Note: Descriptions are shown in the official language in which they were submitted.
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HCV FUSION PROTEINS WITH MODIFIED NS3 DOMAINS
TECHNICAL FIELD
The present invention relates to hepatitis C virus (HCV) constructs. More
particularly, the invention relates to HCV fusion proteins with modified NS3
domains. The proteins are capable of stimulating cell-mediated immune
responses,
such as for priming and/or activating HCV-specific T cells.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) infection is an important health problem with
approximately 1% of the world's population infected with the virus. Over 75%
of
acutely infected individuals eventually progress to a cluonic carrier state
that can
result in cirrhosis, liver failure, and hepatocellular carcinoma. See, Alter
et al. (1992)
N. Engl. J. Med. 327:1899-1905; Resniclc and Koff. (1993) Arch. Intem. Med.
153:1672-1677; Seeff (1995) Gastrointest. Dis. 6:20-27; Tong et al. (1995) N.
Engl. J.
Med. 332:1463-1466.
HCV was first identified and characterized as a cause of NANBH by
Houghton et al. The viral genomic sequence of HCV is l~nown, as are methods
for
obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669;
WO
90/11089; and WO 90/14436. HCV has a 9.5 lcb positive-sense, single-stranded
RNA
genome and is a member of the Flaviridae family of viruses. At least six
distinct, but
related genotypes of HCV, based on phylogenetic analyses, have been identified
(Siimnonds et al., J. Gen. ViYOI. (1993) 74:2391-2399). The virus encodes a
single
polyprotein having more than 3000 amino acid residues (Choo et al., Sciefice
(1989)
244:359-362; Choo et al., P~oc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han
et
al., P~oc. Natl. Acad. Sci. USA (1991) X8:1711-1715). The polyprotein is
processed
co- and post-translationally into both structural and non-structural (NS)
proteins.
In particular, as shown in Figure 1, several proteins are encoded by the HCV
genome. The order and nomenclature of the cleavage products of the HCV
polyprotein is as follows:
NH2_C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NSSa-NSSb-COOH. Tilitial cleavage of the
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polyprotein is catalyzed by host proteases which liberate three structural
proteins, the
N-terminal nucleocapsid protein (termed "core") and two envelope
glycoproteins,
"El" (also known as E) and "E2" (also known as E2/NS1), as well as
nonstructural
(NS) proteins that contain the viral enzymes. The NS regions are tenned'NS2,
NS3,
NS4 and NSS. NS2 is an integral membrane protein with proteolytic activity
and, in
combination with NS3, cleaves the NS2-NS3 sissle bond which in turn generates
the
NS3 N-terminus and releases a large polyprotein that includes both serine
protease
and RNA helicase activities. The NS3 protease serves to process the remaining
polyprotein. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two
proteins
(NS4b and NSSa), and an RNA-dependent RNA polyrnerase (NSSb). Completion of
polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a
junction, catalyzed by the NS3 serine protease.
Despite extensive advances in the development of pharmaceuticals against
certain viruses like HIV, control of acute and chronic HCV infection has had
limited
success (Hoofnagle and di Bisceglie (1997) N. Engl. J. Med. 336:347-356). In
pas-ticular, generation of cellular immune responses, such as strong cytotoxic
T
lymphocyte (CTL) responses, is thought to be important for the control and
eradication of HCV infections. Thus, there is a need in the art for effective
methods
of stimulating cellular immune responses to HCV.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods for
stimulating
a cellular immune response to HCV, such as priming and/or activating T cells
which
recognize epitopes of HCV polypeptides. This and other obj ects of the
invention are
provided by one or more of the embodiments described below.
The invention provides HCV fusion proteins useful for stimulating such
responses. One embodiment of the invention is directed to an HCV fusion
protein
that includes an NS3 polypeptide modified to inhibit protease activity, such
that
cleavage of the fusion is inhibited. The fusion protein includes, in addition
to the
modified NS3 polypeptide, one or more polypeptides from other regions of an
HCV
polyprotein, described in further detail below. These polypeptides are derived
from
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the same HCV isolate as the NS3 polypeptide, or from different strains and
isolates
including isolates having any of the various HCV genotypes, to provide
increased
protection against a broad range of HCV genotypes.
In certain embodiments, the modification to NS3 comprises a substitution of
an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered
relative to the full-length HCV-1 polyprotein.
In further embodiments, the protein comprises a modified NS3 polypeptide, an
NS4 polypeptide, an NSSa polypeptide, and optionally a core polypeptide.
In additional embodiments, the protein further comprises an NSSb
polypeptide, and optionally a core polypeptide.
In yet additional embodiments, the protein further comprises an E2
polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core
polypeptide.
In further embodiments, the protein fiuther comprises an E1 polypeptide, an
E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core
polypeptide.
In additional embodiments, the protein further comprises an E2 polypeptide,
and optionally a core polypeptide.
In yet further embodiments, the protein further comprises an E1 polypeptide,
an E2 polypeptide, and optionally a core polypeptide.
In further embodiments, the protein comprises an E2 polypeptide, a modified
NS3 polypeptide, and optionally a core polypeptide.
In additional embodiments, the protein comprises an E1 polypeptide, an E2
polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.
Another embodiment provides a fusion protein that consists essentially of a
modified NS3, an NS4, an NSSa, and, optionally, a core polypeptide of an HCV.
In
certain embodiments, an NSSb polypeptide is also present.
In the embodiments above, the various regions in the fusion protein need not
be in the order in which they naturally occur in the native HCV polyprotein.
Thus, for
example, the core polypeptide, if present, may be at the N- and/or C-terminus
of the
fusion.
In yet additional embodiments, the invention is directed to an
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immunogenic fusion protein consisting essentially of, in amino terminal to
carboxy
terminal direction:
(a) a modified NS3 polypeptide comprising a substitution of an amino acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, and an NSSa polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide and an NSSb polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited, an NS4 polypeptide, and an NSSa
polypeptide;
(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino
acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, and an NSSa polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited, an NS4 polypeptide, an NSSa
polypeptide and
an NSSb polypeptide;
(fJ an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino
acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide and an NSSb polypeptide;
4
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(g) an E2 polypeptide axed a modified NS3 polypeptide comprising substitution
of an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered
relative to the full-length HCV-1 polyprotein such that protease activity is
inhibited;
(h) an E1 polypeptide, an E2 polypeptide and a modified NS3 polypeptide
comprising a substitution of an amino acid corresponding to His-1083, Asp-1105
and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such
that
protease activity is inhibited;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified
NS3 polypeptide comprising a substitution of an amino acid corresponding to
His-
1083, Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein such that protease activity is inhibited; or
(j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2
polypeptide and a modified NS3 polypeptide comprising a substitution of an
amino
acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to
the
full-length HCV-1 polyprotein such that protease activity is inhibited.
In another embodiment, the invention is directed to an immunogenic fusion
protein consisting essentially of, in amino terminal to carboxy terminal
direction:
(a) a modified NS3 polypeptide comprising a substitution of an amino acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide, and a core polypeptide;
(b) a modified NS3 polypeptide comprising a substitution of an amino acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide, an NSSb polypeptide and a core polypeptide;
(c) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited, an NS4 polypeptide, an NSSa
polypeptide and
a core polypeptide;
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(d) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NSZ
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino
acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide and a core polypeptide;
(e) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited, an NS4 polypeptide, an NSSa
polypeptide, an
NSSb polypeptide and a core polypeptide;
(f) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino
acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
length HCV-1 polyprotein such that protease activity is inhibited, an NS4
polypeptide, an NSSa polypeptide, an NSSb polypeptide and a core polypeptide;
(g) an E2 polypeptide, a modified NS3 polypeptide comprising substitution of
an amino acid corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered
relative to the full-length HCV-1 polyprotein such that protease activity is
inhibited,
and a core polypeptide;
(h) an El polypeptide, an E2 polypeptide, a modified NS3 polypeptide
comprising a substitution of an amino acid corresponding to His-1083, Asp-1105
and/or Ser-1165, numbered relative to the full-length HCV-1 polyprotein such
that
protease activity is inhibited, and a core polypeptide;
(i) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited, and a core polypeptide; or
(j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2
polypeptide, a modified NS3 polypeptide comprising a substitution of an amino
acid
corresponding to His-1083, Asp-1105 and/or Ser-1165, numbered relative to the
full-
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length HCV-1 polyprotein such that protease activity is inhibited, and a core
polypeptide.
In yet a further embodiment, the invention is directed to a modified NS3
polypeptide comprising a substitution of an amino acid corresponding to His-
1083,
Asp-1105 and/or Ser-1165, numbered relative to the full-length HCV-1
polyprotein
such that protease activity is inhibited when the modified NS3 polypeptide is
present
in an HCV fusion protein.
Yet another embodiment of the invention provides an isolated polynucleotide
which encodes any of the proteins detailed above, recombinant vectors
comprising the
same, host cells transformed with the vectors, and methods of recombinantly
producing the fusion proteins.
The invention also provides compositions comprising any of these fusion
proteins, polynucleotides encoding the fusions, or recombinant vectors
including the
polynucleotides, and a pharmaceutically acceptable Garner.
Yet another embodiment of the invention provides a method of stimulating a
cellular irninune response in a vertebrate subject by administering a
composition as
described herein. In certain embodiments, the composition primes and/or
activates T
cells which recognize an epitope of an HCV polypeptide. T cells are contacted
with a
fusion protein comprising a modified NS3 polypeptide and at least one
additional
HCV polypeptide. A population of activated T cells recognizes an epitope of
the NS3
and/or the additional HCV polypeptide(s).
The invention thus provides methods and reagents for stimulating a cellular
immune response to HCV, such as for priming and/or activating T cells which
recognize epitopes of HCV polypeptides. These methods and reagents are
particularly advantageous for identifying epitopes of HCV polypeptides
associated
with a strong CTL response and for immunizing mammals, including humans,
against
HCV.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a diagrammatic representation of the HCV genome, depicting the
various regions of the~HCV polyprotein.
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Figure 2 depicts the DNA and corresponding amino acid sequence (SEQ TD
NOS:3-4) of a representative native, unmodified NS3 protease domain.
Figure 3 shows the DNA and corresponding amino acid sequence (SEQ ID
NOS:S-6) of a representative modified fusion protein, with the NS3 protease
domain
deleted from the N-terminus and including amino acids 1-121 of Core on the
C-terminus.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated,
conventional methods of chemistry, biochemistry, recombinant DNA techniques
and
immunology, within the skill of the art. Such techniques are explained fully
in the
literature. See, e.g., Sambrook, et al., Moleculaf~ Cloning: A Labof~atory
Manual
(2nd Edition); Methods h2 Enzymology (S. Colowick and N. Kaplan eds., Academic
Press, Inc.); DNA Cloning, Vols. I and II (D.N. Glover ed.); Oligon.ucleotide
Synthesis
(M.J. Gait ed.); Nucleic Acid Hyby°idization (B.D. Hames & S.J. Higgins
eds.); Afzimal
Cell Cultu~~e (R.K. Freshney ed.); Perbal, B., A P~~actical Guide to
Moleculaf° Clonifag.
