopes. Therefore, production of a
smaller protein would be a more suitable solution to this problem.
In order to do so, some of the epitopes need to be repositioned
within such a smaller protein. By taking advantage of the knowledge
that exists, ie another CTL epitope (aa 1169-1177) which is not
linked with HCV clearance (Rehermann et al. 1996, 1997), an NS3
molecule was designed to start at aa 1166 and to end at aa 1468
(Table 5). This construct already includes the epitopes described
by Weiner and coworkers, and Diepolder and coworkers. By mutating
the region 1167 to 1180 to the sequence of the region 1071 to 1084,
the non-relevant CTL epitope was changed to the epitope Rehermann
and coworkers found to be linked with viral clearance. The
construct was additionally modified to contain a methionine at
position 1166 to allow initiation of translation. This methionine
will be cleaved off in E. coli since it is followed by an alanine.
In this way, the introduction of new epitopes, which are not
present in the natural NS3, is limited to a minimum. Alternatively,
if the expression of this protein would be cumbersome, the CTL
epitope may be linked to the C-terminus at aa 1468 as depicted in
detail in Table 5.
[0098] The coding sequence of an HCV NS-3 fragment was isolated and
expressed as described in Maertens et al. (PCT/EP99/02547; clone
19b; HCV aa 1188-1468 was used as starting material). The CTL
epitope as described by Rehermann, and not present in the 19b NS-3
fragment was fused to this fragment. Both N-terminal and C-terminal
fusions were constructed, since effects of the fusion on expression
levels, susceptibility to proteolytic breakdown and functionality
may be affected by the position of the epitope.
[0099] Using the pIGRI2NS-3 plasmid, which is an E. coli expression
plasmid expressing the NS-3 19b fragment under control of the
leftward promotor of phage lambda, as a template for PCR, NS3 19b
coding sequences, fused respectively N-- and C-terminally with the
Rehermann CTL epitope (named NS-319bTn and NS-3 l9bTc,
respectively), were first subcloned into the pGEM-T (Promega)
cloning vector giving rise to vectors pGEM-TNS-319bTn and
pGEM-TNS-319bTc. The PCR-amplified sequences were verified by DNA
sequence analysis.
[0100] In the case of fusing the T-cell epitope sequence to the
N-terminal region of NS-3, PCR was carried out with a long sense
primer carrying the CTL epitope and a short antisense primer
homologous to sequences 3' of the NS-3 19b stopcodon. Primer
sequences are depicted below. TABLE-US-00001 Primer 9038 (sense)
(SEQ ID NO 5) 5'-GCCATGGCGACCTGCATCAACGGTGTTTGCTGGACCGTTTACCACGG
TCGTGCGGCTGTTTGCACCCGTGGGGTTGCGAAGGCGGTGG-3' Primer 1901
(antisense) (SEQ ID NO 6) 5'-TTTTATCAGACCGCTTCTGCG-3'
[0101] In the case of the C-terminally fused NS-3, PCR was carried
out with a short sense primer homologous to sequences 5' of the
NS-3 19b startcodon and a long antisense primer carrying the CTL
epitope followed by in-frame stopcodons. Primer sequences are
depicted below. TABLE-US-00002 Primer 1052 (sense) (SEQ ID NO 7)
5'-AGCAAACCACCAAGTGGA-3' Primer 9039 (antisense) (SEQ ID NO 8)
5'-CTCTAGACTATTAACCGTGGTAAACGGTCCAGCAAACACCGTTGATG
CAGGTCGCCAGGCTGAAGTCGACTGTCTGG-3'
[0102] Starting from the coding sequences cloned into the pGEM-T
vectors, the NS-3 19bT sequences are inserted into the pIGRI2 E.
coli expression vector. For the N-terminally fused NS-3 19bT, the
NS-3 19bT coding sequence was isolated as a 379 bp Ncol/SnaBI
fragment and ligated with SnaBI/AllwNI and AlwNI/NcoI fragments
from vector pIGRI2NS-3, resulting in the vector pIGRI2NS-3Tn. For
the C-terminally fused NS-3 19bT, the NS-3 19bT coding sequence was
isolated as a 585 bp SnaBI/Spel fragment and inserted into the
SnaBI/Spel opened vector pIGRI2NS-3, resulting in the vector
pIGRI2NS-3Tc.
[0103] Both pIGRI2NS-3Tn and pIGRI2NS-3Tc vectors were subsequently
transformed to the E.coli expession strain MC1061(pAcI) and after
temperature induction of the lambda P.sub.L promotor, expression
levels were analysed on SDS-PAGE and Western blot, using a
polyclonal rabbit anti NS-3 serum.
[0104] Amino Acid Sequence of the NS-3 19bTn Protein TABLE-US-00003
(SEQ ID NO 1) MATCINGVCWTVYHGRAAVCTRGVAKAVDFVPVESMETTMRSPVFTDNSS
PPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFG
AYMSKAHGVDPNIRTGVRTITTGAPITYSTYGKFLADGGCSGGAYDIIIC
DECHSIDSTSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEV
ALSSTGEIPFYGKAIPIEVIKGGRHLIFCHSKKKCDELAAKLSGFGINAV
AYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFS
[0105] Amino Acid Sequence of the NS-3 19bTc Protein TABLE-US-00004
(SEQ ID NO 2) MGVAKAVDFVPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGK
STKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTIT
TGAPITYSTYGKFLADGGCSGGAYDIIICDECHSIDSTSILGIGTVLDQA
ETAGARLVVLATATPPGSVTVPHPNIEEVALSSTGEIPFYGKAIPIEVIK
GGRHLIFCHSKKKCDELAAKLSGFGINAVAYYRGLDVSVIPTSGDVVVVA
TDALMTGFTGDFDSVIDCNTCVTQTVDFSLATCINGVCWTVYHG
[0106] Nucleotide Sequence of the NS-3 19bTn Coding Region
TABLE-US-00005 (SEQ ID NO 3)
ATGGCGACCTGCATCAACGGTGTTTGCTGGACCGTTTACCACGGTCGTGC
GGCTGTTTGCACCCGTGGGGTTGCGAAGGCGGTGGACTTTGTACCCGTAG
AGTCTATGGAAACCACCATGCGGTCCCCGGTCTTTACGGATAACTCATCT
CCTCCGGCCGTACCGCAGACATTCCAAGTGGCCCATCTACACGCCCCCAC
TGGTAGTGGCAAGAGCACTAAGGTGCCGGCTGCATATGCAGCCCAAGGGT
ACAAGGTACTTGTCCTGAACCCATCCGTTGCCGCCACCTTAGGATTCGGG
GCGTATATGTCTAAAGCACATGGTGTCGACCCTAACATTAGAACTGGGGT
AAGGACCATCACCACGGGCGCCCCCATTACGTACTCCACCTACGGCAAGT
TTCTTGCCGACGGTGGTTGCTCTGGGGGCGCTTACGACATCATAATATGT
GATGAGTGCCACTCGATTGACTCAACCTCCATCTTGGGCATCGGCACCGT
CCTGGATCAGGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACTG
CTACACCTCCGGGGTCGGTCACCGTGCCACATCCCAACATCGAGGAGGTG
GCTCTGTCCAGCACTGGAGAGATCCCCTTTTATGGCAAAGCCATCCCCAT
CGAGGTCATCAAAGGGGGGAGGCACCTCATTTTCTGCCATTCCAAGAAGA
AATGTGACGAGCTCGCCGCAAAGCTATCGGGCTTCGGAATCAACGCTGTA
GCGTATTACCGAGGCCTTGATGTGTCCGTCATACCGACTAGCGGAGACGT
CGTTGTTGTGGCAACAGACGCTCTAATGACGGGCTTTACCGGCGACTTTG
ACTCAGTGATCGACTGTAACACATGCGTCACCCAGACAGTCGACTT CAGCTAA
[0107] Nucleotide Sequence of the NS-3 19bTc Coding Region
TABLE-US-00006 (SEQ ID NO 4)
ATGGGGGTTGCGAAGGCGGTGGACTTTGTACCCGTAGAGTCTATGGAAAC
CACCATGCGGTCCCCGGTCTTTACGGATAACTCATCTCCTCCGGCCGTAC
CGCAGACATTCCAAGTGGCCCATCTACACGCCCCCACTGGTAGTGGCAAG
AGCACTAAGGTGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTACTTGT
CCTGAACCCATCCGTTGCCGCCACCTTAGGATTCGGGGCGTATATGTCTA
AAGCACATGGTGTCGACCCTAACATTAGAACTGGGGTAAGGACCATCACC
ACGGGCGCCCCCATTACGTACTCCACCTACGGCAAGTTTCTTGCCGACGG
TGGTTGCTCTGGGGGCGCTTACGACATCATAATATGTGATGAGTGCCACT
CGATTGACTCAACCTCCATCTTGGGCATCGGCACCGTCCTGGATCAGGCG
GAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACTGCTACACCTCCGGG
GTCGGTCACCGTGCCACATCCCAACATCGAGGAGGTGGCTCTGTCCAGCA
CTGGAGAGATCCCCTTTTATGGCAAAGCCATCCCCATCGAGGTCATCAAA
GGGGGGAGGCACCTCATTTTCTGCCATTCCAAGAAGAAATGTGACGAGCT
CGCCGCAAAGCTATCGGGCTTCGGAATCAACGCTGTAGCGTATTACCGAG
GCCTTGATGTGTCCGTCATACCGACTAGCGGAGACGTCGTTGTTGTGGCA
ACAGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACTCAGTGATCGA
CTGTAACACATGCGTCACCCAGACAGTCGACTTCAGCCTGGCGACCTGCA
TCAACGGTGTTTGCTGGACCGTTTACCACGGTTAA
Example 7b
Purification of the NS-3 19bTn and NS-3 19bTc Proteins
[0108] E. coli cell pasts from erlenmeyer cultures were broken by a
cell disrupter (CSL, model B) at 1.4 kbar in 50 mM TRIS, pH 8. This
lysate was cleared by centrifugation (15000 g, 30 min, 4.degree.
