U.S. patent application number 10/964054 was filed with the patent office on 2005-04-28 for antibodies to hepatitis c virus asialoglycoproteins.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Berger, Kim M., Choo, Qui-Lim, Gervase, Barbara A., Hall, John A., Houghton, Michael, Kuo, George, Marcus, Frank, Ralston, Robert O., Thudium, Kent B..
Application Number | 20050089843 10/964054 |
Document ID | / |
Family ID | 22945252 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089843 |
Kind Code |
A1 |
Ralston, Robert O. ; et
al. |
April 28, 2005 |
Antibodies to hepatitis C virus asialoglycoproteins
Abstract
Two Hepatitis C Virus envelope proteins (E1 and E2) are
expressed without sialylation. Recombinant expression of these
proteins in lower eukaryotes, or in mammalian cells in which
terminal glycosylation is blocked, results in recombinant proteins
which are more similar to native HCV glycoproteins. When isolated
by GNA lectin affinity, the E1 and E2 proteins aggregate into
virus-like particles.
Inventors: |
Ralston, Robert O.;
(Danville, CA) ; Marcus, Frank; (Danville, CA)
; Thudium, Kent B.; (Oakland, CA) ; Gervase,
Barbara A.; (Vallejo, CA) ; Hall, John A.;
(Rohnert Park, CA) ; Berger, Kim M.; (Lafayette,
CA) ; Choo, Qui-Lim; (El Cerrito, CA) ;
Houghton, Michael; (Danville, CA) ; Kuo, George;
(San Francisco, CA) |
Correspondence
Address: |
CHIRON CORPORATION
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
|
Family ID: |
22945252 |
Appl. No.: |
10/964054 |
Filed: |
October 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10964054 |
Oct 12, 2004 |
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09929782 |
Aug 13, 2001 |
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09929782 |
Aug 13, 2001 |
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08249843 |
May 26, 1994 |
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6274148 |
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08249843 |
May 26, 1994 |
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07758880 |
Sep 13, 1991 |
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07758880 |
Sep 13, 1991 |
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07611419 |
Nov 8, 1990 |
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Current U.S.
Class: |
435/5 ; 435/325;
435/456; 435/69.3; 530/395; 536/23.72 |
Current CPC
Class: |
Y10S 977/803 20130101;
Y10S 977/915 20130101; Y10S 977/918 20130101; C07K 14/005 20130101;
Y10S 530/826 20130101; A61P 31/12 20180101; Y10S 530/82 20130101;
Y10S 977/804 20130101; C12N 2770/24222 20130101; Y10S 977/802
20130101; A61P 1/16 20180101; A61K 39/00 20130101 |
Class at
Publication: |
435/005 ;
435/069.3; 435/456; 435/325; 530/395; 536/023.72 |
International
Class: |
C12Q 001/70; C07H
021/04; C07K 014/02; C12N 015/86 |
Claims
1-43. (canceled)
44. An isolated antibody specific for a hepatitis C virus (HCV)
glycoprotein having mannose-terminated glycosylation, wherein less
than about 10% of the total N-linked carbohydrate on said HCV
glycoprotein is sialic acid, wherein said HCV glycoprotein is
selected from the group consisting of a glycoprotein expressed from
the E1 region of HCV, a glycoprotein expressed from the E2 region
of HCV, and aggregates thereof.
45. The antibody of claim 44, wherein said HCV glycoprotein is a
glycoprotein expressed from the E1 region of HCV.
46. The antibody of claim 44, wherein said HCV glycoprotein is a
glycoprotein expressed from the E2 region of HCV.
47. The antibody of claim 44, wherein said HCV glycoprotein is an
aggregate of a glycoprotein expressed from the E1 region of HCV and
a glycoprotein expressed from the E2 region of HCV.
48. The antibody of claim 44, wherein said HCV glycoprotein is an
aggregate of glycoproteins expressed from the E1 region of HCV.
49. The antibody of claim 44, wherein said HCV glycoprotein is an
aggregate of glycoproteins expressed from the E2 region of HCV.
50. The antibody of claim 44, wherein the antibody is a polyclonal
antibody.
51. The antibody of claim 45, wherein the antibody is a polyclonal
antibody.
52. The antibody of claim 46, wherein the antibody is a polyclonal
antibody.
53. The antibody of claim 47, wherein the antibody is a polyclonal
antibody.
54. The antibody of claim 48, wherein the antibody is a polyclonal
antibody.
55. The antibody of claim 49, wherein the antibody is a polyclonal
antibody.
56. An isolated antibody specific for a hepatitis C virus (HCV)
glycoprotein having mannose-terminated glycosylation, wherein less
than about 10% of the total N-linked carbohydrate on said HCV
glycoprotein is sialic acid, wherein said HCV glycoprotein is
selected from the group consisting of a glycoprotein expressed from
the E1 region of HCV, a glycoprotein expressed from the E2 region
of HCV, and aggregates thereof, and further wherein said HCV
glycoprotein is produced by the method comprising the steps of:
growing a host cell transformed with a structural gene encoding an
HCV glycoprotein expressed from the E1 region of HCV or the E2
region of HCV in a suitable culture medium; causing expression of
said structural gene, under conditions inhibiting sialylation; and
isolating said HCV glycoprotein from said cell culture by
contacting said HCV glycoprotein with a mannose-binding protein
specific for mannose-terminated glycoproteins, and isolating the
protein that binds to said mannose-binding protein.
57. The antibody of claim 56, wherein said HCV glycoprotein is a
glycoprotein expressed from the E1 region of HCV.
58. The antibody of claim 56, wherein said HCV glycoprotein is a
glycoprotein expressed from the E2 region of HCV.
59. The antibody of claim 56, wherein said HCV glycoprotein is an
aggregate of a glycoprotein expressed from the E1 region of HCV and
a glycoprotein expressed from the E2 region of HCV.
60. The antibody of claim 56, wherein said HCV glycoprotein is an
aggregate of glycoproteins expressed from the E1 region of HCV.
61. The antibody of claim 56, wherein said HCV glycoprotein is an
aggregate of glycoproteins expressed from the E2 region of HCV.
62. The antibody of claim 56, wherein the antibody is a polyclonal
antibody.
63. The antibody of claim 57, wherein the antibody is a polyclonal
antibody.
64. The antibody of claim 58, wherein the antibody is a polyclonal
antibody.
65. The antibody of claim 59, wherein the antibody is a polyclonal
antibody.
66. The antibody of claim 60, wherein the antibody is a polyclonal
antibody.
67. The antibody of claim 61, wherein the antibody is a polyclonal
antibody.
68. The antibody of claim 56, wherein the structural gene is linked
to a sequence encoding a secretion leader that directs the
glycoprotein to the endoplasmic reticulum and said conditions
inhibiting sialylation comprise inhibiting transport of
glycoproteins from the endoplasmic reticulum to the golgi.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending U.S.
Ser. No. 07/758,880, filed Sep. 13, 1991, which is a
continuation-in-part of U.S. Ser. No. 07/611,419, filed Nov. 8,
1990, now abandoned, the discosures of which are incorporated
herein by reference.
DESCRIPTION
[0002] 1. Technical Field
[0003] This invention relates to the general fields of recombinant
protein expression and virology. More particularly, the invention
relates to glycoproteins useful for diagnosis, treatment, and
prophylaxis of Hepatitis C virus (HCV) infection, and methods for
producing such glycoproteins.