It must be noted that, as used in this specification and the appended claims,
the
singular forms "a", "an" and "the" include plural referents unless the content
clearly
dictates otherwise. Thus, for example, reference to "an antigen" includes a
mixture of
two or more amtigens, and the like.
The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg
(R)
Asparagine: Asn (I~ Aspartic acid:
Asp (D)
Cysteine: Cys (C) Glutamine: Gln
(Q)
Glutamic acid: Glu (E) Glycine: Gly
(G)
Histidine: His (H) Isoleucine: Ile
(I)
Leucine: Leu (L) Lysine: Lys (K)
Methionine: Met (M) Phenylalanine:
Phe (F)
Proline: Pro (P) Serine: Ser (S)
Threonine: Thr (T) Tryptophan: Trp
(~
Tyrosine: Tyr (Y) Valine: Val (V)
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I. Definitions
In describing the present invention, the following teens will be employed, and
are intended to be defined as indicated below.
The terms "polypeptide" and "protein" refer to a polymer of amino acid
residues and are not limited to a minimum length of the product. Thus,
peptides,
oligopeptides, dimers, multimers, and the like, are included within the
definition.
Both full-length proteins and fragments thereof are encompassed by the
definition.
The terms also include postexpression modifications of the polypeptide, for
example,
glycosylation, acetylation, phosphorylation and the like. Furthermore, for
purposes of
the present invention, a "polypeptide" refers to a protein which includes
modifications, such as deletions, additions and substitutions (generally
conservative in
nature), to the native sequence, so long as the protein maintains the desired
activity.
These modifications may be deliberate, as through site-directed mutagenesis,
or may
be accidental, such as through mutations of hosts which produce the proteins
or errors
due to PCR amplification.
An HCV polypeptide is a polypeptide, as defined above, derived from the
HCV polyprotein. The polypeptide need not be physically derived from HCV, but
may be synthetically or recombinantly produced. Moreover, the polypeptide may
be
derived from any of the various HCV strains and isolates including isolates
having
any of the 6 genotypes of HCV described in Simmonds et al., J. Geh.
Vif°ol. (1993)
74:2391-2399 (e.g., strains l, 2, 3, 4 etc.), as well as newly identified
isolates, and
subtypes of these isolates, such as HCVla, HCVlb etc. A number of conserved
and
variable regions are known between these strains and, in general, the amino
acid
sequences of epitopes derived from these regions will have a high degree of
sequence
homology, e.g., amino acid sequence homology of more than 30%, preferably more
than 40%, when the two sequences are aligned. Thus, for example, the term
"NS4"
polypeptide refers to native NS4 from any of the various HCV strains, as well
as NS4
analogs, muteins and immunogenic fragments, as defined further below.
The terms "analog" and "mutein" refer to biologically active derivatives of
the
reference molecule, or fragments of such derivatives, that retain desired
activity, such
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as the ability to stimulate a cell-mediated immune response, as defined below.
hi the
case of a modified NS3, an "analog" or "mutein" refers to an NS3 molecule that
lacks
its native proteolytic activity. In general, the term "analog" refers to
compounds
having a native polypeptide sequence and structure with one or more amino acid
additions, substitutions (generally conservative in nature, or in the case of
modified
NS3, non-conservative in nature at the active proteolytic site) and/or
deletions,
relative to the native molecule, so long as the modifications do not destroy
immunogenic activity. The term "mutein" refers to peptides having one or more
peptide mimics ("peptoids"), such as those described in International
Publication No.
WO 91/04282. Preferably, the analog or mutein has at least the same
immunoactivity
as the native molecule. Methods for making polypeptide analogs and muteins are
lrnown in the art and are described further below.
As explained above, analogs generally include substitutions that are
conservative in nature, i.e., those substitutions that take place within a
family of
amino acids that are related in their side chains. Specifically, amino acids
are
generally divided into four families: (1) acidic -- aspartate and glutamate;
(2) basic --
lysine, argiune, histidine; (3) non-polar -- alanine, valine, leucine,
isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine,
asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For
example, it is reasonably predictable that an isolated replacement of leucine
with
isoleucine or valine, an aspartate with a glutamate, a threonine with a
serine, or a
similar conservative replacement of an amino acid with a structurally related
amino
acid, will not have a major effect on the biological activity. For example,
the
polypeptide of interest may include up to about 5-10 conservative or non-
conservative
amino acid substitutions, or even up to about 15-25 conservative or non-
conservative
amino acid substitutions, or any integer between 5-25, so long as the desired
function
of the molecule remains intact. One of skill in the art may readily determine
regions
of the molecule of interest that can tolerate change by reference to
Hopp/Woods and
Kyte-Doolittle plots, well known in the art.
CA 02491508 2004-12-31
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By "modified NS3" is meant an NS3 polypeptide with a modification such that
protease activity of the NS3 polypeptide is disrupted. The modification can
include
one or more amino acid additions, substitutions (generally non-conservative in
nature)
and/or deletions, relative to the native molecule, wherein the protease
activity of the
NS3 polypeptide is disrupted. Methods of measuring protease activity are
discussed
further below.
By "fragment" is intended a polypeptide consisting of only a part of the
intact
full-length polypeptide sequence and structure. The fragment can include a
C-terminal deletion and/or an N-terminal deletion of the native polypeptide.
An
"immunogenic fragment" of a particular HCV protein will generally include at
least
about 5-10 contiguous amino acid residues of the full-length molecule,
preferably at
least about 15-25 contiguous amino acid residues of the full-length molecule,
and
most preferably at least about 20-50 or more contiguous amino acid residues of
the
full-length molecule, that define an epitope, or any integer between 5 amino
acids and
the full-length sequence, provided that the fragment in question retains
irnlnunogenic
activity, as measured by the assays described herein.
The term "epitope" as used herein refers to a sequence of at least about 3 to
5,
preferably about 5 to 10 or 15, and not more than about 1,000 amino acids (or
any
integer therebetween), which define a sequence that by itself or as part of a
larger
sequence, binds to an antibody generated in response to such sequence. There
is no
critical upper limit to the length of the fragment, which may comprise nearly
the
full-length of the protein sequence, or even a fusion protein comprising two
or more
epitopes from the HCV polyprotein. An epitope for use in the subject invention
is not
limited to a polypeptide having the exact sequence of the portion of the
parent protein
from which it is derived. Indeed, viral genomes are in a state of constant
flux and
contain several variable domains which exhibit relatively high degrees of
variability
between isolates. Thus the term "epitope" encompasses sequences identical to
the
native sequence, as well as modifications to the native sequence, such as
deletions,
additions and substitutions (generally conservative in nature).
Regions of a given polypeptide that include an epitope can be identified using
any number of epitope mapping techniques, well known in the art. See, e.g.,
Epitope
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Mappifzg Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris,
Ed.,
1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be
determined by e.g., concurrently synthesizing large numbers of peptides on
solid
supports, the peptides corresponding to portions of the protein molecule, and
reacting
the peptides with antibodies while the peptides are still attached to the
supports. Such
techniques are lmown in the art and described in, e.g., U.S. Patent No.
4,708,871;
Geysen et al. (1984) P~oc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al.
(1986) Molec. ITnmuf2ol. 23:709-715. Similarly, conformational epitopes are
readily
identified by determining spatial conformation of amino acids such as by,
e.g., x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,
Epitope
Mappifag P~°otocols, supra. Antigenic regions of proteins can also be
identified using
standard antigenicity and hydropathy plots, such as those calculated using,
e.g., the
Omiga version 1.0 software program available fiom the Oxford Molecular Group.
This computer program employs the Hopp/Woods method, Hopp et al.,
Ps°oc. Natl.
Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and
the
Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for
hydropathy
plots.
For a description of various HCV epitopes, see, e.g., Chien et al., Py-oc.
Natl.
Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J. Gast~oefzt. Hepatol.
(1993)
8:533-39; Chien et al., International Publication No. WO 93/00365; Chien,
D.Y.,
International Publication No. WO 94/01778; and U.S. Patent Nos. 6,280,927 and
6,150,087.
As used herein the term "T-cell epitope" refers to a feature of a peptide
structure which is capable of inducing T-cell immunity towards the peptide
structure
or an associated hapten. T-cell epitopes generally comprise linear peptide
determinants that assume extended conformations within the peptide-binding
cleft of
MHC molecules, ((Jnanue et al., Science (1987) 236:551-557). Conversion of
polypeptides to MHC class II-associated linear peptide determinants (generally
between 5-14 amino acids in length) is termed "antigen processing" which is
carried
out by antigen presenting cells (APCs). More particularly, a T-cell epitope is
defined
by local features of a short peptide structure, such as primary amino acid
sequence
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WO 2004/005473 PCT/US2003/020996
properties involving charge and hydrophobicity, and certain types of secondary
structure, such as helicity, that do not depend on the folding of the entire
polypeptide.
Further, it is believed that short peptides capable of recognition by helper T-
cells are
generally amphipathic structures comprising a hydrophobic side (for
interaction with
the MHC molecule) and a hydrophilic side (for interacting with the T-cell
receptor),
(Margalit et al., Computey~ Prediction of T cell Epitopes, New Generation
Vaccines
Marcel-Del~l~er, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116) and further
that
the amphipathic structures have an a-helical configuration (see, e.g., Spouge
et al., J.
Ifsamuhol. (1987) 138:204-212; Berlcower et al., J. Imnaufaol. (1986) 136:2498-
2503).
Hence, segments of proteins that include T-cell epitopes can be readily
predicted using numerous computer programs. (See e.g., Margalit et al.,
Compute~°
Pj°edictioh of T cell Epitopes, New Generation Vaccines Marcel-Dekker,
Inc, ed. G.C.
Woodrow et al., (1990) pp. 109-116). Such programs generally compare the amino
acid sequence of a peptide to sequences known to induce a T-cell response, and
search for patterns of amino acids wluch are believed to be required for a T-
cell
epitope.
A~z "immunological response" to ail HCV antigen (including both polypeptide
and polynucleotides encoding polypeptides that are expressed in vivo) or
composition
is the development in a subject of a humoral and/or a cellular immune response
to
molecules present in the composition of interest. For purposes of the present
invention, a "humoral immune response" refers to an immune response mediated
by
antibody molecules, while a "cellular immune response" is one mediated by T
lymphocytes and/or other white blood cells. One important aspect of cellular
immunity involves an antigen-specific response by cytolytic T cells ("CTLs").
CTLs
have specificity for peptide antigens that are presented in association with
proteins
encoded by the major histocompatibility complex (MHC) and expressed on the
surfaces of cells. CTLs help induce and promote the intracellular destruction
of
intracellular microbes, or the lysis of cells infected with such microbes.
Both CD8+
and CD4+ T cells are capable of killing HCV-infected cells. A~.zother aspect
of
cellular immunity involves an antigen-specific response by helper T cells.