C.). The supernatant was discarded, since both for the N-- and the
C-terminal construct NS3 was recovered in the pellet. This pellet
turned out to be highly stable for the N-terminal construct
allowing thorough washing (first wash with 2% sarcosyl, 0.5 M
guanidiniumchloride and 10 mM DTT, second and third wash with 1%
Triton X-100, 0.5 M guanidiniumchloride and 10 mM EDTA) before
solubilisation. This was not the case for the C-terminal construct.
Purification was further pursued on the N-terminal construct. The
washed pellet was finally dissolved in 6 M guanidiniumchloride/50
mM Na.sub.2HPO.sub.4, at pH 7.2 and was sulfonated as described in
Maertens et al. (PCT/EP99/02547). The sulfonated pellet was first
desalted on a Sephadex G25 column to 6 M Urea/50 mM
triethanolamine, pH 7.5, and finally purified by two sequential
anion-exchange chromatographies in the same buffer composition. The
first anion-exchange was performed on a Hyper DQ (50 .mu.m) column
(BioSpra Inc. Marlborough, Mass. USA) and the NS3 was recovered
between 0.11 and 0.19 M NaCl. After dilution, these fractions were
applied to a second Hyper DQ (20 .mu.m) column (BioSpra Inc.
Marlborough, Mass. USA) and the NS3 was recovered in the fractions
containing 0.125 M NaCl. These fractions were desalted to 6 M Urea
in PBS, pH 7.5. The final purity was estimated >90% based on
SDS-PAGE followed by silver staining. The N-terminal sequencing by
EDMAN degradation showed that this NS3 has an intact N-terminus, in
which the desired epitope is present in the correct sequence. It
was also confirmed that the methionine used for the start of
translation was cleaved of as predicted.
Example 8
Construction of an E2 Protein without Hypervariable Region I
[0109] An immunodominant homologous response has been noted to the
HVR I region of E2. This response will be of little use in a
vaccine approach, since a vaccine approach is a heterologous set-up
(the vaccine strain is always different from the field strains).
Therefore, deletion of this region would be necessary to have an E2
protein inducing antibodies against the more conserved, but less
immunogenic regions of E2. By carefully analyzing the E2 leader
sequence and the E2 hypervariable region the most ideal construct
for expression of an E2 protein without HVR I was designed. This
construct allows expression of an E2 peptide starting at position
aa 409 instead of aa 384. As a leader sequence the C-terminal 20
amino acids of E1 were used. However, since the delineation of this
HVR is not unambiguous, a second version was made (starting at aa
412), which has also a high probability to be cleaved at the right
position.
[0110] Intermediate Construct pvHCV-99 (see also FIGS. 15 and
16)
[0111] In the expression cassette, the coding sequence of E2-715
should be preceded by an E1 leader signal peptide, starting at
Met364. Therefore, in plasmid pvHCV-92 (FIG. 15), which contains
the coding sequence for E2-715 HCV type 1b with the long version of
the E1 signal peptide (starting at Met347), a deletion was made by
a double-digestion with EcoRI and NcoI, followed by a 5'-overhang
fill-in reaction with T4 DNA polymerase. Ligation of the obtained
blunt ends (recircularization of the 6621 bp-fragment), resulted in
plasmid pvHCV-99, which codes for the same protein (E2-715) with a
shorter E1 leader signal peptide (starting at Met364). pvHCV-99 was
deposited in the strain list as ICCG 3635. It should be clear that
HCV or heterologous signal sequences of variable length may be
used.
[0112] The plasmids pvHCV-100 and -101 should contain a deletion in
the E2 sequence, i.e. a deletion of the hypervariable region I
(HVR-I). In plasmid pvHCV-100, amino acids 384(His)-408(Ala) were
deleted, while in plasmid pvHCV-101 aminoacids 384(His)-411(Ile)
were deleted.
[0113] Construction pvHCV-100
[0114] For the construction of pvHCV-100, two oligonucleotides were
designed: TABLE-US-00007 HCV-pr 409 [8749]: (SEQ ID NO 9) 5'-CTT
TGC CGG CGT CGA CGG GCA GAA AAT CCA GCT CGT AA-3' HCV-pr 408
[8750]: (SEQ ID NO 10) 5'-TTA CGA GCT GGA TTT TCT GCC CGT CGA CGC
CGG CAA AG-3'
[0115] PCR amplification (denaturation 5 min 95.degree. C., 30
cycles of amplification consisting of annealing at 55.degree. C.,
polymerization at 72.degree. C., and denaturation at 95.degree. C.
for 1 min each, elongation for 10 min at 72.degree. C.) of the
pvHCV-99 template with Gpt-pr [3757] and HCV-pr 408 [8750] resulted
in a 221 bp fragment, while amplification with HCV-pr 409 [87491]
and TKr-pr [3756] resulted in a 1006 bp fragment. Both PCR
fragments overlap one another by 19 nucleotides. These two
fragments were assembled and amplified by PCR with the Gpt-pr
[3757] and TKr-pr [3756] primers. The resulting 1200 bp fragment
was digested with EcoRI and HinDIII and ligated into the
EcoRI/HinDIII digested pgsATA18 [ICCG 1998] vector (5558 bp). This
construct, pvHCV-100, was analysed by restriction and sequence
analysis, and deposited in the strainlist as ICCG 3636.
[0116] Construction pvHCV-101
[0117] For the construction of pvHCV-101, two oligonucleotides were
designed: TABLE-US-00008 HCV-pr 411 [8747]: (SEQ ID NO 11) 5'-CTT
TGC CGG CGT CGA CGG GCA GCT CGT AAA CAC CAA CG-3' HCV-pr 410
[8748]: (SEQ ID NO 12) 5'-CGT TGG TGT TTA CGA GCT GCC CGT CGA CGC
CGG CAA AG-3'
[0118] PCR amplification of the pvHCV-99 template with Gpt-pr
[3757] and HCV-pr 410 [8748] resulted in a 221 bp fragment, while
amplification with HCV-pr 411 [8747] and TKr-pr [3756] resulted in
a 997 bp fragment. Both PCR fragments overlap one another by 19
nucleotides. These two fragments were assembled and amplified by
PCR with the Gpt-pr [3757] and TKr-pr [3756]. The resulting 1200 bp
fragment was digested with EcoRI and HinDIII and ligated into the
EcoRI/HinDIII digested pgsATA18 [ICCG 1998] vector (5558 bp). This
construct, pvHCV-101, was analysed by restriction and sequence
analysis, and deposited in the strainlist as ICCG 3637.