[0004] 2. Background of the Invention
[0005] Non-A, Non-B hepatitis (NANBH) is a transmissible disease
(or family of diseases) that is believed to be virally induced, and
is distinguishable from other forms of virus-associated liver
disease, such as those caused by hepatitis A virus (HAV), hepatitis
B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV)
or Epstein-Barr virus (EBV). Epidemiologic evidence suggests that
there may be three types of NANBH: the water-borne epidemic type;
the blood or needle associated type; and the sporadically occurring
community acquired type. The number of causative agents is unknown.
However, a new viral species, hepatitis C virus (HCV) has recently
been identified as the primary (if not only) cause of blood-borne
NANBH (BB-NANBH). See for example PCT WO89/046699 and U.S. patent
application Ser. No. 07/355,002, filed 18 May 1989. Hepatitis C
appears to be the major form of transfusion-associated hepatitis in
a number of countries or regions, including the United States,
Europe, and Japan. There is also evidence implicating HCV in
induction of hepatocellular carcinoma. Thus, a need exists for an
effective method for preventing and treating HCV infection.
[0006] The demand for sensitive, specific methods for screening and
identifying carriers of HCV and HCV-contaminated blood or blood
products is significant. Post-transfusion hepatitis (PTH) occurs in
approximately 10% of transfused patients, and HCV accounts for up
to 90% of these cases. The major problem in this disease is the
frequent progression to chronic liver damage (25-55%).
[0007] Patient care, as well as the prevention of transmission of
HCV by blood and blood products or by close personal contact,
requires reliable diagnostic and prognostic tools to detect nucleic
acids, antigens and antibodies related to HCV. In addition, there
is also a need for effective vaccines and immunotherapeutic
therapeutic agents for the prevention and/or treatment of the
disease.
[0008] HCV appears in the blood of infected individuals at very low
rates relative to other infectious viruses, which makes the virus
very difficult to detect. The low viral burden is probably the
primary reason that the causative agent of NANB hepatitis went so
long undetected. Even though it has now been cloned, HCV still
proves difficult to culture and propagate. Accordingly, there is a
strong need for recombinant means of producing
diagnostic/therapeutic/prophylactic HCV proteins.
DISCLOSURE OF THE INVENTION
[0009] It has been found that two HCV proteins, E1 and E2, appear
to be membrane associated asialoglycoproteins when expressed in
recombinant systems. This is surprising because glycoproteins do
not usually remain in mannose-terminated form in mammals, but are
further modified with other carbohydrates: the mannose-terminated
form is typically only transient. In the case of E1 and E2 (as
expressed in our systems), the asialoglycoprotein appears to be the
final form. E1 (envelope protein 1) is a glycoprotein having a
molecular weight of about 35 kD which is translated from the
predicted E1 region of the HCV genome. E2 (envelope protein 2) is a
glycoprotein having a molecular weight of about 72 kD which is
translated from the predicted NS1 (non-structural protein 1) region
of the HCV genome, based on the flaviviral model of HCV. As viral
glycoproteins are often highly immunogenic, E1 and E2 are prime
candidates for use in immunoassays and therapeutic/prophylactic
vaccines.
[0010] The discovery that E1 and E2 are not sialylated is
significant. The particular form of a protein often dictates which
cells may serve as suitable hosts for recombinant expression.
Prokaryotes such as E. coli do not glycosylate proteins, and are
generally not suitable for production of glycoproteins for use as
antigens because glycosylation is often important for full
antigenicity, solubility, and stability of the protein. Lower
eukaryotes such as yeast and fungi glycosylate proteins, but are
generally unable to add terminal sialic acid residues to the
carbohydrate complexes. Thus, yeast-derived proteins may be
antigenically distinct from their natural (non-recombinant)
counterparts. Expression in mammalian cells is preferred for
applications in which the antigenicity of the product is important,
as the glycosylation of the recombinant protein should closely
resemble that of the wild viral proteins.
[0011] New evidence indicates that the HCV virus may gain entry to
host cells during infection through either the asialoglycoprotein
receptor found on hepatocytes, or through the mannose receptor
found on hepatic endothelial cells and macrophages (particularly
Kupffer cells). Surprisingly, it has been found that the bulk of
natural E1 and E2 do not contain terminal sialic acid residues, but
are only core-glycosylated. A small fraction additionally contains
terminal N-acetylglucosamine. Accordingly, it is an object of the
present invention to provide HCV envelope glycoproteins lacking all
or substantially all terminal sialic acid residues.
[0012] Another aspect of the invention is a method for producing
asialo-E1 or E2, under conditions inhibiting addition of terminal
sialic acid, e.g., by expression in yeast or by expression in
mammalian cells using antibiotics to facilitate secretion or
release.
[0013] Another aspect of the invention is a method for purifying E1
or E2 by affinity to lectins which bind terminal mannose residues
or terminal N-acetylglucosamine residues.
[0014] Another aspect of the invention is an immunogenic
composition comprising a recombinant asialoglycoprotein selected
from the group consisting of HCV E1 and E2 in combination with a
pharmaceutically acceptable vehicle. One may optionally include an
immunological adjuvant, if desired.
[0015] Another aspect of the invention is an immunoassay reagent,
comprising a recombinant asialoglycoprotein selected from the group
consisting of HCV E1 and E2 in combination with a suitable support.
Another immunoassay reagent of the invention comprises a
recombinant asialoglycoprotein selected from the group consisting
of HCV E1 and E2 in combination with a suitable detectable
label.
[0016] Another aspect of the invention concerns dimers and
higher-order aggregates of E1 and/or E2. One species of the
invention is an E2 complex. Another species of the invention is an
E1:E2 heterodimer.
[0017] Another aspect of the invention is an HCV vaccine
composition comprising E1:E2 aggregates and a pharmaceutically
acceptable carrier.
[0018] Another aspect of the invention is a method for purifying
E1:E2 complexes.
[0019] Modes of Carrying out the Invention
[0020] A. Definitions
[0021] The term "asialoglycoprotein" refers to a glycosylated
protein which is substantially free of sialic acid moieties.
Asialoglycoproteins may be prepared recombinantly, or by
purification from cell culture or natural sources. Presently
preferred asialoglycoproteins are derived from HCV, preferably the
glycoproteins E1 and E2, most preferably recombinant E1 and E2 (rE1
and rE2). A protein is "substantially free" of sialic acid within
the scope of this definition if the amount of sialic acid residues
does not substantially interfere with binding of the glycoprotein
to mannose-binding proteins such as GNA. This degree of sialylation
will generally be obtained where less than about 40% of the total
N-linked carbohydrate is sialic acid, more preferably less than
about 30%, more preferably less than about 20%, more preferably
less than about 10%, more preferably less than about 5%, and most
preferably less than about 2%.
[0022] The term "E1" as used herein refers to a protein or
polypeptide expressed within the first 400 amino acids of an HCV
polyprotein, sometimes referred to as the E or S protein. In its
natural form it is a 35 kD glycoprotein which is found strongly
membrane-associated. In most natural HCV strains, the E1 protein is
encoded in the viral polyprotein following the C (core) protein.
The E1 protein extends from approximately amino acid 192 to about
aa383 of the full-length polyprotein. The term "E1" as used herein
also includes analogs and truncated mutants which are
immunologically crossreactive with natural E1.