Helper T
cells act to help stimulate the function, and focus the activity of,
nonspecific effector
13
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WO 2004/005473 PCT/US2003/020996
cells against cells displaying peptide antigens in association with MHC
molecules on
their surface. A "cellular immune response" also refers to the production of
antiviral
cytolcines, chemolcines and other such molecules produced by activated T cells
and/or
other white blood cells, including those derived from CD4+ and CD8+ T cells,
including, but not limited to IFN-y and TNF-a.
A composition or vaccine that elicits a cellular immune response may serve to
sensitize a vertebrate subject by the presentation of antigen in association
with MHC
molecules at the cell surface. The cell-mediated immune response is directed
at, or
near, cells presenting antigen at their surface. In addition, antigen-specific
T
lymphocytes can be generated to allow for the future protection of an
immunized host.
The ability of a particular antigen to stimulate a cell-mediated immunological
response may be determined by a number of assays, such as by
lymphoproliferation
(lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for
T
lymphocytes specific for the antigen in a sensitized subject. Such assays are
well
known in the art. See, e.g., Erickson et al., J. Im~2urt.ol. (1993) 151:4189-
4199; Doe et
al., Eur. J. Imr~zuhol. (1994) 24:2369-2376; and the examples below.
Thus, an irmnunological response as used herein may be one which stimulates
the production of CTLs, and/or the production or activation of helper T cells.
The
antigen of interest may also elicit an antibody-mediated immmle response.
Hence, an
immunological response may include one or more of the following effects: the
production of antibodies by B-cells; and/or the activation of suppressor T
cells and/or
y8 T cells directed specifically to an antigen or antigens present in the
composition or
vaccine of interest. These responses may serve to neutralize infectivity,
and/or
mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to
provide protection or alleviation of symptoms to an immunized host. Such
responses
can be determined using standard immunoassays and neutralization assays, well
known in the art.
By "equivalent antigeuc determinant" is meant an antigenic determinant from
different sub-species or strains of HCV, such as from strains 1, 2, 3, etc.,
of HCV
which antigenic determinants axe not necessarily identical due to sequence
variation,
but which occur in equivalent positions in the HCV sequence in question. In
general
14
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WO 2004/005473 PCT/US2003/020996
the amino acid sequences of equivalent antigenic determinants will have a high
degree
of sequence homology, e.g., amino acid sequence homology of more than 30%, ..
usually more than 40%, such as more than 60%, and even more than 80-90%
homology, when the two sequences are aligned.
A "coding sequence" or a sequence which "encodes" a selected polypeptide, is
a nucleic acid molecule which is transcribed (in the case of DNA) and
translated (in
the case of mRNA) into a polypeptide i~2 vitro or in vivo when placed under
the
control of appropriate regulatory sequences. The boundaries of the coding
sequence
are determined by a start codon at the 5' (amino) terminus and a translation
stop codon
at the 3' (carboxy) terminus. A transcription termination sequence may be
located 3'
to the coding sequence.
A "nucleic acid" molecule or "polynucleotide" can include both double- and
single-stranded sequences and refers to, but is not limited to, cDNA from
viral,
procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA
viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA
sequences. The term also captures sequences that include any of the known base
analogs of DNA and RNA.
"Operably linked" refers to an arrangement of elements wherein the
components so described are configured so as to perform their desired
function.
Thus, a given promoter operably linked to a coding sequence is capable of
effecting
the expression of the coding sequence when the proper transcription factors,
etc., are
present. The promoter need not be contiguous with the coding sequence, so long
as it
functions to direct the expression thereof. Thus, for example, intervening
untranslated
yet transcribed sequences can be present between the promoter sequence and the
coding sequence, as can transcribed introns, and the promoter sequence can
still be
considered "operably linked" to the coding sequence.
"Recombinant" as used herein to describe a nucleic acid molecule means a
polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin
which, by
virtue of its origin or manipulation is not associated with all or a portion
of the
polynucleotide with which it is associated in nature. The term "recombinant"
as used
with respect to a protein or polypeptide means a polypeptide produced by
expression
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
of a recombinant polynucleotide. In general, the gene of interest is cloned
and then
expressed in transformed organisms, as described further below. The host
organism
expresses the foreign gene to produce the protein under expression conditions.
A "control element" refers to a polynucleotide sequence which aids in the
expression of a coding sequence to which it is linlced. The temp includes
promoters,
transcription termination sequences, upstream regulatory domains,
polyadenylation
signals, untranslated regions, including 5'-UTRs and 3'-UTRs and when
appropriate,
leader sequences and enhancers, which collectively provide for the
transcription and
translation of a coding sequence in a host cell.
A "promoter" as used herein is a DNA regulatory region capable of binding
RNA polymerise in a host cell and initiating transcription of a downstream (3'
direction) coding sequence operably linlced thereto. For purposes of the
present
invention, a promoter sequence includes the miumum number of bases or elements
necessary to initiate transcription of a gene of interest at levels detectable
above
background. Within the promoter sequence is a transcription initiation site,
as well as
protein binding domains (consensus sequences) responsible for the binding of
RNA
polymerise. Eucaryotic promoters will often, but not always, contain "TATA"
boxes
and "CAT" boxes.
A control sequence "directs the transcription" of a coding sequence in a cell
when RNA polymerise will bind the promoter sequence and transcribe the coding
sequence into mRNA, which is then translated into the polypeptide encoded by
the
coding sequence.
"Expression cassette" or "expression construct" refers to an assembly which is
capable of directing the expression of the sequences) or genes) of interest.
The
expression cassette includes control elements, as described above, such as a
promoter
which is operably linked to (so as to direct transcription of) the sequences)
or genes)
of interest, and often includes a polyadenylation sequence as well. Within
certain
embodiments of the invention, the expression cassette described herein may be
contained within a plasmid construct. In addition to the components of the
expression
cassette, the plasmid construct may also include, one or more selectable
markers, a
signal which allows the plasmid construct to exist as single-stranded DNA
(e.g., a
16
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
M13 origin of replication), at least one multiple cloning site, and a
"mammalian"
origin of replication (e.g., a SV40 or adenovirus origin of replication).
"Transformation," as used herein, refers to the insertion of an exogenous
polynucleotide into a host cell, irrespective of the method used for
insertion: for
example, transformation by direct uptalce, transfection, infection, and the
life. For
particular methods of transfection, see further below. The exogenous
polynucleotide
may be maintained as a nonintegrated vector, for example, an episome, or
alternatively, may be integrated into the host genome.
A "host cell" is a cell which has been transformed, or is capable of
transformation, by an exogenous DNA sequence.
By "isolated" is meant, when referring to a polypeptide, that the indicated
molecule is separate and discrete from the whole organism with which the
molecule is
found in nature or is present in the substantial absence of other biological
macromolecules of the same type. The term "isolated" with respect to a
polynucleotide is a nucleic acid molecule devoid, in whole or part, of
sequences
normally associated with it in nature; or a sequence, as it exists in nature,
but having
heterologous sequences in association therewith; or a molecule disassociated
from the
chromosome.
The teen "purified" as used herein preferably means at least 75% by weight,
more preferably at least 85% by weight, more preferably still at least 95% by
weight,
and most preferably at least 98% by weight, of biological macromolecules of
the same
type are present.
"Homology" refers to the percent identity between two polynucleotide or two
polypeptide moieties. Two DNA, or two polypeptide sequences are "substantially
homologous" to each other when the sequences exhibit at least about 50% ,
preferably
at least about 75%, more preferably at least about 80%-85%, preferably at
least about
90%, and most preferably at least about 95%-98%, or more, sequence identity
over a
defined length of the molecules. As used herein, substantially homologous also
refers
to sequences showing complete identity to the specified DNA or polypeptide
sequence.
17
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WO 2004/005473 PCT/US2003/020996
In general, "identity" refers to an exact nucleotide-to-nucleotide or amino
acid-to-amino acid correspondence of two polynucleotides or polypeptide
sequences,
respectively. Percent identity can be determined by a direct comparison of the
sequence information between two molecules by aligning the sequences, counting
the
exact number of matches between the two aligned sequences, dividing by the
length
of the shorter sequence, and multiplying the result by 100. Readily available
computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff,
M.O.
in Atlas of P~~otein Sequence and Sty~uctu~~e M.O. Dayhoff ed., 5 Suppl. 3:353-
358,
National biomedical Research Foundation, Washington, DC, which adapts the
local
homology algorithm of Smith and Waterman Advances ifz Appl. Math. 2:482-489,
1981 for peptide analysis. Programs for determining nucleotide sequence
identity are
available in the Wisconsin Sequence Analysis Package, Version 8 (available
from
Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and
GAP programs, which also rely on the Smith and Waternan algorithm. These
programs are readily utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis Paclcage
referred to
above. For example, percent identity of a particular nucleotide sequence to a
reference sequence can be determined using the homology algorithm of Smith and
Waterman with a default scoring table and a gap penalty of six nucleotide
positions.
Another method of establishing percent identity in the context of the present
invention is to use the MPSRCH package of programs copyrighted by the
University
of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and
distributed by
IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the
Smith-Waterman algorithm can be employed where default parameters are used for
the scoring table (for example, gap open penalty of 12, gap extension penalty
of one,
and a gap of six). From the data generated the "Match" value reflects
"sequence
identity." Other suitable programs for calculating the percent identity or
similarity
between sequences are generally known in the art, for example, another
alignment
program is BLAST, used with default parameters. For example, BLASTN and
BLASTP can be used using the following default parameters: genetic code =
standard;
filter = none; strand ,-- both; cutoff = 60; expect = 10; Matrix = BLOSUM62;
18
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WO 2004/005473 PCT/US2003/020996
Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant,
GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein +
Spupdate + PIR. Details of these programs can be found at the following
Internet
address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions which form stable duplexes between homologous
regions, followed by digestion with single-stranded-specific nuclease(s), and
size
determination of the digested fragments. DNA sequences that are substantially
homologous can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the art. See,
e.g., Sambroolc
et al., supra; DNA Clohif2g, supra; Nucleic Acid HybridizatiofZ, supra.
By "nucleic acid immunization" is meant the introduction of a nucleic acid
molecule encoding one or more selected immunogens into a host cell, for the in
vivo
expression of the immunogen or immunogens. The nucleic acid molecule can be
introduced directly into the recipient subject, such as by injection,
inhalation, oral,
intra~lasal and mucosal administration, or the lilce, or can be introduced ex
vivo, into
cells which have been removed from the host. In the latter case, the
transformed cells
are reintroduced into the subject where an immune response can be mounted
against
the antigen encoded by the nucleic acid molecule.
As used herein, "treatment" refers to any of (i) the prevention of infection
or
reinfection, as in a traditional vaccine, (ii) the reduction or elimination of
symptoms,
and (iii) the substantial or complete elimination of the pathogen in question.
Treatment may be effected prophylactically (prior to infection) or
therapeutically
(following infection).