[0119] All plasmids were checked by sequence analysis and deposited
in the Innogenetics strainlist. For each plasmid two mini-DNA
preparations (PLASmix) were made under sterile conditions and
pooled. DNA concentration was determined and QA was performed by
restriction analysis. Purified DNA was used to generate recombinant
vaccinia virus as described in Maertens et al. (PCT/EP95/03031).
The recombinant viruses vvHCV-100 and vvHCV-101 were, however,
generated on WHO certified Vero cells. After two rounds of plaque
purification the expression product was analysed by means of
Western-blot analysis as described in Maertens et al.
(PCT/EP95/03031). Proteins were visualised by a specific anti-E2
monoclonal antibody (IGH 212, which can be obtained from the
inventors at Innogenetics N. V., Zwijnaarde, Belgium) of an
estimated molecular weight of 69 and 37 kDa for vvHCV-100 and of 68
and 35 kDa for vvHCV-101. These molecular weights indicate the
presence of both a glycosylated and non-glycosylated E2-protein,
which was confirmed by treatment of the samples prior to
Western-blot analysis with PNGaseF. This treatment results in the
detection of only one single protein of 37 kDa and 35 kDa for
vvHCV-100 and vvHCV-101, respectively.
[0120] Amino Acid Sequence of the Mature E2, Derived from pvHCV-100
TABLE-US-00009 (SEQ ID NO 13)
QKIQLVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKFNSSGCPERLAS
CRSIDKFAQGWGPLTYTEPNSSDQRPYCWHYAPRPCGIVPASQVCGPVYC
FTPSPVVVGTTDRFGVPTYNWGANDSDVLILNNTRPPRGNWFGCTWMNGT
GFTKTCGGPPCNIGGAGNNTLTCPTDCFRKHPEATYARCGSGPWLTPRCM
VHYPYRLWHYPCTVNFTIFKVRMYVGGVEHRFEAACNWTRGERCDLEDRD
RSELSPLLLSTTEWQILPCSFTTLPALSTGLIHLHQNIVDVQYLYGVGSA VVSLVIK
[0121] Amino Acid Sequence of the Mature E2 Derived from pvHCV-101
TABLE-US-00010 (SEQ ID NO 14)
QLVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKFNSSGCPERLASCRS
IDKFAQGWGPLTYTEPNSSDQRPYCWHYAPRPCGIVPASQVCGPVYCFTP
SPVVVGTTDRFGVPTYNWGANDSDVLILNNTRPPRGNWFGCTWMNGTGFT
KTCGGPPCNIGGAGNNTLTCPTDCFRKPEATYARCGSGPWLTPRCMVHYP
YRLWHYPCTVNFTIFKVRMYVGGVEHRFEAACNWTRGERCDLEDRDRSEL
SPLLLSTTEWQILPCSFTTLPALSTGLIHLHQNIVDVQYLYGVGSA VVSLVIK
Example 9
An E1 Particle with Further Improved Immunogenicity
[0122] As set out in example 1, the E1s protein was purified
according to the protocol described in PCT/EP 95/03031 to Maertens
et al. This protocol includes covalent modification of cysteines
(free cysteines and cysteines involved in intermolecular bridging,
the latter after reduction of these cysteine bridges using DTT)
using maleimide derivates (N-ethyl maleimide and biotin-maleimide,
both obtained from Sigma). As an alternative method for maleimide
blocking, active halogens were also evaluated. These compounds, ie
the active halogens, block free cysteines by means of alkylation.
By way of example, an active halogen (iodoacetamide, Merck) was
evaluated. The same protocol was used to purify E1 as described in
Maertens et al. (PCT/EP 95/03031), but instead of maleimide
compounds, iodoacetemide was used. The E1s protein obtained by this
procedure behaved throughout the complete purification procedure
similarly as the maleimide-blocked proteins. Upon final lowering of
the detergent concentration to 0.05% CHAPS or switching to 0.5%
betaine as described in example 1, similar particles were obtained
as determined by DLS. The surprising effect was found, however,
upon immunization of mice with this acetamide-modified E1s.
[0123] In total three series of 6 mice each were immunized with E1s
using three injections with a three week interval, each injection
consisting of 5 .mu.g E1s at 100 .mu.g/ml PBS and mixed with an
equal volume of RIBI adjuvant (R-700). A first series received
E1-maleimide formulated in 0.05% CHAPS, a second series received
E1-acetamide also formulated in 0.05% CHAPS, while a third series
received E1-acetamide formulated with 0.12% betaine. Finally, all
mice were bled 10 days after the third immunization. End point
titers (defined as the dilution of the serum still resulting in a
OD 2 times higher then background values) for each animal
individually were determined against E1-maleimide and E1-acetamide.
FIG. 17 shows these end-point titers, presented as mean with
standard deviations. Mice that received E1-maleimide mounted only
an antibody response which is able to recognize
maleimide-containing epitopes (no reactivity at all on
E1-acetamide), mice that received E1-acetamide clearly mount an
antibody response against true E1 epitopes, since the antibodies
are reactive against both E1-acetamide and E1-maleimide. This was
clearly demonstrated in an additional experiment, in which
antibodies for specific regions of E1 were determined using
peptides which were neither modified with acetamide nor with
maleimide. The results, as shown in FIG. 18, demonstrate that the
mice immunized with E1-acetamide (CHAPS and betaine formulated) do
mount an antibody response which is able to recognize the peptides
V1V2, V2V3, V3V4, V5C4, C4V6. As V6 was not part of E1s, we can
conclude that antibodies were mounted against C4, V3 (V3V4 is
positive while V4V5 is not) and V1V2. Mice immunized with
E1s-maleimide mount only a very low response against the V1V2 and
V2V3 peptides. This stresses once more the fact that the reasonably
high titer measured for these mouse antibodies against the
maleimide-E1s is mainly directed against maleimide-dependent
epitopes. In addition, we were able to prove that the E1s-acetamide
induced response is partially of the Th1 type, since a substantial
amount of the induced antibodies is of the IgG2(a+b) subtype. The
amount of IgG2 is even higher for the betaine formulation compared
to the CHAPS formulation (FIG. 19). From these results it is
concluded that HCV envelope proteins, in which at least one
cysteine (but potentially more than one cysteine) is alkylated, are
extremely immunogenic proteins.
[0124] Consequently, the acetamide-modified E1 formulated in
betaine was also used to reboost the chimpanzees Phil and Ton. Both
chimpanzees were immunized again with two consecutive intramuscular
immunisations with a three week time interval (50 .mu.g E1 mixed
with RIBI adjuvant as for the examples 4 and 5). As can be judged
from FIGS. 20 and 21, the anti-E1 response could indeed be boosted
again, and this to higher levels than obtained in the previous
immunisations after two injections. This titration was performed
against a standard, which is a mixture of three human high titre
anti-E1 sera (obtained from chronic HCV carriers). The anti-E1
titer of these sera was defined as one unit/ml. In chimpanzee Phil
(FIG. 20), titers twice as high as in human carriers were induced
only after two immunizations. In chimpanzee Ton (FIG. 21), titers
up to 140-fold higher were induced. This stresses once more the
high immunogenecity of these E1-particles.
Example 10
Alkylated E1 has Superior Qualities for Diagnostic Use
[0125] The E1s-acetamide as described in example 9 was further
evaluated as antigen for the detection of anti-E1 antibodies in
serum samples from human chronic HCV carriers. By way of example
these antigens were bound to LIA-membranes, and strips were
processed essentially as described in Zrein et al. (1998). Serum
samples from 72 blood donors were evaluated first, in order to
determine the optimal concentration of the E1 antigen which can be
used in the assay in order to exclude "false" positives. For
E1s-maleimide, this concentration proved to be 8 .mu.g/ml, while
for E1s-acetamide a concentration up to 50 .mu.g/ml did not result
in false positive results (no samples showing a relative color
staining above 0.5). Using 8 and 50 .mu.g/ml, respectively, for
E1s-maleimide and E1s-acetamide 24 sera of HCV chronic carriers
were screened for antibodies against E1s. As shown in Table 6, the
E1s-acetamide clearly results in more samples scoring positive (67%
versus 38% for E1s-maleimide). No sample was found which only
scored positive on E1s-maleimide. For samples scoring positive both
on E1s-maleimide and on E1s-acetamide, the reactivity on the latter
is higher. From this example it can be concluded that alkylated
envelope proteins of HCV are better antigens to detect human
antibodies than maleimide-modified envelope proteins.