[0023] The term "E2" as used herein refers to a protein or
polypeptide expressed within the first 900 amino acids of an HCV
polyprotein, sometimes referred to as the NS1 protein. In its
natural form it is a 72 kD glycoprotein which is found strongly
membrane-associated. In most natural HCV strains, the E2 protein
follows the E1 protein. The E2 protein extends from approximately
aa384 to about aa820. The term "E2" as used herein also includes
analogs and truncated mutants which are immunologically
crossreactive with natural E2.
[0024] The term "aggregate" as used herein refers to a complex of
E1 and/or E2 containing more than one E1 or E2 monomer. E1:E1
dimers, E2:E2 dimers, and E1:E2 heterodimers are all "aggregates"
within the scope of this definition. Compositions of the invention
may also include larger aggregates, and may have molecular weights
in excess of 800 kD.
[0025] The term "particle" as used herein refers to an E1, E2, or
E1/E2 aggregate visible by electron microscopy and having a
dimension of at least 20 nm. Preferred particles are those having a
roughly spherical appearance and a diameter of approximately 40 nm
by electron microscopy.
[0026] The term "purified" as applied to proteins herein refers to
a composition wherein the desired protein comprises at least 35% of
the total protein component in the composition. The desired protein
preferably comprises at least 40%, more preferably at least about
50%, more preferably at least about 60%, still more preferably at
least about 70%, even more preferably at least about 80%, even more
preferably at least about 90%, and most preferably at least about
95% of the total protein component. The composition may contain
other compounds such as carbohydrates, salts, lipids, solvents, and
the like, without affecting the determination of percentage purity
as used herein. An "isolated" HCV asialoglycoprotein intends an HCV
asialoglycoprotein composition which is at least 35% pure.
[0027] "Mannose-binding protein" as used herein intends a lectin or
other protein which specifically binds to proteins having
mannose-terminated glycosylation (e.g., asialoglycoproteins), for
example, mannose-binding lectins, antibodies specific for
mannose-terminated glycosylation, mannose receptor protein (R. A.
B. Ezekowitz et al., J Exp Med (1990) 176: 1785-94),
asialoglycoprotein receptor proteins (H. Kurata et al., J Biol Chem
(1990) 265: 1295-98), serum mannose-binding protein (I.
Schuffenecker et al., Cytogenet Cell Genet (1991) 56: 99-102; K.
Sastry et al., J Immunol (1991) 147: 692-97), serum
asialoglycoprotein-binding protein, and the like. Mannose-binding
lectins include, for example, GNA, Concanavalin A (ConA), and other
lectins with similar binding properties.
[0028] The term "GNA lectin" refers to Galanthus nivalus
agglutinin, a commercially available lectin which binds to
mannose-terminated glycoproteins.
[0029] A "recombinant" glycoprotein as used herein is a
glycoprotein expressed from a recombinant polynucleotide, in which
the structural gene encoding the glycoprotein is expressed under
the control of regulatory sequences not naturally adjacent to the
structural gene, or in which the structural gene is modified. For
example, one may form a vector in which the E1 structural gene is
placed under control of a functional fragment of the yeast
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. A
presently preferred promoter for use in yeast is the hybrid
ADH2/GAP promoter described in U.S. Pat. No. 4,880,734
(incorporated herein by reference), which employs a fragment of the
GAPDH promoter in combination with the upstream activation sequence
derived from alcohol dehydrogenase 2. Modifications of the
structural gene may include substitution of different codons with
degenerate codons (e.g., to utilize host-preferred codons,
eliminate or generate restriction enzyme cleavage sites, to control
hairpin formation, etc.), and substitution, insertion or deletion
of a limited number of codons encoding different amino acids
(preferably no more than about 10%, more preferably less than about
5% by number of the natural amino acid sequence should be altered),
and the like. Similarly, a "recombinant" receptor refers to a
receptor protein expressed from a recombinant polynucleotide, in
which the structural gene encoding the receptor is expressed under
the control of regulatory sequences not naturally adjacent to the
structural gene, or in which the structural gene is modified.
[0030] The term "isolated polypeptide" refers to a polypeptide
which is substantially free of other HCV viral components,
particularly polynucleotides. A polypeptide composition is
"substantially free" of another component if the weight of the
polypeptide in the composition is at least 70% of the weight of the
polypeptide and other component combined, more preferably at least
about 80%, still more preferably about 90%, and most preferably 95%
or greater. For example, a composition containing 100 .mu.g/ml E1
and only 3 .mu.g/ml other HCV components (e.g., DNA, lipids, etc.)
is substantially free of "other HCV viral components", and thus is
a composition of an isolated polypeptide within the scope of this
definition.
[0031] The term "secretion leader" refers to a polypeptide which,
when encoded at the N-terminus of a protein, causes the protein to
be secreted into the host ceU's culture medium following
translation. The secretion leader will generally be derived from
the host cell employed. For example, suitable secretion leaders for
use in yeast include the Saccharomyces cerevisiae .alpha.-factor
leader (see U.S. Pat. No. 4,870,008, incorporated herein by
reference).
[0032] The term "lower eukaryote" refers to host cells such as
yeast, fungi, and the like. Lower eukaryotes are generally (but not
necessarily) unicellular. Preferred lower eukaryotes are yeasts,
particularly species within Saccharomyces, Schizosaccharomyces,
Kluveromyces, Pichia, Hansenula, and the like. Saccharomyces
cerevisiae, S. carlsbergensis and K. lactis are the most commonly
used yeast hosts, and are convenient fungal hosts.
[0033] The term "higher eukaryote" refers to host cells derived
from higher animals, such as mammals, reptiles, insects, and the
like. Presently preferred higher eukaryote host cells are derived
from Chinese hamster (e.g., CHO), monkey (e.g., COS cells), human,
and insect (e.g., Spodoptera frugiperda). The host cells may be
provided in suspension or flask cultures, tissue cultures, organ
cultures, and the like.
[0034] The term "calcium modulator" refers to a compound capable of
sequestering or binding calcium ions within the endoplasmic
reticulum, or affects calcium ion concentration within the ER by
its effect on calcium regulatory proteins (e.g., calcium channel
proteins, calcium pumps, etc.). Suitable calcium modulators
include, for example thapsigargin, EGTA (ethylene glycol
bis[.beta.-aminoethyl ether] N,N,N',N'-tetraacetic acid). The
presently preferred modulator is thapsigargin (see e.g., O.
Thastrup et al, Proc Nat Acad Sci USA (1990) 87: 2466-70).
[0035] The term "immunogenic" refers to the ability of a substance
to cause a humoral and/or cellular immune response, whether alone
or when linked to a carrier, in the presence or absence of an
adjuvant. "Neutralization" refers to an immune response that blocks
the infectivity, either partially or fully, of an infectious agent.
A "vaccine" is an immunogenic composition capable of eliciting
protection against HCV, whether partial or complete, useful for
treatment of an individual.
[0036] The term "biological liquid" refers to a fluid obtained from
an organism, such as serum, plasma, saliva, gastric secretions,
mucus, and the like. In general, a biological liquid will be
screened for the presence of HCV particles. Some biological fluids
are used as a source of other products, such as clotting factors
(e.g., Factor VIII:C), serum albumin, growth hormone, and the like.