By "vertebrate subject" is meant any member of the subphylum cordata,
including, without limitation, humans and other primates, including non-human
primates such as chimpanzees and other apes and monkey species; farm animals
such
as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and
cats;
laboratory animals including rodents such as mice, rats and guinea pigs;
birds,
including domestic, wild and game birds such as chiclcens, turkeys and other
19
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WO 2004/005473 PCT/US2003/020996
gallinaceous birds, duclcs, geese, and the lilce. The term does not denote a
particular
age. Thus, both adult and newborn individuals are intended to be covered. The
invention described herein is intended for use in any of the above vertebrate
species,
since the innnune systems of all of these vertebrates operate similarly.
II. Modes of Carrying out the Invention
Before describing the present invention in detail, it is to be understood that
this
invention is not limited to particular formulations or process parameters as
such may,
of course, vary. It is also to be understood that the terminology used herein
is for the
purpose of describing particular embodiments of the invention only, and is not
intended to be limiting.
Although a number of compositions and methods similar or equivalent to
those described herein can be used in the practice of the present invention,
the
preferred materials and methods are described herein.
The present invention pertains to fusion proteins and polynucleotides encoding
the same, comprising a modified NS3 polypeptide and at least one other HCV
polypeptide from the HCV polyprotein. The fusion proteins of the present
invention
can be used to stimulate a cellular immune response, such as to activate HCV-
specific
T cells, i.e., T cells which recognize epitopes of these polypeptides and/or
to elicit the
production of helper T cells and/or to stimulate the production of antiviral
cytol~ines,
chemolcines, and the lilce. Activation of HCV-specific T cells by such fusion
proteins
provides both ih vitro and ifa vivo model systems for the development of HCV
vaccines, particularly for identifying HCV polypeptide epitopes associated
with a
response. The fusion proteins can also be used to generate an immune response
against HCV in a mammal, for example a CTL response, and/or to prime CD~+ and
CD4+ T cells to produce antiviral agents, for either therapeutic or
prophylactic
purposes.
In order to further an understanding of the invention, a more detailed
discussion is provided below regarding fusion proteins for use in the subject
compositions, as well as production of the proteins, compositions comprising
the
same and methods of using the proteins.
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
Fusion Pf°oteirzs
The genomes of HCV strains contain a single open reading frame of
approximately 9,000 to 12,000 nucleotides, which is transcribed into a
polyprotein.
S As shown in Figure 1 and Table 1, an HCV polyprotein, upon cleavage,
produces at
least ten distinct products, in the order of NH2-
Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NSSa-NSSb-COOH. The core polypeptide
occurs at positions 1-191, numbered relative to HCV-1 (see, Choo et al. (1991)
P~oc.
Natl. Acad. Sci. USA 88:2451-2455, for the HCV-1 genome). This polypeptide is
further processed to produce an HCV polypeptide with approximately amino acids
1-173. The envelope polypeptides, E1 and E2, occur at about positions 192-383
and
384-746, respectively. The P7 domain is found at about positions 747-809. NS2
is an
integral membrane protein with proteolytic activity and is found at about
positions
810-1026 of the polyprotein. NS2, in combination with NS3, (found at about
positions 1027-1657), cleaves the NS2-NS3 sissle bond which in turn generates
the
NS3 N-terminus and releases a large polyprotein that includes both serine
protease
and RNA helicase activities. The NS3 protease, found at about positions 1027-
1207,
serves to process the remaining polyprotein. The helicase activity is found at
about
positions 1193-1657. NS3 liberates an NS3 cofactor (NS4a, found about
positions
1658-1711), two proteins (NS4b found at about positions 1712-1972, and NSSa
found
at about positions 1973-2420), and an RNA-dependent RNA polymerase (NSSb found
at about positions 2421-3011). Completion of polyprotein maturation is
initiated by
autocatalytic cleavage at the NS3-Ns4a junction, catalyzed by the NS3 serine
protease.
Table 1
omain Approximate Boundaries*
C (core) 1-191
E1 192-383
E2 3 84-746
7 747-809
S2 810-1026
21
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S3 1027-1657
S4a 1658-1711
S4b 1712-1972
NSSa 1973-2420
NSSb 2421-3011
*Numbered relative to HCV-1. See, Choo et al. (1991) Pf°oc. Natl. Acad.
Sci.
USA 88:2451-2455.
Fusion proteins of the invention include an NS3 polypeptide modified to
inhibit protease activity, such that further cleavage of the fusion is
inhibited. The NS3
polypeptide can be modified by deletion of all or a portion of the NS3
protease
domain. Alternatively, proteolytic activity can be inhibited by substitutions
of amino
acids within active regions of the protease domain. Finally, additions of
amino acids
to active regions of the domain, such that the catalytic site is modified,
will also serve
to inhibit proteolytic activity.
As explained above, the protease activity is found at about amino acid
positions 1027-1207, numbered relative to the full-length HCV-1 polyprotein
(see,
Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455), positions 2-182
of
Figure 2 (SEQ ID N0:4). The structure of the NS3 protease and active site are
known. See, e.g., De Francesco et al., Antivif°. Tlrer. (1998) 3:99-
109; Koch et al.,
Biochemistry (2001) 40:631-640. Thus, deletions or modifications to the native
sequence will typically occur at or near the active site of the molecule.
Particularly, it
is desirable to modify or make deletions to one or more amino acids occurnng
at
positions 1- or 2-182, preferably 1- or 2-170, or 1- or 2-155 of Figure 2 (SEQ
ID
N0:4). Preferred modifications are to the catalytic triad at the active site
of the
protease, i.e., H, D and/or S residues, in order to inactivate the protease.
These
residues occur at positions 1083, 1105 and 1165, respectively, numbered
relative to
the full-length HCV polyprotein (positions 58, 80 and 140, respectively, of
Figure 2
(SEQ ID N0:4)). Such modifications will suppress proteolytic cleavage while
maintaining T-cell epitopes.
One of skill in the art can readily determine portions of the NS3 protease to
delete in order to disrupt activity. The presence or absence of activity can
be
determined using methods known to those of skill in the art.
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WO 2004/005473 PCT/US2003/020996
For example, protease activity or lacy thereof may be determined using the
procedure described below in the examples, as well as using assays well known
in the
art. See, e.g., Tal~eshita et al., Anal. Biochem. (1997) 247:242-246;
Kalciuchi et al., J.
Biochem. (1997) 122:749-755; Sali et al., Biochemistp (1998) 37:3392-3401; Cho
et
al., J. Virol. Meth. (1998) 72:109-115; Cerretani et al., Anal. Biochem..
(1999)
266:192-197; Zhang et al., Anal. Biochena. (1999) 270:268-275; Kal~iuchi et
al., J.
Viol. Meth. (1999) X0:77-84; Fowler et al., J. Biomol. Screen. (2000) 5:153-
158; and
Kim et al., Anal. Biochem. (2000) 284:42-48.
The fusion protein of the present invention includes, in addition to the
modified NS3 polypeptide, one or more polypeptides from one or more other
regions
of an HCV polyprotein. In fact, the fusion can include all the regions of the
HCV
polyprotein. These polypeptides may derived from the same HCV isolate as the
NS3 polypeptide, or from different strains and isolates including isolates
having any
of the various HCV genotypes, to provide increased protection against a broad
range
of HCV genotypes. Additionally, polypeptides can be selected based on the
particular
viral Glades endemic in specific geograpluc regions where vaccine compositions
containing the fusions will be used. It is readily apparent that the subject
fusions
provide an effective means of treating HCV infection in a wide variety of
contexts.
In certain embodiments, the fusion protein comprises a modified NS3 (also
referred to herein as NS3*), an NS4 (NS4a and NS4b), an NSSa and, optionally,
a
core polypeptide of an HCV (NS3*NS4NSSa or NS3*NS4NSSaCore fusion proteins,
also termed "NS3*45a" and "NS3*45aCore" herein). These regions need not be in
the order in which they naturally occur in the native HCV polyprotein. Thus,
for
example, the core polypeptide may be at the N- and/or C-terminus of the
fusion.
Another embodiment provides a fusion protein that includes an NS3*, an NS4,
a~i NSSa, an NSSb, and optionally, a core polypeptide of an HCV
(NS3*NS4NSSaNSSb or NS3*NS4NSSaNSSbCore fusion proteins, also termed
"NS3*45ab" and "NS3*45abCore" herein). These regions need not be in the order
in
which they naturally occur in the native HCV polyprotein. Thus, for example,
the
core polypeptide may be at the N- and/or C-terminus of the fusion.
23
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WO 2004/005473 PCT/US2003/020996
Yet other embodiments are directed to a fusion protein comprising an NS3*
combined with an NS2, an NS3* combined with an NS2, p7 and E2, an NS3*
combined with an NS2, p7 and an E1, an NS3* combined with an NS2, p7 and an E1
and an E2, an NS3* combined with an E2, an NS3* combined with an E1 and an E2,
all with or without a core polypeptide. As with those fusions described above,
these
regions need not be in the order in which they occur naturally. Moreover, each
of
these regions can be derived from the same or a different HCV isolate.
Figure 3 (SEQ ID NOS:S-6) shows a representative modified fusion protein,
with the NS3 protease domain deleted from the N-terminus and including amino
acids
1-121 of Core on the C-terminus.
The various HCV polypeptides present in the various fusions described above
can either be full-length polypeptides or portions thereof. The portions of
the HCV
polypeptides malting up the fusion protein comprise at least one epitope,
which is
recognized by a T cell receptor on an activated T cell, such as
2152-HEYPVGSQL-2160 (SEQ ID NO:1) and/or
2224-AELIEANLLWRQEMG-2238 (SEQ ID N0:2). Epitopes ofNS2, p7, El, E2,
NS3, NS4 (NS4a and NS4b), NSSa, NSSb, NS3NS4NSSa, and NS3NS4NSSaNSSb
can be identified by several methods. For example, the individual polypeptides
or
fusion proteins comprising any combination of the above, can be isolated, by,
e.g.,
immunoaffinity purification using a monoclonal antibody for the polypeptide or
protein. The isolated protein sequence can then be screened by preparing a
series of
short peptides by proteolytic cleavage of the purified protein, which together
span the
entire protein sequence. By starting with, for example, 100-mer polypeptides,
each
polypeptide can be tested for the presence of epitopes recognized by a T-cell
receptor
on an HCV-activated T cell, progressively smaller and overlapping fragments
can
then be tested from an identified 100-mer to map the epitope of interest.
Epitopes recognized by a T-cell receptor on an HCV-activated T cell can be
identified by, for example, 51 Cr release assay (see Example 4) or by
lymphoproliferation assay (see Example 6). In a SlCr release assay, target
cells can
be constructed that display the epitope of interest by cloning a
polynucleotide
encoding the epitope into an expression vector and transforming the expression
vector
24
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WO 2004/005473 PCT/US2003/020996
into the target cells. HCV-specific CD8+ T cells will lyse target cells
displaying, for
example, one or more epitopes from one or more regions of the HCV polyprotein
found in the fusion, and will not lyse cells that do not display such an
epitope. In a
lynphoproliferation assay, HCV-activated CD4+ T cells will proliferate when
cultured with, for example, one or more epitopes from one or more regions of
the
HCV polyprotein found in the fusion, but not in the absence of an HCV epitopic
peptide.