Example 11
Production of Mixed Particles Containing E1 and E2
[0126] E1s and E2s (vvHCV-44) were produced and purified as
described in Maertens et al. PCT/EP95/030301 except for the fact
the maleimide-modificiation was replaced by alkylation using
iodoacetamide. E1s and E2s in 3% empigen alone or as an equimolar
mixture were injected on a Superdex-200 PC 3.2/30 column
equilibrated in PBS/0.2% CHAPS. This column is designed to use with
the SMART.TM. HPLC equipment from Pharmacia LKB (Sweden). The
fractions were screened by means of three different sandwich
ELISAs. For these ELISAs, E1-(IGH 207) and E2-(IGH 223) specific
monoclonals were coated at 2 .mu.g/ml. Fractions of the gel
filtration were incubated in a 1/2500 dilution. Two other E1 (IGH
200) and E2 (IGH 212) monoclonals, conjugated with biotin were used
for detection of the bound antigen. The streptavidin-HRP/TMB system
was used to develop the bound biotin into a yellow color which was
measured at 450 nm.
[0127] This ELISA system was used in a homologous (anti-E1
coating/anti-E1 detection or anti-E2 coating/anti-E2 detection) and
a heterologous set-up (anti-E1 coating/anti-E2 detection). The
latter theoretically only detects particles in which both E1 and E2
are incorporated. The reactive fractions were pooled, concentrated
on a 10 kDa filter, and again chromatographed on Superdex-200 in
PBS/0.05% CHAPS. All these fractions were tested for reactivity by
using the different ELISA set-ups. As can be judged from FIG. 22,
the addition of E2 to E1 does not result in a major shift in the
retention time, compared to E1 alone, indicating that particles are
indeed still present. These particles contain both E1 and E2, since
only in this set-up the heterologous ELISA scores positive.
Example 12
Production of Mixed Particles Containing E1 from 2 Different
Genotypes
[0128] E1s of genotype 1b and of genotype 4 (vvHCV-72) were
produced and purified as described in Maertens et al.,
PCT/EP95/030301 except for the fact the maleimide-modificiation was
replaced by alkylation using iodoacetamide for the genotype 1b.
E1s-1b and E1s-4 in 3% empigen alone or as an equimolar mixture
were injected on a Superdex-200 PC 3.2/30 column equilibrated in
PBS/0.2% CHAPS. This column is designed to use with the SMART.TM.
HPLC equipment from Pharmacia LKB (Sweden). The major protein
containing fractions were pooled, concentrated on a 10 kDa filter,
and again chromatographed on Superdex-200 in PBS/0.05% CHAPS. All
these fractions were tested for reactivity by using an ELISA set-up
which should only detect particles containing E1 from both
genotypes. For this ELISA streptavidin was coated at 2 .mu.g/ml.
Fractions of the gel filtration were incubated in a 1/2500
dilution. An E1 monoclonal antibody (IGH 200) which only recognizes
E1 from genotype 1 and 10 was used for detection of the bound
antigen. The goat-anti-mouse-HRP/TMB system was used for
development of the assay into a yellow color which was measured at
450 nm. As can be judged from FIG. 23, the addition of E1-4 to
E1-1b does not result in a major shift in the retention time of the
proteins, indicating that particles are indeed still present. These
particles contain both E1 proteins, ie E1s of genotype 1b and
genotype 4, since only in this set-up the ELISA scores
positive.
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TABLE-US-00011 TABLE 1 The E1s consensus sequence of HCV-B AA
Position* 200 233 235 251 253 271 293 298 304 313 314 322 Region V1
V3 V3 V4 V4 HR HR V5 C4 C4 C4 C4 HCV-J I S F S I L F Y -- V S --
HCV-B con M N S A V F I H C I T M HCCl9A -- -- -- -- -- L -- -- --
-- -- -- HCCl9B -- D -- -- -- -- -- -- Y -- -- -- HCCl9C -- -- --
-- -- -- -- -- -- -- -- T HCCl10A -- -- -- -- -- -- -- -- -- -- --
-- HCCl10B -- D -- -- I -- -- -- -- -- -- -- HCCl11A -- -- -- -- --
-- -- -- -- -- -- -- HCCl11B -- -- -- -- -- -- -- -- -- -- -- --
HCCl14 -- -- -- -- -- -- -- -- -- -- -- -- HCCl17 -- -- -- -- I --
-- -- -- -- -- -- *Positions between aa 192 and 326 of E1s which
are completely conserved are not indicated
[0147] TABLE-US-00012 TABLE 2 The E1s vaccine sequence aligned with
the HCV sequence of the virus present in the chronic carriers 192
259 Ton (1a)
YQVRNSTGLYHVTNDCPNSSIVYEAADAILHTPGCVPCVREGNASRCWVAMTPTVATRDGKLPTT-
QLR (SEQ ID NO 15) * ** * * * * * * * * * **** ** E1 vaccin
YEVRNVSGMYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRENNSSRCWVALTPTLAARNASVPT-
TTIR (SEQ ID NO 16) * * * * Phi1 (1b)
YEVRNVSGVYHVTNDCSNASIVYEAADMIMHTPGCVPCVREGNSSRCWVALTPTLAARNVSVPT-
TTIR (SEQ ID NO 17) 260 326 Ton (1a)
RHIDLLVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTTQECNCSMYPGHITGHRMAWDMMM-
NW * * ** * * * * * * E1 vaccin
RHVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPRRHETVQDCNCSIYPGHITGHRMAWDMM-
MNW * * ** Phi1 (1b)
RHVDLIVGAAAFCSAMYVGDLCGSVFLVSQLFTFSPRRHETVQDCNCSIYPGHVSGHRMAWDMM-
MNW
[0148] TABLE-US-00013 TABLE 3 Changes induced by therapeutic
vaccination (6 .times. 50 .mu.g E1s) Ton (subtype 1a) Phil (subtype
1b) Before After Before After Serum E1Ab titer 0 30000 0 14500 HCV
RNA titer (10.sup.5) 2-3 no change 2-4 no change ALT (IU) 85 .+-.