In such cases, it is important that the source biological fluid be
free of contamination by virus such as HCV.
[0037] B. General Method
[0038] The E1 region of the HCV genome is described in EP 388,232
as region E1, while E2 is described as "NS1." The E1 region
comprises approximately amino acids 192-383 in the full-length
viral polyprotein. The E2 region comprises approximately amino
acids 384-820. The complete sequences of prototypes of these
proteins (strain HCV-1) are available in the art (see EP 388,232),
as are general methods for cloning and expressing the proteins.
Both E1 and E2 may be expressed from a polynucleotide encoding the
first 850-900 amino acids of the HCV polyprotein:
post-translational processing in most eukaryotic host cells cleaves
the initial polyprotein into C, E1, and E2. One may truncate the 5'
end of the coding region to reduce the amount of C protein
produced.
[0039] Expression of asialoglycoproteins may be achieved by a
number of methods. For example, one may obtain expression in lower
eukaryotes (such as yeast) which do not normally add sialic acid
residues to glycosylated proteins. In yeast expression systems, it
is presently preferred to employ a secretion leader such as the S.
cerevisiae .alpha.-factor leader, so that the protein is expressed
into the culture medium following translation. It is also presently
preferred to employ glycosylation-deficient mutants such as pmr1,
as these mutants supply only core glycosylation, and often secrete
heterologous proteins with higher efficiency (H. K. Rudolph et al,
Cell (1989) 58:133-45). Alternatively, one may employ other species
of yeast, such as Pichia pastoris, which express glycoproteins
containing 8-9 mannose residues in a pattern believed to resemble
the core glycosylation pattern observed in mammals and S.
cerevisiae.
[0040] Alternatively, one may arrange expression in mammalian
cells, and block terminal glycosylation (addition of sialic acid).
Recombinant constructs will preferably include a secretion signal
to insure that the protein is directed toward the endoplasmic
reticulum. Transport to the golgi appears to be blocked by E1 and
E2 themselves: high-level expression of E1 or E2 in mammalian cells
appears to arrest secretion of all cellular proteins at the
endoplasmic reticulum or cis golgi. One may additionally employ a
glycosylation defective mutant. See for example, P. Stanley, Ann
Rev Genet (1984) 18: 525-52. In the event a glycosylation or
transport mutant expresses E1 or E2 with sialylation, the terminal
sialic acid residues may be removed by treatment with
neuraminidase.
[0041] Yield should be further increased by use of a calcium
modulator to obtain release of protein from within the endoplasmic
reticulum. Suitable modulators include thapsigargin, EGTA, and
A23817 (see e.g., O. Thastrup et al, Proc Nat Acad Sci USA (1990)
87: 2466-70). For example, one may express a large amount of E1 or
E2 intracellularly in mammalian cells (e.g., CHO, COS, HeLa cells,
and the like) by transfection with a recombinant vaccinia virus
vector. After allowing time for protein expression and accumulation
in the endoplasmic reticulum, the cells are exposed to a calcium
modulator in concentration large enough to cause release of the ER
contents. The protein is then recovered from the culture medium,
which is replaced for the next cycle.
[0042] Additionally, it may be advantageous to express a truncated
form of the envelope protein. Both E1 and E2 appear to have a
highly hydrophobic domain, which apparently anchors the protein
within the endoplasmic reticulum and prevents efficient release.
Thus, one may wish to delete portions of the sequence found in one
or more of the regions aa170-190, aa260-290 or aa330-380 of E1
(numbering from the beginning of the polyprotein), and aa660-830 of
E2 (see for example FIG. 20-1 of EP 388,232). It is likely that at
least one of these hydrophobic domains forms a transmembrane region
which is not essential for antigenicity of the protein, and which
may thus be deleted without detrimental effect. The best region to
delete may be determined by conducting a small number of deletion
experiments within the skill of the ordinary practitioner. Deletion
of the hydrophobic 3' end of E2 results in secretion of a portion
of the E2 expressed, with sialylation of the secreted protein.
[0043] One may use any of a variety of vectors to obtain
expression. Lower eukaryotes such as yeast are typically
transformed with plasmids using the calcium phosphate precipitation
method, or are transfected with a recombinant virus. The vectors
may replicate within the host cell independently, or may integrate
into the host cell genome. Higher eukaryotes may be transformed
with plasmids, but are typically infected with a recombinant virus,
for example a recombinant vaccinia virus. Vaccinia is particularly
preferred, as infection with vaccinia halts expression of host cell
proteins. Presently preferred host cells include HeLa and
plasmacytoma cell lines. In the present system, this means that E1
and E2 accumulate as the major glycosylated species in the host ER.
As the rE1 and rE2 will be the predominant glycoproteins which are
mannose-terminated, they may easily be purified from the cells by
using lectins such as Galanthus nivalus agglutinin (GNA) which bind
terminal mannose residues.
[0044] Proteins which are naturally expressed as mannose-terminated
glycoproteins are relatively rare in mammalian physiology. In most
cases, a mammalian glycoprotein is mannose-terminated only as a
transient intermediate in the glycosylation pathway. The fact that
HCV envelope proteins, expressed recombinantly, contain
mannose-terminated glycosylation or (to a lesser degree)
N-acetylglucosamine means that HCV proteins and whole virions may
be separated and partially purified from endogenous proteins using
lectins specific for terminal mannose or N-acetylglucosamine. The
recombinant proteins appear authentic, and are believed essentially
identical to the envelope proteins found in the mature, free
virion, or to a form of cell-associated envelope protein. Thus, one
may employ lectins such as GNA for mannose-terminated proteins, and
WGA (wheat germ agglutinin) and its equivalents for
N-acetylglucosamine-terminated proteins. One may employ lectins
bound to a solid phase (e.g., a lectin-Sepharose.RTM. column) to
separate E1 and E2 from cell culture supernatants and other fluids,
e.g., for purification during the production of antigens for
vaccine or immunoassay use.
[0045] Alternatively, one may provide a suitable lectin to isolate
E1, E2, or HCV virions from fluid or tissue samples from subjects
suspected of HCV infection. As mannose-terminated glycoproteins are
relatively rare, such a procedure should serve to purify the
proteins present in a sample, substantially reducing the
background. Following binding to lectin, the HCV protein may be
detected using anti-HCV antibodies. If whole virions are present,
one may alternatively detect HCV nucleic acids using PCR techniques
or other nucleic acid amplification methods directed toward
conserved regions of the HCV genome (for example, the 5' non-coding
region). This method permits isolation and characterization of
differing strains of HCV without regard for antigenic drift or
variation, e.g., in cases where a new strain is not immunologically
crossreactive with the strain used for preparing antibodies. There
are many other ways to take advantage of the unique recognition of
mannose-terminated glycoproteins by particular lectins. For
example, one may incubate samples suspected of containing HCV
virions or proteins with biotin or avidin-labeled lectins, and
precipitate the protein-lectin complex using avidin or biotin. One
may also use lectin affinity for HCV proteins to target compounds
to virions for therapeutic use, for example by conjugating an
antiviral compound to GNA. Alternatively, one may use suitable
lectins to remove mannose-terminated glycoproteins from serum or
plasma fractions, thus reducing or eliminating the risk of HCV
contamination.