The various HCV polypeptides can occur in any order in the fusion protein. If
desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of one or more of the
polypeptides
may occur in the fusion protein. Multiple viral strains of HCV occur, and HCV
polypeptides of any of these strains can be used in a fusion protein.
Nucleic acid and amino acid sequences of a number of HCV strains and
isolates, including nucleic acid and amino acid sequences of the various
regions of the
HCV polyprotein, including Core, NS2, p7, E1, E2, NS3, NS4, NSSa, NSSb genes
and polypeptides have been determined. For example, isolate HCV J1.1 is
described
in I~ubo et al. (1989) Japan. Nucl. Acids Res. 17:10367-10372; Talceuchi et
al. (1990)
Gene 91:287-291; Tal~euchi et al. (1990) J. Gen. Virol. 71:3027-3033; and
Talceuchi
et al. (1990) Nucl. Acids Res. 18:4626. The complete coding sequences of two
independent isolates, HCV-J and BK, are described by Nato et al., (1990) Proc.
Natl.
Acad. Sci. USA 87:9524-9528 and Talcamizawa et al., (1991) J. Virol. 65:1105-
1113
respectively.
Publications that describe HCV-1 isolates include Choo et al. (1990) Brit.
Med. Bull. 46:423-441; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-
2455
and Han et al. (1991) Proc. Natl. Acad. Sci. USA 88:1711-1715. HCV isolates HC-
J1
and HC-J4 are described in Okamoto et al. (1991) Japan J. Exp. Med. 60:167-
177.
HCV isolates HCT 18~, HCT 23, Th, HCT 27, EC1 and EC10 are described in
Weiner et al. (1991) Virol. 180:842-848. HCV isolates Pt-1, HCV-Kl and HCV-K2
are described in Enomoto et al. (1990) Biochem. Biophys. Res. Commun.
170:1021-1025. HCV isolates A, C, D & E are described in Tsulciyama-I~ohara et
al.
(1991) Virus Genes 5:243-254.
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
Each of the components of a fusion protein can be obtained from the same
HCV strain or isolate or from different HCV strains or isolates. Fusion
proteins
comprising HCV polypeptides from, for example, the NS3 polypeptide can be
derived
from a first strain of HCV, and the other HCV polypeptides present can be
derived
from a second strain of HCV. Alternatively, one or more of the other HCV
polypeptides, for example NS2, NS4, Core, p7, E1 and/or E2, if present, can be
derived from a first strain ofHCV, and the remaining HCV polypeptides can be
derived from a second strain of HCV. Additionally, each or the HCV
polypeptides
present can be derived from different HCV strains.
As explained above, it may be desirable to include polypeptides derived from
the core region of the HCV polyprotein in the fusions of the invention. This
region
occurs at amino acid positions 1-191 of the HCV polyprotein, numbered relative
to
HCV-1. Either the full-length protein, fragments thereof, such as amino acids
1-160,
e.g., amino acids 1-150, 1-140, 1-130, 1-120, for example, amino acids 1-121,
1-122,
1-123...1-151, etc., or smaller fragments containing epitopes of the full-
length protein
may be used in the subject fusions, such as those epitopes found between amino
acids
10-53, amino acids 10-45, amino acids 67-88, amino acids 120-130, or any of
the core
epitopes identified in, e.g., Houghton et al., U.S. Patent No. 5,350,671;
Chien et al.,
Ps°oe. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J.
Gastf°oeyat.
Flepatol. (1993) 8:533-39; Chien et al., International Publication No. WO
93/00365;
Chien, D.Y., International Publication No. WO 94/01778; and U.S. Patent Nos.
6,280,927 and 6,150,087. Moreover, a protein resulting from a frameshift in
the core
region of the polyprotein, such as described in International Publication No.
WO
99/63941, may be used.
If a core polypeptide is present, it can occur at the N- terminus, the
C-terminus and/or internal to the fusion. Particularly preferred is a core
polypeptide
on the C-terminus as this allows for the formation of complexes with certain
adjuvants, such as ISCOMs, described further below.
As described above, useful polypeptides in the HCV fusion include T-cell
epitopes derived from any of the various regions in the polyprotein. In this
regard,
E1, E2, p7 and NS2 are known to contain human T-cell epitopes (both CD4+ and
26
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
CD8+) and including one or more of these epitopes serves to increase vaccine
efficacy as well as to increase protective levels against multiple HCV
genotypes.
Moreover, multiple copies of specific, conserved T-cell epitopes can also be
used in
the fusions, such as a composite of epitopes from different genotypes.
For example, polypeptides from the HCV E1 and/or E2 regions can be used in
the fusions of the present invention. E2 exists as multiple species (Spaete et
al., Iji~ol.
(1992) 188:819-830; Selby et al., J. Yif-ol. (1996) 70:5177-5182; Gralcoui et
al., J.
Ijif°ol.~(1993) 67:1385-1395; Tomei et al., J. Vi~~ol. (1993) 67:4017-
4026) and clipping
and proteolysis may occur at the N- and C-termini of the E2 polypeptide. Thus,
an
E2 polypeptide for use herein may comprise amino acids 405-661, e.g., 400,
401,
402... to 661, such as 383 or 384-661, 383 or 384-715, 383 or 384-746, 383 or
384-749 or 383 or 384-809; or 383 or 384 to any C-terminus between 661-809, of
an
HCV polyprotein, numbered relative to the full-length HCV-1 polyprotein.
Similarly,
E1 polypeptides for use herein can comprise amino acids 192-326, 192-330, 192-
333,
192-360, 192-363, 192-383, or 192 to any C-terminus between 326-383, of an HCV
polyprotein.
Immunogenic fragments of El and/or E2 which comprise epitopes may be
used in the subject fusions. For example, fragments of El polypeptides can
comprise
from about 5 to nearly the full-length of the molecule, such as 6, 10, 25, 50,
75, 100,
125, 150, 175, 185 or more amino acids of an E1 polypeptide, or any integer
between
the stated numbers. Similarly, fragments of E2 polypeptides can comprise 6,
10, 25,
50, 75, 100, 150, 200, 250, 300, or 350 amino acids of an E2 polypeptide, or
any
integer between the stated numbers.
For example, epitopes derived from, e.g., the hypervariable region of E2, such
as a region spanning amino acids 384-410 or 390-410, can be included in the
fusions.
A particularly effective E2 epitope to incorporate into an E2 polypeptide
sequence is
one which includes a consensus sequence derived from this region, such as the
consensussequence
Gly-S er-Ala-Ala-Arg-Thr-Thr-S er-Gly-Phe-V al-S er-Leu-Phe-Ala-Pro-Gly-
Ala-Lys-Gln-Asn (SEQ ID N0:7),'which represents a consensus sequence for amino
acids 390-410 of the HCV type 1 genome. Additional epitopes of E1 and E2 are
27
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
l~nown and described in, e.g., Chien et al., International Publication No. WO
93/00365.
Moreover, the E1 and/or E2 polypeptides may lacy all or a portion of the
membrane spanning domain. With E1, generally polypeptides terminating with
about
amino acid position 370 and higher (based on the numbering of HCV-1 E1) will
be
retained by the ER and hence not secreted into growth media. With E2,
polypeptides
terminating with about amino acid position 731 and higher (also based on the
numbering of the HCV-1 E2 sequence) will be retained by the ER and not
secreted.
(See, e.g., International Publication No. WO 96/04301, published February 15,
1996).
It should be noted that these amino acid positions are not absolute and may
vary to
some degree. Thus, the present invention contemplates the use of El and/or E2
polypeptides which retain the transmembrane binding domain, as well as
polypeptides
which laclc all or a portion of the transmembrane binding domain, including E1
polypeptides terminating at about amino acids 369 and lower, and E2
polypeptides,
terminating at about amino acids 730 and lower. Furthermore, the C-terminal
truncation can extend beyond the transmembrane spanning domain towards the
N-terminus. Thus, for example, E1 truncations occurring at positions lower
than, e.g.,
360 and E2 truncations occurring at positions lower than, e.g., 715, are also
encompassed by the present invention. All that is necessary is that the
truncated E1
and E2 polypeptides remain functional for their intended purpose. However,
particularly preferred truncated E1 constructs are those that do not extend
beyond
about amino acid 300. Most preferred are those terminating at position 360.
Preferred truncated E2 constructs are those with C-terminal truncations that
do not
extend beyond about amino acid position 715. Particularly preferred E2
truncations
are those molecules truncated after any of amino acids 715-730, such as 725.
For a description of various HCV epitopes from these and other
HCV regions, see, e.g., Chien et al., Proc. Natl. Acad. Sci. USA (1992)
89:10011-10015; Chien et al., J. Gast~~eyat. Hepatol. (1993) 8:533-39; Chien
et al.,
International Publication No. WO 93/00365; Cluen, D.Y., International
Publication
No. WO 94/01778; and U.S. Patent Nos. 6,280,927 and 6,150,087.
28
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
Preferably, the above-described fusion proteins, as well as the individual
components of these proteins, are produced recombinantly. A polynucleotide
encoding these proteins can be introduced into an expression vector which can
be
expressed in a suitable expression system. A variety of bacterial, yeast,
mammalian
and insect expression systems are available in the art and any such expression
system
can be used. Optionally, a polynucleotide encoding these proteins can be
translated in
a cell-free translation system. Such methods are well known in the art. The
proteins
also can be constructed by solid phase protein synthesis.
If desired, the fusion proteins, or the individual components of these
proteins,
also can contain other amino acid sequences, such as amino acid linkers or
signal
sequences, as well as ligands useful in protein purification, such as
glutathione-S-transferase and staphylococcal protein A.
Polyrtucleotides Etacoditzg the Fusion Proteihs
Polynucleotides contain less than an entire HCV genome, or alternatively can
include the sequence of the entire polyprotein with a mutated NS3 domain, as
described above. The polynucleotides can be RNA or single- or double-stranded
DNA. Preferably, the polynucleotides are isolated free of other components,
such as
proteins and lipids. The polynucleotides encode the fusion proteins described
above,
and thus comprise coding sequences for NS3* and at least one other HCV
polypeptide
from a different region of the HCV polyprotein, such as polypeptides derived
from
NS2, p7, E1, E2, NS4, NSSa, NSSb, core, etc. Polynucleotides of the invention
can
also comprise other nucleotide sequences, such as sequences coding for
linkers, signal
sequences, or ligands useful in protein purification such as glutathione-S-
transferase
~ and staphylococcal protein A.