11 62 .+-. 6 44 .+-. 4 37 .+-. 6 Liver Antigen staining strongly
positive negative strongly positive negative Histology CAH CPH CAH
CPH Portal inflammation light none severe moderate Lobular
hepatitis moderate minimal severe moderate Interface hepatitis + -
+ - Histological activity 4 1-2 6-8 2-3
[0149] TABLE-US-00014 TABLE 4 E1 peptides SEQ ID NO Genotype name #
aa YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGC 18 1b V1V2 888 192-226
IVYEAADMIMHTPGCVPCVRENNSSRCWV 19 1b V2V3 1036 212-244
VRENNSSRCWVALTPTLAARNASVPTTTIRRHVD 20 1b V3V4 1022 230-263
HVDLLVGAAAFCSAMYVGDLCGSVFLVSQL 21 1b HR 1150 261-290
SQLFTISPRRHETVQDCNCSIYPGHITGHRMAWDMMMNWS 22 1b V5C4 1176 288-327
SIYPGHITGHRMAWDMMMNWSPTTALVVSQLLRI 23 1b C4V6 1039 307-340
[0150] TABLE-US-00015 TABLE 5 aa 1188 * (SEQ ID NO 24)
MATCINGVCWTVYHGRAAVCTRGVAK . . . proposed sequence (SEQ ID NO 25)
GGPLLCPAGHAVGIFRAAVCTRGVAK . . . natural sequence double
underlined: minimal CTL epitope single underlined: additional
surrounding natural amino acids At the C-terminus the epitope and
its surroundings may be linked directly. VDFSLATCINGVCWTVYHG
Proposed se- (SEQ ID NO 26) quence VDFSLDPTFTIETITLPQD Natural
sequence (SEQ ID NO 27) * aa 1468
[0151] TABLE-US-00016 TABLE 6 antigen E1s-maleimide E1s-acetamide
.mu.g/ml 8 50 17758 2 4 17761 0 0.5 17763 0 0.5 17766 0 1 17767 0 1
17771 0.5 2 17773 0 0 17775 0 0.5 17776 0 0.5 17777 0.5 2 17779 0 0
17784 3 4 17785 0.5 2 17786 0 2 17789 2 4 17790 2 4 17794 2 4 17795
0 1 17796 0 0 17798 0 0.5 17820 2 4 17825 2 3 17827 2 4 17842 1 2
#pos 9 16 % pos 38 67
[0152]
Sequence CWU 1
1
30 1 300 PRT Hepatitis C virus 1 Met Ala Thr Cys Ile Asn Gly Val
Cys Trp Thr Val Tyr His Gly Arg 1 5 10 15 Ala Ala Val Cys Thr Arg
Gly Val Ala Lys Ala Val Asp Phe Val Pro 20 25 30 Val Glu Ser Met
Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn 35 40 45 Ser Ser
Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala His Leu His 50 55 60
Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala 65
70 75 80 Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val Ala
Ala Thr 85 90 95 Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly
Val Asp Pro Asn 100 105 110 Ile Arg Thr Gly Val Arg Thr Ile Thr Thr
Gly Ala Pro Ile Thr Tyr 115 120 125 Ser Thr Tyr Gly Lys Phe Leu Ala
Asp Gly Gly Cys Ser Gly Gly Ala 130 135 140 Tyr Asp Ile Ile Ile Cys
Asp Glu Cys His Ser Ile Asp Ser Thr Ser 145 150 155 160 Ile Leu Gly
Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala 165 170 175 Arg
Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val 180 185
190 Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Ser Thr Gly Glu Ile
195 200 205 Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Val Ile Lys Gly
Gly Arg 210 215 220 His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp
Glu Leu Ala Ala 225 230 235 240 Lys Leu Ser Gly Phe Gly Ile Asn Ala
Val Ala Tyr Tyr Arg Gly Leu 245 250 255 Asp Val Ser Val Ile Pro Thr
Ser Gly Asp Val Val Val Val Ala Thr 260 265 270 Asp Ala Leu Met Thr
Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp 275 280 285 Cys Asn Thr
Cys Val Thr Gln Thr Val Asp Phe Ser 290 295 300 2 294 PRT Hepatitis
C virus 2 Met Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser
Met Glu 1 5 10 15 Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser
Ser Pro Pro Ala 20 25 30 Val Pro Gln Thr Phe Gln Val Ala His Leu
His Ala Pro Thr Gly Ser 35 40 45 Gly Lys Ser Thr Lys Val Pro Ala
Ala Tyr Ala Ala Gln Gly Tyr Lys 50 55 60 Val Leu Val Leu Asn Pro
Ser Val Ala Ala Thr Leu Gly Phe Gly Ala 65 70 75 80 Tyr Met Ser Lys
Ala His Gly Val Asp Pro Asn Ile Arg Thr Gly Val 85 90 95 Arg Thr
Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr Gly Lys 100 105 110
Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile 115
120 125 Cys Asp Glu Cys His Ser Ile Asp Ser Thr Ser Ile Leu Gly Ile
Gly 130 135 140 Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu
Val Val Leu 145 150 155 160 Ala Thr Ala Thr Pro Pro Gly Ser Val Thr
Val Pro His Pro Asn Ile 165 170 175 Glu Glu Val Ala Leu Ser Ser Thr
Gly Glu Ile Pro Phe Tyr Gly Lys 180 185 190 Ala Ile Pro Ile Glu Val
Ile Lys Gly Gly Arg His Leu Ile Phe Cys 195 200 205 His Ser Lys Lys
Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser Gly Phe 210 215 220 Gly Ile
Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile 225 230 235
240 Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr
245 250 255 Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr
Cys Val 260 265 270 Thr Gln Thr Val Asp Phe Ser Leu Ala Thr Cys Ile
Asn Gly Val Cys 275 280 285 Trp Thr Val Tyr His Gly 290 3 903 DNA
Hepatitis C virus 3 atggcgacct gcatcaacgg tgtttgctgg accgtttacc
acggtcgtgc ggctgtttgc 60 acccgtgggg ttgcgaaggc ggtggacttt
gtacccgtag agtctatgga aaccaccatg 120 cggtccccgg tctttacgga
taactcatct cctccggccg taccgcagac attccaagtg 180 gcccatctac
acgcccccac tggtagtggc aagagcacta aggtgccggc tgcatatgca 240
gcccaagggt acaaggtact tgtcctgaac ccatccgttg ccgccacctt aggattcggg
300 gcgtatatgt ctaaagcaca tggtgtcgac cctaacatta gaactggggt
aaggaccatc 360 accacgggcg cccccattac gtactccacc tacggcaagt
ttcttgccga cggtggttgc 420 tctgggggcg cttacgacat cataatatgt
gatgagtgcc actcgattga ctcaacctcc 480 atcttgggca tcggcaccgt
cctggatcag gcggagacgg ctggagcgcg gcttgtcgtg 540 ctcgccactg
ctacacctcc ggggtcggtc accgtgccac atcccaacat cgaggaggtg 600
gctctgtcca gcactggaga gatccccttt tatggcaaag ccatccccat cgaggtcatc
660 aaagggggga ggcacctcat tttctgccat tccaagaaga aatgtgacga
gctcgccgca 720 aagctatcgg gcttcggaat caacgctgta gcgtattacc
gaggccttga tgtgtccgtc 780 ataccgacta gcggagacgt cgttgttgtg
gcaacagacg ctctaatgac gggctttacc 840 ggcgactttg actcagtgat
cgactgtaac acatgcgtca cccagacagt cgacttcagc 900 taa 903 4 885 DNA
Hepatitis C virus 4 atgggggttg cgaaggcggt ggactttgta cccgtagagt
ctatggaaac caccatgcgg 60 tccccggtct ttacggataa ctcatctcct
ccggccgtac cgcagacatt ccaagtggcc 120 catctacacg cccccactgg
tagtggcaag agcactaagg tgccggctgc atatgcagcc 180 caagggtaca
aggtacttgt cctgaaccca tccgttgccg ccaccttagg attcggggcg 240
tatatgtcta aagcacatgg tgtcgaccct aacattagaa ctggggtaag gaccatcacc
300 acgggcgccc ccattacgta ctccacctac ggcaagtttc ttgccgacgg
tggttgctct 360 gggggcgctt acgacatcat aatatgtgat gagtgccact
cgattgactc aacctccatc 420 ttgggcatcg gcaccgtcct ggatcaggcg
gagacggctg gagcgcggct tgtcgtgctc 480 gccactgcta cacctccggg
gtcggtcacc gtgccacatc ccaacatcga ggaggtggct 540 ctgtccagca
ctggagagat ccccttttat ggcaaagcca tccccatcga ggtcatcaaa 600
ggggggaggc acctcatttt ctgccattcc aagaagaaat gtgacgagct cgccgcaaag
660 ctatcgggct tcggaatcaa cgctgtagcg tattaccgag gccttgatgt
gtccgtcata 720 ccgactagcg gagacgtcgt tgttgtggca acagacgctc
taatgacggg ctttaccggc 780 gactttgact cagtgatcga ctgtaacaca
tgcgtcaccc agacagtcga cttcagcctg 840 gcgacctgca tcaacggtgt
ttgctggacc gtttaccacg gttaa 885 5 88 DNA Hepatitis C virus 5
gccatggcga cctgcatcaa cggtgtttgc tggaccgttt accacggtcg tgcggctgtt
60 tgcacccgtg gggttgcgaa ggcggtgg 88 6 21 DNA Hepatitis C virus 6