[0046] It is presently preferred to isolate E1 and/or E2
asialoglycoproteins from crude cell lysates by incubation with an
immobilized mannose-binding protein, particularly a lectin such as
ConA or GNA. Cells are lysed, e.g., by mechanical disruption in a
hypotonic buffer followed by centrifugation to prepare a
post-nuclear lysate, and further centrifuged to obtain a crude
microsomal membrane fraction. The crude membrane fraction is
subsequently solubilized in a buffer containing a detergent, such
as Triton X-100, NP40, or the like. This detergent extract is
further clarified of insoluble particulates by centrifugation, and
the resulting clarified lysate incubated in a chromatography column
comprising an immobilized mannose-binding protein, preferably GNA
bound to a solid support such as agarose or Sepharose.RTM. for a
period of time sufficient for binding, typically 16 to 20 hours.
The suspension is then applied to the column until E1/E2 begins to
appear in the eluent, then incubated in the column for a period of
time sufficient for binding, typically about 12-24 hours. The bound
material is then washed with additional buffer containing detergent
(e.g., Triton X-100, NP40, or the like), and eluted with mannose to
provide purified asialoglycoprotein. On elution, it is preferred to
elute only until protein begins to appear in the eluate, at which
point elution is halted and the column permitted to equilibrate for
2-3 hours before proceeding with elution of the protein. This is
believed to allow sufficient time for the slow off-rate expected of
large protein aggregates. In cases wherein E1 and E2 are expressed
together in native form (i.e., without truncation of the
membrane-binding domain), a substantial fraction of the
asialoglycoproteins appear as E1:E2 aggregates. When examined by
electron microscopy, a significant portion of these aggregates
appear as roughly spherical particles having a diameter of about 40
nm, which is the size expected for intact virus. These particles
appear to be self-assembling subviral particles. These aggregates
are expected to exhibit a quaternary structure very similar to the
structure of authentic HCV virion particles, and thus are expected
to serve as highly immunogenic vaccines.
[0047] The E1/E2 complexes may be further purified by gel
chromatography on a basic medium, for example, Fractogel-DEAE or
DEAE-Sepharose.RTM.. Using Fractogel-DEAE gel chromatography, one
may obtain E1/E2 complexes of approximately 60-80% purity. One may
further purify E1 by treatment with lysine protease, because E1 has
0-1 Lys residues. Treatment of the complex with lysine protease
destroys E2, and permits facile separation of E1.
[0048] The tissue specificity of HCV, in combination with the
observation that HCV envelope glycoproteins are mannose-terminated,
suggests that the virus employs the mannose receptor or the
asialoglycoprotein receptor (ASGR) in order to gain entry into host
cells. Mannose receptors are found on macrophages and hepatic
sinusoidal cells, while the ASGR is found on parenchymal
hepatocytes. Thus, it should be possible to culture HCV by
employing host cells which express one or both of these receptors.
One may either employ primary cell cultures which naturally express
the receptor, using conditions under which the receptor is
maintained, or one may transfect another cell line such as HeLa,
CHO, COS, and the like, with a vector providing for expression of
the receptor. Cloning of the mannose receptor and its transfection
and expression in fibroblasts has been demonstrated by M. E. Taylor
et al, J Biol Chem (1990) 265: 12156-62. Cloning and sequencing of
the ASGR was described by K. Drickamer et al, J Biol Chem (1984)
259: 770-78 and M. Spiess et al, Proc Nat Acad Sci USA (1985) 82:
6465-69; transfection and expression of functional ASGR in rat HTC
cells was described by M. McPhaul and P. Berg, Proc Nat Acad Sci
USA (1986) 83: 8863-67 and M. McPhaul and P. Berg, Mol Cell Biol
(1987) 7: 1841-47. Thus, it is possible to transfect one or both
receptors into suitable cell lines, such as CHO, COS, HeLa, and the
like, and to use the resulting cells as hosts for propagation of
HCV in culture. Serial passaging of HCV in such cultures should
result in development of attenuated strains suitable for use as
live vaccines. It is presently preferred to employ an immortalized
cell line transfected with one or both recombinant receptors.
[0049] Immunogenic compositions can be prepared according to
methods known in the art. The present compositions comprise an
immunogenic amount of a polypeptide, e.g., E1, E2, or E1/E2
particle compositions, usually combined with a pharmaceutically
acceptable carrier, preferably further comprising an adjuvant. If a
"cocktail" is desired, a combination of HCV polypeptides, such as,
for example, E1 plus E2 antigens, can be mixed together for
heightened efficacy. The virus-like particles of E1/E2 aggregates
are expected to provide a particularly useful vaccine antigen.
Immunogenic compositions may be administered to animals to induce
production of antibodies, either to provide a source of antibodies
or to induce protective immunity in the animal.
[0050] Pharmaceutically acceptable carriers include any carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition. Suitable carriers are
typically large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers; and inactive virus
particles. Such carriers are well known to those of ordinary skill
in the art.
[0051] Preferred adjuvants to enhance effectiveness of the
composition include, but are not limited to: aluminum hydroxide
(alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as
found in U.S. Pat. No. 4,606,918,
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-di-
palmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE)
and RIBI, which contains three components extracted from bacteria,
monophosphoryl lipid A, trehalose dimycolate, and cell wall
skeleton (MPL+TDM+CWS) in a 2% squalene/Tween.RTM. 80 emulsion.
Additionally, adjuvants such as Stimulon (Cambridge Bioscience,
Worcester, Mass.) may be used. Further, Complete Freund's Adjuvant
(CFA) and Incomplete Freund's Adjuvant (IFA) may be used for
non-human applications and research purposes.
[0052] The immunogenic compositions typically will contain
pharmaceutically acceptable vehicles, such as water, saline,
glycerol, ethanol, etc. Additionally, auxiliary substances, such as
wetting or emulsifying agents, pH buffering substances, and the
like, may be included in such vehicles.
[0053] Typically, the immunogenic compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection may also be prepared. The preparation also may be
emulsified or encapsulated in liposomes for enhanced adjuvant
effect.
[0054] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of the HCV polypeptide, as well as
any other of the above-mentioned components, as needed.
"Immunologically effective amount", means that the administration
of that amount to an individual, either in a single dose or as part
of a series, is effective for treatment, as defined above. This
amount varies depending upon the health and physical condition of
the individual to be treated, the taxonomic group of individual to
be treated (e.g., nonhuman primate, primate, etc.), the capacity of
the individual's immune system to synthesize antibodies, the degree
of protection desired, the formulation of the vaccine, the treating
doctor's assessment of the medical situation, the strain of
infecting HCV, and other relevant factors. It is expected that the
amount will fall in a relatively broad range that can be determined
through routine trials.
[0055] The self-assembling E1/E2 aggregates may also serve as
vaccine carriers to present heterologous (non-HCV) haptens, in the
same manner as Hepatitis B surface antigen (See European Patent
Application 174,444). In this use, the E1/E2 aggregates provide an
immunogenic carrier capable of stimulating an immune response to
haptens or antigens conjugated to the aggregate. The antigen may be
conjugated either by conventional chemical methods, or may be
cloned into the gene encoding E1 and/or E2 at a location
corresponding to a hydrophilic region of the protein.
[0056] The immunogenic compositions are conventionally administered
parenterally, typically by injection, for example, subcutaneously
or intramuscularly. Additional formulations suitable for other
modes 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.
C. EXAMPLES
[0057] The examples presented below are provided as a further guide
to the practitioner of ordinary skill in the art, and are not to be
construed as limiting the invention in any way.