To aid expression yields, it may be desirable to split the polyprotein into
fragments for expression. These fragments can be used in combination in
compositions as described herein. Alternatively, these fragments can be joined
subsequent to expression. Thus, for example, NS3*NS4Core can be expressed as
one
construct and NSSaNSSbCore can be expressed as a second construct. Similarly,
NS3*NS4NSSa can be expressed as one construct and a second construct with,
e.g.,
29
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
NS3*NS4NSSaCore can be expressed as a second construct. For example,
NS2p7E2NS3~NS4 can be expressed as a single construct, and
NS3(unmodified)NS4NSSb can be expressed as an additional construct for use in
the
subject compositions. It is to be understood that the above combinations axe
merely
representative and any combination of fusions can be expressed separately.
Polynucleotides encoding the various HCV polypeptides can be isolated from
a genomic library derived from nucleic acid sequences present in, for example,
the
plasma, serum, or liver homogenate of an HCV infected individual or can be
synthesized in the laboratory, for example, using an automatic synthesizer. An
amplification method such as PCR, can be used to amplify polynucleotides from
either
HCV genomic DNA or cDNA encoding therefor.
Polynucleotides can comprise coding sequences for these polypeptides which
occur naturally or can be artificial sequences which do not occur in nature.
These
polynucleotides can be ligated to form a coding sequence for the fusion
proteins using
standard molecular biology techniques. If desired, polynucleotides can be
cloned into
an expression vector and transformed into, for example, bacterial, yeast,
insect, or
mammalian cells so that the fusion proteins of the invention can be expressed
in and
isolated from a cell culture.
The expression constructs of the present invention, including the desired
fusion, or individual expression constructs comprising the individual
components of
these fusions, may be used for nucleic acid immunization, to stimulate a
cellular
immune response, using standard gene delivery protocols. Methods for gene
delivery
are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859,
5,589,466.
Genes can be delivered either directly to the vertebrate subject or,
alternatively,
delivered ex vivo, to cells derived from the subject and the cells reimplanted
in the
subject. For example, the constructs can be delivered as plasmid DNA, e.g.,
contained within a plasmid, such as pBR322, pUC, or ColEl
Additionally, the expression constructs can be paclcaged in liposomes prior to
delivery to the cells. Lipid encapsulation is generally accomplished using
liposomes
which are able to stably bind or entrap and retain nucleic acid. The ratio of
condensed
DNA to lipid preparation can vary but will generally be around 1:1 (mg
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes
as
carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim.
Biophys. Acta.
(1991) 1097:1-17; Straubinger et al., inMetIZOds ofEfazysraology (1983), Vol.
101, pp.
512-527.
Liposomal preparations for use with the present invention include cationic
(positively charged), anionic (negatively charged) and neutral preparations,
with
cationic liposomes particularly preferred. Cationic liposomes are readily
available.
For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA)
liposomes are available under the trademark Lipofectin, from GIBCO BBL, Grand
Island, NY. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)
84:7413-7416). Other commercially available lipids include transfectace
(DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be
prepared from readily available materials using techniques well known in the
art.
See, e.g., Szoka et al., P~oc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT
Publication No. WO 90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. The various
liposome-nucleic acid complexes are prepared using methods known in the art.
See,
e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp.
512-527; Szolca et al., P~oc. Natl. Acad. Sci. USA (1978) 75:4194-4198;
Papahadjopoulos et al., Biochim. Biop7Zys. Acta (1975) 394:483; Wilson et al.,
Cell
(1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629;
Ostro
et al., Bioclaem. Biophys. Res. Commufa. (1977) 76:836; Fraley et al.,
Pf°oc. Natl.
Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Pi°oc. Natl.
Acad. Sci. USA
(1979) 76:145); Fraley et al., J. Biol. Claerya. (1980) 255:10431; Szoka and
Papahadjopoulos, PYOC. Natl. Acad. Sci. USA (1978) 75:145; a~ld Schaefer-
Bidder et
al., Science (1982) 215:166.
The DNA can also be delivered in cochleate lipid compositions similar to
those described by Papahadjopoulos et al., Bioclaena. Biophys. Acta. (1975)
394:483-491. See, also, U.S. Patent Nos. 4,663,161 and 4,871,488.
A number of viral based systems have been developed for gene transfer into
mammalian cells. For example, retroviruses provide a convenient platform for
gene
31
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
delivery systems, such as murine sarcoma virus, mouse mammary tumor virus,
Moloney murine leukemia virus, and Rous sarcoma virus. A selected gene can be
inserted into a vector and paclcaged in retroviral particles using techniques
known in
the art. The recombinant virus can then be isolated and delivered to cells of
the
subject either irr. vivo or ex vivo. A number of retroviral systems have been
described
(LJ.S. Patent No. 5,219,740; Miller and Rosman, BioTechyaiques (1989) 7:980-
990;
Miller, A.D., Human Gefae Thef°apy (1990) 1:5-14; Scarpa et al.,
Virology (1991)
180:849-852; Burns et al., P~oc. Natl. Acad. Sci. USA (1993) 90:8033-8037;
a~zd
Boris-Lawrie and Temin, Cup. Opih. Genet. Develop. (1993) 3:102-109. Briefly,
retroviral gene delivery vehicles of the present invention may be readily
constructed
from a wide variety of retroviruses, including for example, B, C, and D type
retroviruses as well as spumaviruses and lentiviruses such as FIV, HIV, HIV-1,
HIV-2 arid SIV (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor
Laboratory, 1985). Such retroviruses may be readily obtained from depositories
or
collections such as the American Type Culture Collection ("ATCC"; 10801
University Blvd., Manassas, VA 20110-2209), or isolated from known sources
using
commonly available techniques.
A number of adenovirus vectors have also been described, such as adenovirus
Type 2 and Type 5 vectors. Unlilce retroviruses which integrate into the host
genome,
adenoviruses persist extrachromosomally thus minimizing the rislcs associated
with
insertional mutagenesis (Haj-Ahmad and Graham, J. Viol. (1986) 57:267-274;
Bett et
al., J. Yirol. (1993) 67:5911-5921; Mittereder et al., Human Gehe Therapy
(1994)
5:717-729; Seth et al., J. Tri~ol. (1994) 68:933-940; Barr et al., Gef~e
Tlaef°apy (1994)
1:51-58; Berkner, I~.L. BioTeclz~iques (1988) 6:616-629; and Rich et al.,
Humaya
Gehe TheYapy (1993) 4:461-476).
Molecular conjugate vectors, such as the adenovirus chimeric vectors
described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et
al.,
Pf°oc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for
gene delivery.
Members of the Alphavirus genus, such as but not limited to vectors derived
from the Sindbis and Semliki Forest viruses, VEE, will also find use as viral
vectors
for delivering the gene of interest. For a description of Sindbis-virus
derived vectors
32
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
useful for the practice of the instant methods, see, Dubensky et al., J.
Pirol. (1996)
70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072.
Other vectors can be used, including but not limited to simian virus 40 and
cytomegalovirus. Bacterial vectors, such as Salmonella ssp. Ye~sifaia
entef°ocolitica,
Shigella spp., Vibrio chole~ae, Mycobacteriuan strain BCG, and Listeria
moraocytogeraes can be used. Minicluomosomes such as MC and MC1,
bacteriophages, cosmids (plasmids into which phage lambda cos sites have been
inserted) and replicons (genetic elements that are capable of replication
under their
own control in a cell) can also be used.
The expression constructs may also be encapsulated, adsorbed to, or
associated with, particulate carriers. Such carriers present multiple copies
of a
selected molecule to the immune system and promote trapping and retention of
molecules in local lymph nodes. The particles can be phagocytosed by
macrophages
and can enhance antigen presentation through cytol~ine release. Examples of
particulate Garners include those derived from polymethyl methacrylate
polymers, as
well as microparticles derived from poly(lactides) and poly(lactide-co-
glycolides),
known as PLG. See, e.g., Jeffery et al., Plaa~m. Res. (1993) 10:362-368; and
McGee
et al., J. Mic~~oencap. (1996).
A wide variety of other methods can be used to deliver the expression
constructs to cells. Such methods include DEAF dextran-mediated transfection,
calcium phosphate precipitation, polylysine- or polyornithine-mediated
transfection,
or precipitation using other insoluble inorganic salts, such as strontium
phosphate,
aluminum silicates including bentonite and kaolin, chromic oxide, magnesium
silicate, talc, and the like. Other useful methods of transfection include
electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery,
or
microinjection. See, e.g., Sambrook et al., supYa, for a discussion of
techniques for
transforming cells of interest; and Felgner, P.L., Advafaced D~°ug
Delivery Reviews
(1990) 5:163-187, for a review of delivery systems useful for gene transfer.
One
particularly effective method of delivering DNA using electroporation is
described in
International Publication No. WO/0045823.
33
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
Additionally, biolistic delivery systems employing particulate carriers such
as
gold and tungsten, are especially useful for delivering the expression
constructs of the
present invention. The particles are coated with the construct to be delivered
and
accelerated to high velocity, generally under a reduced atmosphere, using a
gun
powder discharge from a "gene gun." For a description of such techniques, and
apparatuses useful therefore, see, e.g., U.S. Patent Nos. 4,945,050;
5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,47,744.
Corrapositions Cofnprisifzg Fusion P~oteiras o~° Polyfaucleotides
The invention also provides compositions comprising the fusion proteins or
polynucleotides. The compositions may include one or more fusions, so long as
one
of the fusions includes a mutated NS3 domain as described herein. Compositions
of
the invention may also comprise a pharmaceutically acceptable carrier. The
carrier
should not itself induce the production of antibodies harmful to the host.
Pharmaceutically acceptable carriers are well known to those in the art. Such
carriers
include, but are not limited to, large, slowly metabolized, macromolecules,
such as
proteins, polysaccharides such as latex functionalized sepharose, agarose,
cellulose,
cellulose beads and the like, polylactic acids, polyglycolic acids, polymeric
amino
acids such as polyglutamic acid, polylysine, and the like, amino acid
copolymers, and
inactive virus particles.
Pharmaceutically acceptable salts can also be used in compositions of the
invention, for example, mineral salts such as hydrochlorides, hydrobromides,
phosphates, or sulfates, as well as salts of organic acids such as acetates,
proprionates,
malonates, or benzoates. Especially useful protein substrates are serum
albumins,
keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin,
tetanus toxoid, and other proteins well known to those of skill in the art.
Compositions of the invention can also contain liquids or excipients, such as
water,
saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as
well as
substances such as wetting agents, emulsifying agents, or pH buffering agents.
The
proteins of the invention can also be adsorbed to, entrapped within or
otherwise
34
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
associated with liposomes and particulate carriers such as PLG. Liposomes and
other
pas-ticulate carriers are described above.