ttttatcaga ccgcttctgc g 21 7 18 DNA Hepatitis C virus 7 agcaaaccac
caagtgga 18 8 77 DNA Hepatitis C virus 8 ctctagacta ttaaccgtgg
taaacggtcc agcaaacacc gttgatgcag gtcgccaggc 60 tgaagtcgac tgtctgg
77 9 38 DNA Hepatitis C virus 9 ctttgccggc gtcgacgggc agaaaatcca
gctcgtaa 38 10 38 DNA Hepatitis C virus 10 ttacgagctg gattttctgc
ccgtcgacgc cggcaaag 38 11 38 DNA Hepatitis C virus 11 ctttgccggc
gtcgacgggc agctcgtaaa caccaacg 38 12 38 DNA Hepatitis C virus 12
cgttggtgtt tacgagctgc ccgtcgacgc cggcaaag 38 13 307 PRT Hepatitis C
virus 13 Gln Lys Ile Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile
Asn Arg 1 5 10 15 Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln Thr Gly
Phe Phe Ala Ala 20 25 30 Leu Phe Tyr Lys His Lys Phe Asn Ser Ser
Gly Cys Pro Glu Arg Leu 35 40 45 Ala Ser Cys Arg Ser Ile Asp Lys
Phe Ala Gln Gly Trp Gly Pro Leu 50 55 60 Thr Tyr Thr Glu Pro Asn
Ser Ser Asp Gln Arg Pro Tyr Cys Trp His 65 70 75 80 Tyr Ala Pro Arg
Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly 85 90 95 Pro Val
Tyr Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp 100 105 110
Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val 115
120 125 Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly
Cys 130 135 140 Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly
Gly Pro Pro 145 150 155 160 Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr
Leu Thr Cys Pro Thr Asp 165 170 175 Cys Phe Arg Lys His Pro Glu Ala
Thr Tyr Ala Arg Cys Gly Ser Gly 180 185 190 Pro Trp Leu Thr Pro Arg
Cys Met Val His Tyr Pro Tyr Arg Leu Trp 195 200 205 His Tyr Pro Cys
Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr 210 215 220 Val Gly
Gly Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg 225 230 235
240 Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro
245 250 255 Leu Leu Leu Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys Ser
Phe Thr 260 265 270 Thr Leu Pro Ala Leu Ser Thr Gly Leu Ile His Leu
His Gln Asn Ile 275 280 285 Val Asp Val Gln Tyr Leu Tyr Gly Val Gly
Ser Ala Val Val Ser Leu 290 295 300 Val Ile Lys 305 14 304 PRT
Hepatitis C virus 14 Gln Leu Val Asn Thr Asn Gly Ser Trp His Ile
Asn Arg Thr Ala Leu 1 5 10 15 Asn Cys Asn Asp Ser Leu Gln Thr Gly
Phe Phe Ala Ala Leu Phe Tyr 20 25 30 Lys His Lys Phe Asn Ser Ser
Gly Cys Pro Glu Arg Leu Ala Ser Cys 35 40 45 Arg Ser Ile Asp Lys
Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr Thr 50 55 60 Glu Pro Asn
Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro 65 70 75 80 Arg
Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val Tyr 85 90
95 Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly
100 105 110 Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu
Ile Leu 115 120 125 Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly
Cys Thr Trp Met 130 135 140 Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly
Gly Pro Pro Cys Asn Ile 145 150 155 160 Gly Gly Ala Gly Asn Asn Thr
Leu Thr Cys Pro Thr Asp Cys Phe Arg 165 170 175 Lys His Pro Glu Ala
Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu 180 185 190 Thr Pro Arg
Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro 195 200 205 Cys
Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly 210 215
220 Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg
225 230 235 240 Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro
Leu Leu Leu 245 250 255 Ser Thr Thr Glu Trp Gln Ile Leu Pro Cys Ser
Phe Thr Thr Leu Pro 260 265 270 Ala Leu Ser Thr Gly Leu Ile His Leu
His Gln Asn Ile Val Asp Val 275 280 285 Gln Tyr Leu Tyr Gly Val Gly
Ser Ala Val Val Ser Leu Val Ile Lys 290 295 300 15 135 PRT
Hepatitis C virus 15 Tyr Gln Val Arg Asn Ser Thr Gly Leu Tyr His
Val Thr Asn Asp Cys 1 5 10 15 Pro Asn Ser Ser Ile Val Tyr Glu Ala
Ala Asp Ala Ile Leu His Thr 20 25 30 Pro Gly Cys Val Pro Cys Val
Arg Glu Gly Asn Ala Ser Arg Cys Trp 35 40 45 Val Ala Met Thr Pro
Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Thr 50 55 60 Thr Gln Leu
Arg Arg His Ile Asp Leu Leu Val Gly Ser Ala Thr Leu 65 70 75 80 Cys
Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val 85 90
95 Gly Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Glu
100 105 110 Cys Asn Cys Ser Met Tyr Pro Gly His Ile Thr Gly His Arg
Met Ala 115 120 125 Trp Asp Met Met Met Asn Trp 130 135 16 135 PRT
Hepatitis C virus 16 Tyr Glu Val Arg Asn Val Ser Gly Met Tyr His
Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Tyr Glu Ala
Ala Asp Met Ile Met His Thr 20 25 30 Pro Gly Cys Val Pro Cys Val
Arg Glu Asn Asn Ser Ser Arg Cys Trp 35 40 45 Val Ala Leu Thr Pro
Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr 50 55 60 Thr Thr Ile
Arg Arg His Val Asp Leu Leu Val Gly Ala Ala Ala Phe 65 70 75 80 Cys
Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val 85 90
95 Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val Gln Asp
100 105 110 Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
Met Ala 115 120 125 Trp Asp Met Met Met Asn Trp 130 135 17 135 PRT
Hepatitis C virus 17 Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His
Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ala Ser Ile Val Tyr Glu Ala
Ala Asp Met Ile Met His Thr 20 25 30 Pro Gly Cys Val Pro Cys Val
Arg Glu Gly Asn Ser Ser Arg Cys Trp 35 40 45 Val Ala Leu Thr Pro
Thr Leu Ala Ala Arg Asn Val Ser Val Pro Thr 50 55 60 Thr Thr Ile
Arg Arg His Val Asp Leu Ile Val Gly Ala Ala Ala Phe 65 70 75 80 Cys
Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val 85 90
95 Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His Glu Thr Val Gln Asp
100 105 110 Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His Arg
Met Ala 115 120 125 Trp Asp Met Met Met Asn Trp 130 135 18 35 PRT
Hepatitis C virus 18 Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His
Val Thr Asn Asp Cys 1 5 10 15 Ser Asn Ser Ser Ile Val Tyr Glu Ala
Ala Asp Met Ile Met His Thr 20 25 30 Pro Gly Cys 35 19 29 PRT
Hepatitis C virus 19 Ile Val Tyr Glu Ala Ala Asp Met Ile Met His
Thr Pro Gly Cys Val 1 5 10 15 Pro Cys Val Arg Glu Asn Asn Ser Ser
Arg Cys Trp Val 20 25 20 34 PRT Hepatitis C virus 20 Val Arg Glu
Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 1 5 10 15 Leu
Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His 20 25
30 Val Asp 21 30 PRT Hepatitis C virus 21 His Val Asp Leu Leu Val
Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr 1 5 10 15 Val Gly Asp Leu
Cys Gly Ser Val Phe Leu Val Ser Gln Leu 20 25 30 22 40 PRT
Hepatitis C virus 22 Ser Gln Leu Phe Thr Ile Ser Pro Arg Arg His
Glu Thr Val Gln Asp 1 5 10 15 Cys Asn Cys Ser Ile Tyr Pro Gly His
Ile Thr Gly His Arg Met Ala 20 25 30 Trp Asp Met Met Met Asn Trp
Ser 35 40 23 34 PRT Hepatitis C virus 23 Ser Ile Tyr Pro Gly His
Ile Thr Gly His Arg Met Ala Trp Asp Met 1 5 10 15 Met Met Asn Trp
Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu 20 25 30 Arg Ile 24
26 PRT Hepatitis C virus 24 Met Ala Thr Cys Ile Asn Gly Val Cys Trp