Example 1
Cloning and Expression
[0058] (A) Vectors were constructed from plasmids containing the 5'
portion of the HCV genome, as described in EP 318,216 and EP
388,232. Cassette HCV(S/B) contains a StuI-BglII DNA fragment
encoding the 5' end of the polyprotein from Met.sub.1 up to
Leu.sub.906, beginning at nucleotide -63 relative to Met.sub.1.
This includes the core protein (C), the E1 protein (also sometimes
referred to as S), the E2 protein (also referred to as NS1), and a
5' portion of the NS2a region. Upon expression of the construct,
the individual C, E1 and E2 proteins are produced by proteolytic
processing.
[0059] Cassette HCV(A/B) contains a ApaLI-BglII DNA fragment
encoding the 5' end of the polyprotein from Met.sub.1 up to
Leu.sub.906, beginning at nucleotide-6 relative to Met.sub.1. This
includes the core protein (C), the E1 protein (also sometimes
referred to as S), the E2 protein (also referred to as NS1), and a
5' portion of the NS2a region. Upon expression of the construct,
the individual C, E1 and E2 proteins are produced by proteolytic
processing.
[0060] Cassette C-E1(S/B) (a StuI-BamHI portion) contains the 5'
end from Met.sub.1 up to Ile.sub.340 (a BamHI site in the gene).
Expression of this cassette results in expression of C and a
somewhat truncated E1 (E1'). The portion truncated from the 3' end
is a hydrophobic region believed to serve as a translocation
signal.
[0061] Cassette NS1(B/B) (a BamHI-BglII portion) contains a small
3' portion of E1 (from Met.sub.364), all of E2, and a portion of
NS2a (to Leu.sub.906). In this construct, the E1 fragment serves as
a translocation signal.
[0062] Cassette TPA-NS1 employs a human tissue plasminogen
activator (tPA) leader as a translocation signal instead of the 3'
portion of E1. The cassette contains a truncated form of E2, from
Gly.sub.406 to Glu.sub.661, in which the hydrophobic 3' end is
deleted.
[0063] Each cassette was inserted into the vector pGEM3Z (Promega)
with and without a synthetic .beta.-globin 5' non-coding sequence
for transcription and translation using T7 and rabbit reticulocyte
expression in vitro. Recombinant vaccinia virus (rVV) vectors were
prepared by inserting the cassettes into the plasmid pSC11
(obtained from Dr. B. Moss, NIH) followed by recombination with
vaccinia virus, as described by Charkrabarty et al., Mol Cell Biol
(1985) 5: 3403-09.
[0064] (B) An alternate expression vector was constructed by
inserting HCV(A/B) between the StuI and SpeI sites of pSC59
(obtained from Dr. B. Moss, NIH) followed by recombination with
vaccinia virus, as described by Charkrabarty et al, Mol Cell Biol
(1985) 5: 3403-09.
[0065] (C) HeLa S3 cells were collected by centrifugation for 7
minutes at 2000 rpm at room temperature in sterile 500 ml
centrifuge bottles (JA-10 rotor). The pellets were resuspended at a
final concentration of 2.times.10.sup.7 cells/ml in additional
culture medium (Joklik modified MEM Spinner medium +5% horse serum
and Gentamycin) ("spinner medium"). Sonicated crude vv/SC59-HCV
virus stock was added at a multiplicity of infection of 8 pfu/cell,
and the mixture stirred at 37.degree. C. for 30 minutes. The
infected cells were then transferred to a spinner flask containing
8 liters spinner medium and incubated for 3 days at 37.degree.
C.
[0066] The cultured cells were then collected by centrifugation,
and the pellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15
ml). The cells were then homogenized using a 40 ml Dounce
Homogenizer (50 strokes), and the nuclei pelleted by centrifugation
(5 minutes, 1600 rpm, 4.degree. C., JA-20 rotor). The nuclear
pellets were resuspended in Tris buffer (4 ml), rehomogenized, and
pelleted again, pooling all supernatants.
[0067] The pooled lysate was divided into 10 ml aliquots and
sonicated 3.times.30 minutes in a cuphorn sonicator at medium
power. The sonicated lysate (15 ml) was layered onto 17 ml sucrose
cushions (36%) in SW28 centrifuge tubes, and centrifuged at 13,500
rpm for 80 minutes at 4.degree. C. to pellet the virus. The virus
pellet was resuspended in 1 ml of Tris buffer (1 mM Tris HCl, pH
9.0) and frozen at -80.degree. C.
Example 2
Comparison of in vitro and in vivo Products
[0068] (A) E1 and E2 were expressed both in vitro and in vivo and
.sup.35S-Met labeled using the vectors described in Example 1
above. BSC-40 and HeLa cells were infected with the rVV vectors for
in vivo expression. Both the medium and the cell lysates were
examined for recombinant proteins. The products were
immunoprecipitated using human HCV immune serum, while in vitro
proteins were analyzed directly. The resulting proteins were
analyzed by SDS-PAGE.
[0069] The reticulocyte expression system (pGEM3Z with HCV(S/B) or
HCV(A/B)) produced C, E1 and E2 proteins having molecular weights
of approximately 18 kD, 35 kD, and 72 kD, respectively. Lysates
from BSC-40 and HeLa cells transfected with rVV containing
HCV(S/B), HCV(A/B) or C-E1(S/B) exhibited the same proteins.
Because the reticulocyte system does not provide efficient golgi
processing and therefore does not provide sialic acid, the fact
that both in vitro and in vivo products exhibited identical
mobilities suggests that the proteins are not sialylated in vivo.
Only the rVV vector containing TPA-NS1 resulted in any
extracellular secretion of E2, which exhibited an altered mobility
consistent with sialylation.
[0070] (B) HCV(S/B) was expressed in vitro and incubated with a
panel of biotinylated lectins: GNA, SNA, PNA, WGA, and ConA.
Following incubation, the complexes were collected on
avidin-acrylic beads, washed, eluted with Laemmli sample buffer,
and analyzed by SDS-PAGE. The results showed that E1 and E2 bound
to GNA and ConA, which indicates the presence of mannose. GNA binds
to terminal mannose groups, while ConA binds to any .alpha.-linked
mannose. The lack of binding to SNA, PNA, and WGA indicates that
none of the proteins contained sialic acid,
galactose-N-acetylgalactosamine, or N-acetylglucosamine.
[0071] (C) Radiolabeled E1 and E2 were produced in BSC-40 cells by
infection with rVV containing HCV(S/B) (vv/SC11-HCV), and
immunoprecipitated with human HCV.sup.+ immune serum. One half of
the immunoprecipitated material was treated overnight with
neuraminidase to remove any sialic acid. Following treatment, the
treated and untreated proteins were analyzed by SDS-PAGE. No
significant difference in mobility was observed, indicating lack of
sialylation in vivo.
[0072] (D) Radiolabeled E1 and E2 were produced in BSC-40 cells by
infection with rVV containing HCV(A/B) (vv/SC59-HCV), and either
immunoprecipitated with human HCV.sup.+ serum, or precipitated
using biotinylated GNA lectin linked to acrylic beads, using
vv/SC11 free of HCV sequences as control. The precipitates were
analyzed by SDS-PAGE. The data demonstrated that E1 and E2 were the
major species of mannose-terminated proteins in vv/SC59-HCV
infected cells. GNA was as efficient as human antisera in
precipitating E1 and E2 from cell culture medium. A 25 kD component
was observed, but appears to be specific to vaccinia-infected
cells.