If desired, co-stimulatory molecules which improve immunogen presentation
to lymphocytes, such as B7-1 or B7-2, or cytolcines, lympholcines, and
chemokines,
including but not limited to cytolcines such as IL-2, modified IL-2
(cys125~ser125),
GM-CSF, IL-12, 'y- interferon, IP-10, MIP 1 (3, FLP-3, ribavirin and RANTES,
may be
included in the composition. Optionally, adjuvants can also be included in a
composition. Adjuvants which can be used include, but are not limited to:
(1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, '
aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without
other
specific immunostimulating agents such as muramyl peptides (see below) or
bacterial
cell wall components), such as for example (a) MF59 (PCT Publ. No. WO
90/14837),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing
various amounts of MTP-PE ), formulated into submicron particles using a
microfluidizer such as Model 1 l0Y microfluidizer (Microfluidics, Newton, MA),
(b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer
L121, and thr-MDP (see below) either microfluidized into a submicron emulsion
or
vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant
system
(R.AS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80,
and one or more bacterial cell wall components from the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton
(CWS), preferably MPL + CWS (DetoxTM); (3) saponin adjuvants, such as QS21 or
StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles
generated therefrom such as ISCOMs (immunostunulating complexes), which
ISCOMs may be devoid of additional detergent (see, e.g., International
Publication
No. WO 00/07621); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds
Adjuvant (IFA); (5) cytokines, such as interleulcins, such as IL-1, IL-2, IL-
4, IL-5,
IL-6, IL-7, IL-12 etc. (see, e.g., International Publication No. WO 99/44636),
interferons, such as gamma interferon, macrophage colony stimulating factor
(M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified mutants of a
bacterial
ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT),
or an E.
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
coli heat-labile toxin (LT), particularly LT-K63 (where lysine is substituted
for the
wild-type amino acid at position 63) LT-R72 (where arginine is substituted for
the
wild-type amino acid at position 72), CT-S 109 (where serine is substituted
for the
wild-type amino acid at position 109), and PT-K9/G129 (where lysine is
substituted
for the wild-type amino acid at position 9 and glycine substituted at position
129)
(see, e.g., International Publication Nos. W093/13202 and W092/19265); (7)
monophosporyl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB
2220221; EPA 0689454), optionally in the substantial absence of alum (see,
e.g.,
International Publication No. WO 00/56358); (8) combinations of 3dMPL with,
for
example, QS21 and/or oil-in-water emulations (see, e.g., EPA 0835318; EPA
0735898; EPA 0761231); (9) a polyoxyethylene ether or a polyoxyethylene ester
(see,
e.g., International Publication No. WO 99/52549); (10) an immunostimulatory
oligonucleotide such as a CpG oligonucleotide, or a saponin and an
immunostimulatory oligonucleotide, such as a CpG oligonucleotide (see, e.g.,
International Publication No. WO 00/62800); (11) an immunostimulant and a
particle
of a metal salt (see, e.g., International Publication No. WO 00/23105); (12) a
saponin
and an oil-in-water emulsion (see, e.g., International Publication No. WO
99/11241;
(13) a saponin (e.g., QS21) + 3dMPL + IL-12 (optionally + a sterol) (see,
e.g.,
International Publication No. WO 98/57659); (14) the MPL derivative RC529; and
(15) other substances that act as immunostimulating agents to enhance the
effectiveness of the composition. Ahun and MF59 are preferred.
As mentioned above, muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), -
acetyl-normuramyl-z-alanyl-D-isoglutamine (CGP 11637, referred to nor-MDP),
N-acetylmuramyl-z-alanyl-D-isoglutaminyl-z-alanine-2-(1'-2'-dipalmitoyl-sf~-
glycero-
3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), etc.
Moreover, the fusion protein can be adsorbed to, or entrapped within, an
ISCOM. Classic ISCOMs are formed by combination of cholesterol, saponin,
phospholipid, and immunogens, such as viral envelope proteins. Generally,
irnrnunogens (usually with a hydrophobic region) are solubilized in detergent
and
added to the reaction mixture, whereby ISCOMs are formed with the immunogen
36
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
incorporated therein. ISCOM matrix compositions are formed identically, but
without
viral proteins. Proteins with high positive charge may be electrostatically
bound in
the ISCOM particles, rather than through hydrophobic forces. For a more
detailed
general discussion of saponins and ISCOMs, and methods of formulating ISCOMs,
see Barr et al. (1998) Adv. Ds°ug Delivery Reviews 32:247-271 (1998).
ISCOMs for use with the present invention are produced using standard
techniques, well known in the art, and are described in e.g., U.S. Patent Nos.
4,981,684, 5,178,860, 5,679,354 and 6,027,732; European Publ. Nos. EPA
109,942;
180,564 and 231,039; Coulter et al. (1998) Yaccihe 16:1243. Typically, the
term
"ISCOM" refers to immunogenic complexes formed between glycosides, such as
triterpenoid saponins (particularly Quil A), and antigens which contain a
hydrophobic
region. See, e.g., European Publ. Nos. EPA 109,942 and 180,564. In this
embodiment, the HCV fusions (usually with a hydrophobic region) are
solubilized in
detergent and added to the reaction mixture, whereby ISCOMs are formed with
the
fusions incorporated therein. The HCV polypeptide ISCOMs are readily made with
HCV polypeptides which show amphipathic properties. However, proteins and
peptides which lack the desirable hydrophobic properties may be incorporated
into the
immunogenic complexes after coupling with peptides having hydrophobic amino
acids, fatty acid radicals, alkyl radicals and the like.
As explained in European Publ. No. EPA 231,039, the presence of antigen is
not necessary in order to form the basic ISCOM structure (referred to as a
matrix or
ISCOMATRIX), which may be formed from a sterol, such as cholesterol, a
phospholipid, such as phosphatidylethanolamine, and a glycoside, such as Quil
A.
Thus, the HCV fusion of interest, rather than being incorporated into the
matrix, is
present on the outside of the matrix, for example adsorbed to the matrix via
electrostatic interactions. For example, HCV fusions with high positive charge
may
be electrostatically bound to the ISCOM particles, rather than through
hydrophobic
forces. For a more detailed general discussion of saponins and ISCOMs, and
methods
of formulating ISCOMs, see Barn et al. (1998) Adv. DYUg Delivery Reviews
32:247-271 (1998).
37
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The ISCOM matrix may be prepared, for example, by mixing together
solubilized sterol, glycoside and (optionally) phospholipid. If phospholipids
are not
used, two dimensional structures are formed. See, e.g., European Publ. No. EPA
231,039. The teen "ISCOM matrix" is used to refer to both the 3-dimensional
and 2-
dimensional structures. The glycosides to be used are generally glycosides
which
display amphipathic properties and comprise hydrophobic and hydrophilic
regions in
the molecule. Preferably saponins are used, such as the saponin extract from
Quillaja
saponaria Molina and Quil A. Other preferred saponins are aescine from
Aesculus
hippocastanum (Patt et al. (1960) Arzneinaittelforschurzg 10:273-275 and
sapoalbin
from Gypsophilla struthium (Vochten et al. (1968) J. Pharnz. Belg. 42:213-226.
In order to prepare the ISCOMs, glycosides are used in at least a critical
micelle-forming concentration. In the case of Quil A, this concentration is
about
0.03% by weight. The sterols used to produce ISCOMs may be known sterols of
animal or vegetable origin, such as cholesterol, lanosterol, lumisterol,
stigmasterol
and sitosterol. Suitable phospholipids include phosphatidylcholine and
phosphatidylethanolamine. Generally, the molar ratio of glycoside (especially
when it
is Quil A) to sterol (especially when it is cholesterol) to phospholipid is
1:1:0-1, +
20% (preferably not more than +10%) for each figure. This is equivalent to a
weight
ratio of about 5:1 for the Quil A:cholesterol.
A solubilizing agent may also be present and may be, for example a detergent,
urea or guanidine. Generally, a non-ionic, ionic or zwitter-ionic detergent or
a cholic
acid based detergent, such as sodium desoxycholate, cholate and CTAB
(cetyltriammonium bromide), can be used for this purpose. Examples of suitable
detergents include, but are not limited to, octylglucoside, nonyl N-methyl
glucamide
or decanoyl N-methyl glucamide, alkylphenyl polyoxyethylene ethers such as a
polyethylene glycol p-isooctyl-phenylether having 9 to 10 oxyethylene groups
(commercialized under the trade name TRITON X-100RTM), acylpolyoxyethylene
esters such as acylpolyoxyethylene sorbitane esters (commercialized under the
trade
name TWEEN 20TM , TWEEN 80TM, and the like). The solubilizing agent is
generally removed for formation of the ISCOMs, such as by ultrafiltration,
dialysis,
38
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WO 2004/005473 PCT/US2003/020996
ultracentrifugation or chromatography, however, in certain methods, this step
is
unnecessary. (See, e.g., U.S. Patent No. 4,981,684).
Generally, the ratio of glycoside, such as QuilA, to HCV fusion by weight is
in the range of 5:1 to 0.5:1. Preferably the ratio by weight is approximately
3:1 to 1:1,
and more preferably the ratio is 2:1.
Once the ISCOMs are formed, they may be formulated into compositions arid
administered to animals, as described herein. If desired, the solutions of the
irnmunogenic complexes obtained may be lyophilized and then reconstituted
before
use.
Methods of Pf~oducing HCIT Specific Atatibodies
The HCV fusion proteins can be used to produce HCV-specific polyclonal and
monoclonal mtibodies. HCV-specific polyclonal and monoclonal antibodies
specifically bind to HCV antigens. Polyclonal antibodies can be produced by
administering the fusion protein to a mammal, such as a mouse, a rabbit, a
goat, or a
horse. Serum from the immunized animal is collected and the antibodies are
purified
from the plasma by, for example, precipitation with ammonium sulfate, followed
by
chromatography, preferably affinity chromatography. Techniques for producing
and
processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against HCV-specific epitopes present in the
fusion proteins can also be readily produced. Normal B cells from a mammal,
such as
a mouse, immunized with an HCV fusion protein, can be fused with, for example,
HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing
HCV-specific antibodies can be identified using RIA or ELISA and isolated by
cloning in semi-solid agar or by limiting dilution. Clones producing HCV-
specific
antibodies are isolated by another round of screening.
Antibodies, either monoclonal and polyclonal, which are directed against
HCV epitopes, are particularly useful for detecting the presence of HCV or HCV
antigens in a sample, such as a serum sample from an HCV-infected human. An
immunoassay for an HCV antigen may utilize one antibody or several antibodies.
An
immunoassay for an HCV antigen may use, for example, a monoclonal antibody
39
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directed towards an HCV epitope, a combination of monoclonal antibodies
directed
towards epitopes of one HCV polypeptide, monoclonal antibodies directed
towards
epitopes of different HCV polypeptides, polyclonal antibodies directed towards
the
same HCV antigen, polyclonal antibodies directed towards different HCV
antigens, or
a combination of monoclonal and polyclonal antibodies. Immunoassay protocols
may
be based, for example, upon competition, direct reaction, or sandwich type
assays
using, for example, labeled antibody. The labels may be, for example,
fluorescent,
chemiluminescent, or radioactive.
The polyclonal or monoclonal antibodies may further be used to isolate HCV
particles or antigens by immunoaffinity columns. The antibodies can be affixed
to a
solid support by, for example, adsorption or by covalent linlcage so that the
antibodies
retain their immunoselective activity. Optionally, spacer groups may be
included so
that the antigen binding site of the antibody remains accessible. The
immobilized
antibodies can then be used to bind HCV particles or antigens from a
biological
sample, such as blood or plasma. The bound HCV particles or antigens are
recovered
from the column matrix by, for example, a change in pH.