Thr Val Tyr His Gly Arg 1 5 10 15 Ala Ala Val Cys Thr Arg Gly Val
Ala Lys 20 25 25 26 PRT Hepatitis C virus 25 Gly Gly Pro Leu Leu
Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg 1 5 10 15 Ala Ala Val
Cys Thr Arg Gly Val Ala Lys 20 25 26 19 PRT Hepatitis C virus 26
Val Asp Phe Ser Leu Ala Thr Cys Ile Asn Gly Val Cys Trp Thr Val 1 5
10 15 Tyr His Gly 27 19 PRT Hepatitis C virus 27 Val Asp Phe Ser
Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu 1 5 10 15 Pro Gln
Asp 28 6710 DNA Hepatitis C virus 28 agcttttgcg atcaataaat
ggatcacaac cagtatctct taacgatgtt cttcgcagat 60 gatgattcat
tttttaagta tttggctagt caagatgatg aatcttcatt atctgatata 120
ttgcaaatca ctcaatatct agactttctg ttattattat
tgatccaatc aaaaaataaa 180 ttagaagccg tgggtcattg ttatgaatct
ctttcagagg aatacagaca attgacaaaa 240 ttcacagact ttcaagattt
taaaaaactg tttaacaagg tccctattgt tacagatgga 300 agggtcaaac
ttaataaagg atatttgttc gactttgtga ttagtttgat gcgattcaaa 360
aaagaatcct ctctagctac caccgcaata gatcctgtta gatacataga tcctcgtcgc
420 aatatcgcat tttctaacgt gatggatata ttaaagtcga ataaagtgaa
caataattaa 480 ttctttattg tcatcatgaa cggcggacat attcagttga
taatcggccc catgttttca 540 ggtaaaagta cagaattaat tagacgagtt
agacgttatc aaatagctca atataaatgc 600 gtgactataa aatattctaa
cgataataga tacggaacgg gactatggac gcatgataag 660 aataattttg
aagcattgga agcaactaaa ctatgtgatg tcttggaatc aattacagat 720
ttctccgtga taggatactc ataaatccag ttgccgccac ggtagccaat caccgtatcg
780 tataaatcat cgtcggtacg ttcggcatcg ctcatcacaa tacgtgcctg
gacgtcgagg 840 atttcgcgtg ggtcaatgcc gcgccagatc cacatcagac
ggttaatcat gcgataccag 900 tgagggatgg ttttaccatc aagggccgac
tgcacaggcg gttgtgcgcc gtgattaaag 960 cggcggacta gcgtcgaggt
ttcaggatgt ttaaagcggg gtttgaacag ggtttcgctc 1020 aggtttgcct
gtgtcatgga tgcagcctcc agaatactta ctggaaacta ttgtaacccg 1080
cctgaagtta aaaagaacaa cgcccggcag tgccaggcgt tgaaaagatt agcgaccgga
1140 gattggcggg acgaatacga cgcccatatc ccacggctgt tcaatccagg
tatcttgcgg 1200 gatatcaaca acatagtcat caaccagcgg acgaccagcc
ggttttgcga agatggtgac 1260 aaagtgcgct tttggataca tttcacgaat
cgcaaccgca gtaccaccgg tatccaccag 1320 gtcatcaata acgatgaagc
cttcgccatc gccttctgcg cgtttcagca ctttaagctc 1380 gcgctggttg
tcgtgatcgt agctggaaat acaaacggta tcgacatgac gaatacccag 1440
ttcacgcgcc agtaacgcac ccggtaccag accgccacgg cttacggcaa taatgccttt
1500 ccattgttca gaaggcatca gtcggcttgc gagtttacgt gcatggatct
gcaacatgtc 1560 ccaggtgacg atgtattttt cgctcatgtg aagtgtccca
gcctgtttat ctacggctta 1620 aaaagtgttc gaggggaaaa taggttgcgc
gagattatag ggccttactt tgtaatataa 1680 tgatatatat tttcacttta
tctcatttga gaataaaaat gtttttgttt aaccactgca 1740 tgatgtcaat
tccgatccta gaagcgatgc tacgctagtc acaatcacca ctttcatatt 1800
tagaatatat gtatgtaaaa atatagtaga atttcatttt gtttttttct atcgattaaa
1860 tagaattcga gctcggtacc cggggatccc acaagctgtc gtggacatgg
tggcgggggc 1920 ccattgggga gtcctggcgg gtctcgccta ctattccatg
gtggggaact gggctaaggt 1980 tttgattgtg atgctactct ttgccggcgt
cgacgggcat acccgcgtgt caggaggggc 2040 agcagcctcc gataccaggg
gccttgtgtc cctctttagc cccgggtcgg ctcagaaaat 2100 ccagctcgta
aacaccaacg gcagttggca catcaacagg actgccctga actgcaacga 2160
ctccctccaa acagggttct ttgccgcact attctacaaa cacaaattca actcgtctgg
2220 atgcccagag cgcttggcca gctgtcgctc catcgacaag ttcgctcagg
ggtggggtcc 2280 cctcacttac actgagccta acagctcgga ccagaggccc
tactgctggc actacgcgcc 2340 tcgaccgtgt ggtattgtac ccgcgtctca
ggtgtgcggt ccagtgtatt gcttcacccc 2400 gagccctgtt gtggtgggga
cgaccgatcg gtttggtgtc cccacgtata actggggggc 2460 gaacgactcg
gatgtgctga ttctcaacaa cacgcggccg ccgcgaggca actggttcgg 2520
ctgtacatgg atgaatggca ctgggttcac caagacgtgt gggggccccc cgtgcaacat
2580 cgggggggcc ggcaacaaca ccttgacctg ccccactgac tgttttcgga
agcaccccga 2640 ggccacctac gccagatgcg gttctgggcc ctggctgaca
cctaggtgta tggttcatta 2700 cccatatagg ctctggcact acccctgcac
tgtcaacttc accatcttca aggttaggat 2760 gtacgtgggg ggcgtggagc
acaggttcga agccgcatgc aattggactc gaggagagcg 2820 ttgtgacttg
gaggacaggg atagatcaga gcttagcccg ctgctgctgt ctacaacaga 2880
gtggcagata ctgccctgtt ccttcaccac cctgccggcc ctatccaccg gcctgatcca
2940 cctccatcag aacatcgtgg acgtgcaata cctgtacggt gtagggtcgg
cggttgtctc 3000 ccttgtcatc aaataagctt aattaattag cttgggatcg
gctgtgagcg tatggcaaac 3060 gaaggaaaaa tagttatagt agccgcactc
gatgggacat ttcaacgtaa accgtttaat 3120 aatattttga atcttattcc
attatctgaa atggtggtaa aactaactgc tgtgtgtatg 3180 aaatgcttta
aggaggcttc cttttctaaa cgattgggtg aggaaaccga gatagaaata 3240
ataggaggta atgatatgta tcaatcggtg tgtagaaagt gttacatcga ctcataatat
3300 tatatttttt atctaaaaaa ctaaaaataa acattgatta aattttaata
taatacttaa 3360 aaatggatgt tgtgtcgtta gataaaccgt ttatgtattt
tgaggaaatt gataatgagt 3420 tagattacga accagaaagt gcaaatgagg
tcgcaaaaaa actgccgtat caaggacagt 3480 taaaactatt actaggagaa
ttattttttc ttagtaagtt acagcgacac ggtatattag 3540 atggtgccac
cgtagtgtat ataggatctg ctcccggatc gatcctgcat taatgaatcg 3600
gccaacgcgc ggggagaggc ggtttgcgta ttgggcttcc tcgctgcgct cggtcgttcg
3660 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
cagaatcagg 3720 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
aaggccagga accgtaaaaa 3780 ggccgcgttg ctggcgtttt tccataggct
ccgcccccct gacgagcatc acaaaaatcg 3840 acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg cgtttccccc 3900 tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3960
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
4020 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 4080 ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg acttatcgcc 4140 actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg gtgctacaga 4200 gttcttgaag tggtggccta
actacggcta cactagaaga acagtatttg gtatctgcgc 4260 tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4320
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
4380 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 4440 acgttaaggg attttggtca tgagattatc aaaaaggatc
ttcacctaga tccttttaaa 4500 ttaaaaatga agttttaaat caatctaaag
tatatatgag taaacttggt ctgacagtta 4560 ccaatgctta atcagtgagg
cacctatctc agcgatctgt ctatttcgtt catccatagt 4620 tgcctgactc
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 4680
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
4740 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
ccatccagtc 4800 tattaattgt tgccgggaag ctagagtaag tagttcgcca
gttaatagtt tgcgcaacgt 4860 tgttgccatt gctacaggca tcgtggtgtc
acgctcgtcg tttggtatgg cttcattcag 4920 ctccggttcc caacgatcaa
ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4980 tagctccttc
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5040
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
5100 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
cgagttgctc 5160 ttgcccggcg tcaatacggg ataataccgc gccacatagc
agaactttaa aagtgctcat 5220 cattggaaaa cgttcttcgg ggcgaaaact
ctcaaggatc ttaccgctgt tgagatccag 5280 ttcgatgtaa cccactcgtg
cacccaactg atcttcagca tcttttactt tcaccagcgt 5340 ttctgggtga
gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5400
gaaatgttga atactcatac tcctcctttt tcaatattat tgaagcattt atcagggtta