Example 3
Purification using Lectin
[0073] (A) HeLa S3 cells were inoculated with purified high-titer
vv/SC59-HCV virus stock at a multiplicity of infection of 5
pfu/cell, and the mixture stirred at 37.degree. C. for 30 minutes.
The infected cells were then transferred to a spinner flask
containing 8 liters spinner medium and incubated for 3 days at
37.degree. C. The cells were collected again by centrifugation and
resuspended in hypotonic buffer (20 mM HEPES, 10 mM NaCl, 1 mM
MgCl.sub.2, 120 ml) on ice. The cells were then homogenized by
Dounce Homogenizer (50 strokes), and the nuclei pelleted by
centrifugation (5 minutes, 1600 rpm, 4.degree. C., JA-20 rotor).
The pellets were pooled, resuspended in 48 ml hypotonic buffer,
rehomogenized, recentrifuged, pooled again, and frozen at
-80.degree. C.
[0074] The frozen supernatants were then thawed, and the microsomal
membrane fraction of the post-nuclear Iysate isolated by
centrifuging for 20 minutes in a JA-20 rotor at 13,500 rpm at
4.degree. C. The supernatant was removed by aspiration.
[0075] The pellets were taken up in 96 ml detergent buffer (20 mM
Tris-HCl, 100 .mu.M NaCl, 1 mM EDTA, 1 mM DDT, 0.5% Triton X-100,
pH 7.5) and homogenized (50 strokes). The product was clarified by
centrifugation for 20 minutes at 13,500 rpm, 4.degree. C., and the
supernatants collected.
[0076] A GNA-agarose column (0.1 cm.times.3 cm, 3 mg. GNA/ml beads,
6 ml bed volume, Vector Labs, Burlingame, Calif.) was
pre-equilibrated with detergent buffer. The supernatant sample was
applied to the column with recirculation at a flow rate of 1 ml/min
for 16-20 hours at 4.degree. C. The column was then washed with
detergent buffer.
[0077] The purified E1/E2 proteins were eluted with
.alpha.-D-mannoside (0.9 M in detergent buffer) at a flow rate of
0.5 ml/minute. Elution was halted at the appearance of E1/E2 in the
eluent, and the column allowed to reequilibrate for 2-3 hours.
Fractions were analyzed by Western blot and silver staining. Peak
fractions were pooled and UV-irradiated to inactivate any residual
vaccinia virus.
[0078] (B) GNA-agarose purified E1 and E2 asialoglycoproteins were
sedimented through 20-60% glycerol gradients. The gradients were
fractionated and proteins were analyzed by SDS-PAGE and western
blotting. Blots were probed with GNA for identification of E1 and
E2. The results indicate the presence of a E1:E2 heterodimer which
sediments at the expected rate (i.e., a position characteristic of
a 110 kD protein). Larger aggregates of HCV envelope proteins also
are apparent. E2:E2 homodimers also were apparent. E2 appeared to
be over-represented in the larger species relative to E1, although
discrete E1:E2 species also were detected. The larger aggregates
sedimented significantly faster than the thyroglobulin marker.
[0079] (C) GNA-agarose purified E1 and E2 were sedimented through
20-60% glycerol gradients containing 1 mM EDTA. Fractions were
analyzed by SDS-PAGE with and without .beta.-mercaptoethanol
(.beta.ME). Little or no difference in the apparent abundance of E1
and E2 in the presence or absence of .beta.ME was observed,
indicating the absence of disulfide links between heterodimers.
[0080] (D) E1/E2 complexes (approximately 40% pure) were analyzed
on a Coulter DM-4 sub-micron particle analyzer. Material in the
20-60 nm range was detected.
[0081] (E) E1/E2 complexes (approximately 40% pure) were analyzed
by electron microscopy using negative staining with phosphotungstic
acid. The electron micrograph revealed the presence of particles
having a spherical appearance and a diameter of about 40 nm. E1/E2
complexes were incubated with HCV+human immune serum, then analyzed
by EM with negative staining. Antibody complexes containing large
aggregates and smaller particles were observed.
Example 4
Chromatographic Purification
[0082] (A) The GNA lectin-purified material prepared as described
in Example 3 (0.5-0.8 ml) was diluted 10.times. with buffer A (20
mM Tris-Cl buffer, pH 8.0, 1 mM EDTA), and applied to a
1.8.times.1.5 cm column of Fractogel EMD DEAE-650 (EM Separations,
Gibbstown, N.J., cat. no. 16883) equilibrated in buffer A. The
protein fraction containing E1/E2 was eluted with the same buffer
at a flow rate of 0.2 ml/minute, and 1 ml fractions collected.
Fractions containing E1 and E2 (determined by SDS-PAGE) were pooled
and stored at -80.degree. C.
[0083] (B) The material purified in part (A) above has a purity of
60-80%, as estimated by SDS-PAGE. The identification of the
putative E1 and E2 bands was confirmed by N-terminal sequence
analysis after using a transfer technique. For the purpose, the
fractogel-DEAE purified E1/E2 material was reduced by addition of
Laemmli buffer (pH 6.8, 0.06 M Tris-Cl, 2.3% SDS, 10% glycerol,
0.72 M .beta.-mercaptoethanol) and boiled for 3 minutes. The sample
was then loaded onto a 10% polyacrylamide gel. After SDS-PAGE, the
protein was transferred to a polyvinylidene difluoride (PVDF) 0.2
.mu.m membrane (Bio-Rad Laboratories, Richmond, Calif.). The
respective putative E1 and E2 protein bands were excised from the
blot and subjected to N-terminal amino acid analysis, although no
special care was taken to prevent amino-terminal blockage during
preparation of the material. The first 15 cycles revealed that the
E1 sample had a sequence Tyr-Gln-Val-Arg-X-Ser-T-
hr-Gly-X-Tyr-His-Val-X-Asn-Asp, while the sequence of E2 was
Thr-His-Val-Thr-Gly-X-X-Ala-Gly-His-X-Val-X-Gly-Phe. This amino
acid sequence data is in agreement with that expected from the
corresponding DNA sequences.
[0084] The E1/E2 product purified above by fractogel-DEAE
chromatography is believed to be aggregated as evidenced by the
fact that a large amount of E1 and E2 coelutes in the void volume
region of a gel permeation chromatographic Bio-Sil TSK-4000 SW
column. This indicates that under native conditions a significant
amount of the E1/E2 complex has a molecular weight of at least 800
kD. E1/E2 material having a molecular weight of about 650 kD was
also observed.
Example 5
Additional Cloning and Expression
[0085] (A) The following cassettes containing 5' portions of the
HCV polyprotein were inserted into the vector pGEM4Z (Promega) with
and without a synthetic yellow fever virus 5' non-coding sequence
and also into recombinant vaccinia virus (rVV) vectors (as
described in Example 1A). Cassette C5p-1 contains a fragment
encoding the 5' end of the polyprotein from Met.sub.1 to
Trp.sub.1079 beginning at nucleotide -275 relative to Met.sub.1,
with EcoRI linkers on the 5' and 3' ends. Cassette C5p-3 contains
an fragment encoding the 5' end of the polyprotein from Met.sub.1
to Trp.sub.1079, with EcoRI linkers on the 5' and 3' ends. Both
cassettes encode C, E1 and E2 proteins and a 5' portion of the NS2
protein.
[0086] (B) The following cassettes containing 5' portions of the
HCV polyprotein were inserted into the vector pSC59 followed by
recombination with vaccinia virus (described in Example 1B).
Cassette HCV(Poly) contains a blunt-ended StuI-Bg III fragment
encoding the 5' end of the polyprotein from Met.sub.1 to
Asp.sub.966 beginning at nucleotide -65 relative to Met.sub.1. This
construct expresses C, E1 and E2 proteins, and a 5' portion of the
NS2 protein.
[0087] Cassette HCV(5C/SB) contains a blunt-ended StuI-BamHI
fragment encoding the 5' end of the polyprotein from Met.sub.1 to
Ile.sub.340 beginning at nucleotide -65 relative to Met.sub.1. This
construct expresses the C protein and a truncated E1 protein.
[0088] Cassette HCV(6C/SS) contains a SalI(blunted)-EcoRI fragment
encoding the 5' end of the polyprotein from Met.sub.1 to
ASP.sub.382 wherein Ser.sub.2 is replaced with Gly.sub.2. This
construct expresses the C protein and a truncated E1 protein.
[0089] Cassette HCV(E12C/B) contains a blunt-ended ClaI-BglII
fragment encoding a portion of the polyprotein from Met.sub.134 to
ASP.sub.966 inserted into an EcoRI blunted SC59 vector.
[0090] Cassette HCV(E1/S) contains a blunt-ended ClaI/SalI fragment
encoding a portion of the polyprotein from Met.sub.134 to
Val.sub.381 inserted into an EcoRI blunted SC59 vector.
[0091] (C) HeLa S3 cells were collected by centrifugation for 7
minutes at 2000 rpm at 4.degree. C. in sterile 250 ml centrifuge
bottles. The pellets were resuspended at a final concentration of
5.times.10.sup.6 cells/ml in Gey's balanced Salt Solution (GBSS).
Sonicated crude vv/SC59-HCV virus stock was added at a multiplicity
of infection of 0.5 pfu/cell, and the mixture stirred at 37.degree.
C. for 1-2 hours. The infected cells were then transferred at a
final concentration of 10.sup.6 cells/ml to a spinner flask
containing 1 liter culture medium (Joklik MEM+10% fetal bovine
serum+non-essential amino acids, vitamins, pen/strep) and incubated
for 3 days at 37.degree. C.
[0092] The cultured cells were then collected by centrifugation,
and the pellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15
ml). The cells were then homogenized using a 40 ml Dounce
Homogenizer (50 strokes), and the nuclei pelleted by centrifugation
(5 minutes, 1600 rpm, 4.degree. C., JA-20 rotor). The nuclear
pellets were resuspended in Tris buffer (4 ml), rehomogenized, and
pelleted again, pooling all supernatants.
[0093] The pooled lysate was divided into 5 ml aliquots, and 0.1
volume of 2.5 mg/ml trypsin added and incubated at 37.degree. C.
for 30 minutes. The aliquots were then sonicated 3.times.30 seconds
in a cuphorn sonicator at medium power. The sonicated lysate was
used as the crude stock.
Example 6
Additional Comparison of in vitro and in vivo Products
[0094] (A) E1 and E2 were expressed both in vitro and in vivo and
.sup.35S-Met labeled using the vectors described in Example 5 above
and the procedures described in Example 2 above. BSC-40 and HeLa
cells were infected with the rVV vectors for in vivo expression.
Both the medium and the cell lysates were examined for recombinant
proteins. The products were immunoprecipitated using human HCV
immune serum or rabbit or goat anti-HCV antiserum, while in vitro
proteins were analyzed directly. The resulting proteins were
analyzed by SDS-PAGE and EndoH digestion.
Example 7
Additional Lectin Purification
[0095] (A) HeLa S3 cells were inoculated with purified high-titer
vv/SC59-HCV virus stock (HCV(Poly) or HCV(E12C/B) as described in
Example 5 above) at a multiplicity of infection of 1 pfu/cell, and
the mixture stirred at 37.degree. C. for 1-2 hours. The infected
cells were then transferred to spinner flasks containing 1 liter
culture medium (see Example 5, supra) and incubated for 2 days at
37.degree. C. A total of 10 liters of cells were collected by
centrifugation and resuspended in hypotonic buffer (20 mM HEPES, 10
mM NaCl, 1 mM MgCl.sub.2, 120 ml) containing protease inhibitors
(PMSF and pepstatin A) on ice. The cells were then homogenized in a
40 ml homogenizer in two batches, pelleted by centrifugation (20
minutes, 12,000 rpm), and re-suspended and re-homogenized. Each
pellet was resuspended in approximately 10 ml 25 mM NaPO.sub.4 (pH
6.8) in a homogenizer, and an equal volume of 4% Triton X-100 in
100 mM NaPO.sub.4 (pH 6.8) added. The pelleted cells were
homogenized with 20 strokes, spun at 12,000 rpm for 15 minutes (4
tubes/pellet), and the supernatant saved. The resuspension, Triton
addition and centrifugation steps were repeated, and the saved
supernatants combined, and frozen at -80.degree. C.
[0096] The frozen supernatants were thawed, spun at 12,000 rpm for
15 minutes, combined and held at 4.degree. C. A GNA-agarose column
(1 cm.times.3 cm, 3 mg GNA/ml beads, 6 ml bed volume, Vector Labs,
Burlingame, Calif.) was pre-equilibrated with detergent buffer (2%
Triton X-100 in 50 mM NaPO.sub.4, pH 6.8). The supernatant sample
was applied to the column with recirculation at a flow rate of 1
ml/min for 16-20 hours at 4.degree. C. The column was then washed
with detergent buffer, followed by 30 ml each of: Buffer A (1M
NaCl, 20 mM NaPO.sub.4 (pH 6.0), 0.1% Triton X-100); Buffer B (20
mM NaPO.sub.4 (pH 6.0), 0.1% Triton X-100); Buffer D (0.2M
methyl-.alpha.-D-mannopyranoside (mmp), 20 mM NaPO.sub.4 (H 6.0),
0.1% Triton X-100); Buffer E (1M mmp, 20 mM NaPO.sub.4 (pH 6.0),
0.1% Triton X-100); Buffer F (1M mmp, 1M NaCl, 20 mM NaPO.sub.4 (pH
6.0), 0.1% Triton X-100). Purified E1/E2 proteins come off as
eluted material in Buffers D and E, which were collected separately
and analyzed by SDS-PAGE.
Example 8
Additional Chromatographic Purification
[0097] (A) The GNA lectin-purified material prepared as described
in Example 7 was applied to a column of S-Sepharose Fast Flow
(Pharmacia) equilibrated in buffer B (see Example 7). The column
was washed with Buffer B, then eluted with Buffer 1 (0.5M NaCl, 20
mM NaPO.sub.4 (pH 6.0), 0.1% Triton X-100) and Buffer 2 (1M NaCl,
20 mM NaPO.sub.4 (pH 6.0), 0.1% Triton X-100). Fractions containing
E1 and E2 (determined by SDS-PAGE) were pooled and stored at
-80.degree. C.
* * * * *