5460 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
taggggttcc 5520 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
accattatta tcatgacatt 5580 aacctataaa aataggcgta tcacgaggcc
ctttcgtctc gcgcgtttcg gtgatgacgg 5640 tgaaaacctc tgacacatgc
agctcccgga gacggtcaca gcttgtctgt aagcggatgc 5700 cgggagcaga
caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 5760
taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
5820 gcacagatgc gtaaggagaa aataccgcat caggcgattc cgttgcaatg
gctggcggta 5880 atattgttct ggatattacc agcaaggccg atagtttgag
ttcttctact caggcaagtg 5940 atgttattac taatcaaaga agtattgcga
caacggttaa tttgcgtgat ggacagactc 6000 ttttactcgg tggcctcact
gattataaaa acacttctca ggattctggc gtaccgttcc 6060 tgtctaaaat
ccctttaatc ggcctcctgt ttagctcccg ctctgattct aacgaggaaa 6120
gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
6180 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
cctagcgccc 6240 gctcctttcg ctttcttccc ttcctttctc gccacgttcg
ccggctttcc ccgtcaagct 6300 ctaaatcggg ggctcccttt agggttccga
tttagtgctt tacggcacct cgaccccaaa 6360 aaacttgatt agggtgatgg
ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 6420 cctttgacgt
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 6480
ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
6540 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
aatattaacg 6600 cttacaattt aaatatttgc ttatacaatc ttcctgtttt
tggggctttt ctgattatca 6660 accggggtac atatgattga catgctagtt
ttacgattac cgttcatcgg 6710 29 1110 DNA Hepatitis C virus CDS
(1)..(1110) 29 atg gtg gcg ggg gcc cat tgg gga gtc ctg gcg ggt ctc
gcc tac tat 48 Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu
Ala Tyr Tyr 1 5 10 15 tcc atg gtg ggg aac tgg gct aag gtt ttg att
gtg atg cta ctc ttt 96 Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile
Val Met Leu Leu Phe 20 25 30 gcc ggc gtc gac ggg cat acc cgc gtg
tca gga ggg gca gca gcc tcc 144 Ala Gly Val Asp Gly His Thr Arg Val
Ser Gly Gly Ala Ala Ala Ser 35 40 45 gat acc agg ggc ctt gtg tcc
ctc ttt agc ccc ggg tcg gct cag aaa 192 Asp Thr Arg Gly Leu Val Ser
Leu Phe Ser Pro Gly Ser Ala Gln Lys 50 55 60 atc cag ctc gta aac
acc aac ggc agt tgg cac atc aac agg act gcc 240 Ile Gln Leu Val Asn
Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala 65 70 75 80 ctg aac tgc
aac gac tcc ctc caa aca ggg ttc ttt gcc gca cta ttc 288 Leu Asn Cys
Asn Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe 85 90 95 tac
aaa cac aaa ttc aac tcg tct gga tgc cca gag cgc ttg gcc agc 336 Tyr
Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser 100 105
110 tgt cgc tcc atc gac aag ttc gct cag ggg tgg ggt ccc ctc act tac
384 Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr
115 120 125 act gag cct aac agc tcg gac cag agg ccc tac tgc tgg cac
tac gcg 432 Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His
Tyr Ala 130 135 140 cct cga ccg tgt ggt att gta ccc gcg tct cag gtg
tgc ggt cca gtg 480 Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val
Cys Gly Pro Val 145 150 155 160 tat tgc ttc acc ccg agc cct gtt gtg
gtg ggg acg acc gat cgg ttt 528 Tyr Cys Phe Thr Pro Ser Pro Val Val
Val Gly Thr Thr Asp Arg Phe 165 170 175 ggt gtc ccc acg tat aac tgg
ggg gcg aac gac tcg gat gtg ctg att 576 Gly Val Pro Thr Tyr Asn Trp
Gly Ala Asn Asp Ser Asp Val Leu Ile 180 185 190 ctc aac aac acg cgg
ccg ccg cga ggc aac tgg ttc ggc tgt aca tgg 624 Leu Asn Asn Thr Arg
Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp 195 200 205 atg aat ggc
act ggg ttc acc aag acg tgt ggg ggc ccc ccg tgc aac 672 Met Asn Gly
Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn 210 215 220 atc
ggg ggg gcc ggc aac aac acc ttg acc tgc ccc act gac tgt ttt 720 Ile
Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe 225 230
235 240 cgg aag cac ccc gag gcc acc tac gcc aga tgc ggt tct ggg ccc
tgg 768 Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro
Trp 245 250 255 ctg aca cct agg tgt atg gtt cat tac cca tat agg ctc
tgg cac tac 816 Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu
Trp His Tyr 260 265 270 ccc tgc act gtc aac ttc acc atc ttc aag gtt
agg atg tac gtg ggg 864 Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val
Arg Met Tyr Val Gly 275 280 285 ggc gtg gag cac agg ttc gaa gcc gca
tgc aat tgg act cga gga gag 912 Gly Val Glu His Arg Phe Glu Ala Ala
Cys Asn Trp Thr Arg Gly Glu 290 295 300 cgt tgt gac ttg gag gac agg
gat aga tca gag ctt agc ccg ctg ctg 960 Arg Cys Asp Leu Glu Asp Arg
Asp Arg Ser Glu Leu Ser Pro Leu Leu 305 310 315 320 ctg tct aca aca
gag tgg cag ata ctg ccc tgt tcc ttc acc acc ctg 1008 Leu Ser Thr
Thr Glu Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu 325 330 335 ccg
gcc cta tcc acc ggc ctg atc cac ctc cat cag aac atc gtg gac 1056
Pro Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp 340
345 350 gtg caa tac ctg tac ggt gta ggg tcg gcg gtt gtc tcc ctt gtc
atc 1104 Val Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val Val Ser Leu
Val Ile 355 360 365 aaa taa 1110 Lys 370 30 369 PRT Hepatitis C
virus 30 Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala
Tyr Tyr 1 5 10 15 Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile Val
Met Leu Leu Phe 20 25 30 Ala Gly Val Asp Gly His Thr Arg Val Ser
Gly Gly Ala Ala Ala Ser 35 40 45 Asp Thr Arg Gly Leu Val Ser Leu
Phe Ser Pro Gly Ser Ala Gln Lys 50 55 60 Ile Gln Leu Val Asn Thr
Asn Gly Ser Trp His Ile Asn Arg Thr Ala 65 70 75 80 Leu Asn Cys Asn
Asp Ser Leu Gln Thr Gly Phe Phe Ala Ala Leu Phe 85 90 95 Tyr Lys
His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser 100 105 110
Cys Arg Ser Ile Asp Lys Phe Ala Gln Gly Trp Gly Pro Leu Thr Tyr 115
120 125 Thr Glu Pro Asn Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr
Ala 130 135 140 Pro Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys
Gly Pro Val 145 150 155 160 Tyr Cys Phe Thr Pro Ser Pro Val Val Val
Gly Thr Thr Asp Arg Phe 165 170 175 Gly Val Pro Thr Tyr Asn Trp Gly
Ala Asn Asp Ser Asp Val Leu Ile 180 185 190 Leu Asn Asn Thr Arg Pro
Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp 195 200 205 Met Asn Gly Thr
Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn 210 215 220 Ile Gly
Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe 225 230 235
240 Arg Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp
245 250 255 Leu Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp
His Tyr 260 265 270 Pro Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg
Met Tyr Val Gly 275 280 285 Gly Val Glu His Arg Phe Glu Ala Ala Cys
Asn Trp Thr Arg Gly Glu 290 295 300 Arg Cys Asp Leu Glu Asp Arg Asp
Arg Ser Glu Leu Ser Pro Leu Leu 305 310 315 320 Leu Ser Thr Thr Glu
Trp Gln Ile Leu Pro Cys Ser Phe Thr Thr Leu 325 330 335 Pro Ala Leu
Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp 340 345 350 Val
Gln Tyr Leu Tyr Gly Val Gly Ser Ala Val Val Ser Leu Val Ile 355 360
365 Lys
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