HCTV Specific T cells
HCV-specific T cells that are activated by the above-described fusions,
including the NS3*NS4NSSa fusion protein or NS3*NS4NSSaNSSb fusion protein,
with or without a core polypeptide, as well as any of the other various
fusions
described herein, expressed i~z vivo or ifZ vitf°o, preferably
recognize an epitope of an
HCV polypeptide such as an NS2, p7, El, E2, NS3, NS4, NSSa or NSSb
polypeptide,
including an epitope of a fusion of one or more of these peptides with an
NS3*, with
or without a core polypeptide. HCV-specific T cells can be CD8+ or CD4+.
HCV-specific CD8+ T cells can be cytotoxic T lymphocytes (CTL) which can
bill HCV-infected cells that display any of these epitopes complexed with an
MHC
class I molecule. HCV-specific CD8+ T cells can be detected by, for example,
SlCr
release assays (see Example 4). SICr release assays measure the ability of
HCV-specific CD8+ T cells to lyse target cells displaying one or more of these
CA 02491508 2004-12-31
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epitopes. HCV-specific CD8+ T cells which express antiviral agents, such as
IFN-y,
are also contemplated herein and can also be detected by immunological
methods,
preferably by intracellular staining for IFN-y or lilce cytolcine after in
vitro stimulation
with one or more of the HCV polypeptides, such as but not limited to an NS3,
an
S NS4, an NSSa, or an NSSb polypeptide (see Example 5).
HCV-specific CD4+ cells activated by the above-described fusions, such as
but not limited to an NS3*NS4NSSa or NS3*NS4NSSaNSSb fusion protein, with or
without a core polypeptide, expressed ifa vivo or ira vitro, preferably
recognize an
epitope of an HCV polypeptide, such as but not limited to an NS2, p7, E1, E2,
NS3,
NS4, NSSa, or NSSb polypeptide, including an epitope of fusions thereof, such
as but
not limited to an NS3NS4NSSa or NS3NS4NSSaNSSb fusion protein, that is bound
to
an MHC class II molecule on an HCV-infected cell and proliferate in response
to
stimulating, e.g., NS3*NS4NSSa or NS3*NS4NSSaNSSb peptides, with or without a
core polypeptide.
HCV-specific CD4+ T cells can be detected by a lymphoproliferation assay
(see Example 6). Lymphoproliferation assays measure the ability of HCV-
specific
CD4+ T cells to proliferate in response to, e.g., an NS2, p7, El, E2, NS3, an
NS4, an
NSSa, and/or an NSSb epitope.
Methods of Activatihg HChSpecific T Cells.
The HCV fusion proteins or polynucleotides can be used to activate
HCV-specific T cells either ih vitf°o or in. vivo. Activation of HCV-
specific T cells
can be used, ifzte~ alia, to provide model systems to optimize CTL responses
to HCV
and to provide prophylactic or therapeutic treatment against HCV infection.
For ih
vitro activation, proteins are preferably supplied to T cells via a plasmid or
a viral
vector, such as an adenovirus vector, as described above.
Polyclonal populations of T cells can be derived from the blood, and
preferably from peripheral lymphoid organs, such as lymph nodes, spleen, or
thymus,
of mammals that have been infected with an HCV. Preferred mammals include
mice,
chimpanzees, baboons, and humans. The HCV serves to expand the number of
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activated HCV-specific T cells in the mammal. The HCV-specific T cells derived
from the mammal can then be restimulated iy2 vitro by adding, an HCV fusion
protein
as described herein, such as but not limited to HCV NS3NS4NSSa or
NS3NS4NSSaNSSb epitopic peptides, with or without a core polypeptide, to the T
cells. The HCV-specific T cells can then be tested for, irttef- alia,
proliferation, the
production of IFN-y, and the ability to lyse target cells displaying, for
example,
NS3NS4NSSa or NS3NS4NSSaNSSb epitopes ih vitro.
In a lymphoproliferation assay (see Example 6), HCV-activated CD4+ T cells
proliferate when cultured with an HCV polypeptide, such as but not limited to
an
NS3, NS4, NSSa, NSSb, NS3NS4NSSa, or NS3NS4NSSaNSSb epitopic peptide, but
not in the absence of an epitopic peptide. Thus, particular HCV epitopes, such
as
NS2, p7, E1, E2, NS3, NS4, NSSa, NSSb, and fusions of these epitopes, such as
but
not limited to NS3NS4NSSa and NS3NS4NSSaNSSb epitopes that are recognized by
HCV-specific CD4+ T cells can be identified using a lymphoproliferation assay.
Similarly, detection of IFN-y in HCV-specific CD4+ and/or CD8+ T cells
after i~a vitf°o stimulation with the above-described fusion proteins,
can be used to
identify, for example, fusion protein epitopes, such as but not limited to
NS2, p7, E1,
E2, NS3, NS4, NSSa, NSSb, and fusions of these epitopes, such as but not
limited to
NS3NS4NSSa, and NS3NS4NSSaNSSb epitopes that are particularly effective at
stimulating CD4+ andlor CD8+ T cells to produce IFN-y (see Example 5).
Further, SlCr release assays are useful for determining the level of CTL
response to HCV. See Cooper et al. limnunity 10:439-449. For example,
HCV-specific CD8+ T cells can be derived from the liver of an HCV infected
mammal. These T cells can be tested in 5lCr release assays against target
cells
displaying, e.g., NS3NS4NSSa NS3NS4NSSaNSSb epitopes. Several target cell
populations expressing different NS3NS4NSSa or NS3NS4NSSaNSSb epitopes can
be constructed so that each target cell population displays different epitopes
of
NS3NS4NSSa or NS3NS4NSSaNSSb. The HCV-specific CD8+ cells cm be assayed
against each of these target cell populations. The results of the 51 Cr
release assays
can be used to determine which epitopes of NS3NS4NSSa or NS3NS4NSSaNSSb are
42
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WO 2004/005473 PCT/US2003/020996
responsible for the strongest CTL response to HCV. NS3*NS4NSSa fusion proteins
or NS3*NS4NSSaNSSb fusion proteins, with or without core polypeptides, which
contain the epitopes responsible for the strongest CTL response can then be
constructed using the information derived from the 5lCr release assays.
An HCV fusion protein as described above, or polynucleotide encoding such a
fusion protein, can be administered to a marmnal, such as a mouse, baboon,
chimpanzee, or human, to activate HCV-specific T cells ifa vivo.
Administration can
be by any means known in the art, including parenteral, intranasal,
intramuscular or
subcutaneous injection, including injection using a biological ballistic gun
("gene
gun"), as discussed above.
Preferably, injection of an HCV polynucleotide is used to activate T cells. In
addition to the practical advantages of simplicity of construction and
modification,
injection of the polynucleotides results in the synthesis of a fusion protein
in the host.
Thus, these immunogens are presented to the host immune system with native
post-translational modifications, structure, and conformation. The
polynucleotides
are preferably injected intramuscularly to a large mammal, such as a human, at
a dose
of 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mglkg.
A composition of the invention comprising an HCV fusion protein or
polynucleotide is administered in a manner compatible with the particular
composition used and in an amount which is effective to activate HCV-specific
T
cells as measured by, inter alia, a SlCr release assay, a lymphoproliferation
assay, or
by intracellular staining for IFN-y. The proteins and/or polynucleotides can
be
administered either to a mammal which is not infected with an HCV or can be
administered to an HCV-infected mammal. The particular dosages of the
polynucleotides or fusion proteins in a composition will depend on many
factors
including, but not limited to the species, age, and general condition of the
mammal to
which the composition is administered, and the mode of administration of the
composition. An effective amount of the composition of the invention can be
readily
determined using only routine experimentation. In vitro and in vivo models
described
above can be employed to identify appropriate doses. The amount of
polynucleotide
used in the example described below provides general guidance which can be
used to
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WO 2004/005473 PCT/US2003/020996
optimize the activation of HCV-specific T cells either in vivo or ih
vitf°o. Generally,
0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg of an HCV fusion protein or
polynucleotide,
with or without a core polypeptide, will be administered to a large mammal,
such as a
baboon, chimpanzee, or human. If desired, co-stimulatory molecules or
adjuvants can
also be provided before, after, or together with the compositions.
Immune responses of the mammal generated by the delivery of a composition
of the invention, including activation of HCV-specific T cells, can be
enhanced by
varying the dosage, route of administration, or boosting regimens.
Compositions of
the invention may be given in a single dose schedule, or preferably in a
multiple dose
schedule in which a primary course of vaccination includes 1-10 separate
doses,
followed by other doses given at subsequent time intervals required to
maintain and/or
reinforce an immune response, for example, at 1-4 months for a second dose,
and if
needed, a subsequent dose or doses after several months.
III. Experimental
Below are examples of specific embodiments for carrying out the present
invention. The examples are offered for illustrative purposes only, and are
not
intended to limit the scope of the present invention in any way. Those of
slcill in the
art will readily appreciate that the invention may be practiced in a variety
of ways
given the teaching of this disclosure.
Efforts have been made to ensure accuracy with respect to numbers used (e.g.,
amounts, temperatures, etc.), but some experimental error and deviation
should, of
course, be allowed for.
EXAMPLE 1
Production of NS3*NS4NSSaCore Polynucleotides
NS3~= in the following examples represents a modified NS3 molecule. A
polynucleotide encoding NS3NS4NSSa (approximately amino acids 1027 to 2399,
numbered relative to HCV-1) (also termed "NS345a" herein) is isolated from an
HCV. The NS3 portion of the molecule is mutagenzied by mutating the coding
sequence for the His, Asp and Ser residues found at the protease active site,
such that
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WO 2004/005473 PCT/US2003/020996
the resulting molecule codes for amino acids other than His, Asp and Ser at
these
positions and lacks NS3 protease activity. This construct is fused with a
polynucleotide encoding a core polypeptide wluch includes amino acids 1-122 of
the
full-length polyprotein The core-encoding polynucleotide sequence is fused
downstream from the NSSa-encoding portion of the construct such that the
resulting
fusion protein includes the core polypeptide at its C-terminus. The construct
is cloned
into plasmid, vaccinia virus, and adenovirus vectors. Additionally, the
construct is
inserted into a recombinant expression vector and used to transform host cells
to
produce the NS3*NS4NSSaCore fusion protein.
Protease enzyme activity is determined as follows. An NS4A peptide
(KKGSVVIVGRIVLSGKPAIIPKK) (SEQ ID N0:8), and the fusion protein are
diluted in 90 ~,1 of reaction buffer (25 mM Tris, pH 7.5, O.15M NaCI, 0.5 mM
EDTA,
10% glycerol, 0.05 n-Dodecyl B-D-Maltoside, 5 mM DTT) and allowed to mix for
30
minutes at room temperature. 90 wl of the mixture is added to a microtiter
plate
(Costar, Inc., Corning, NY) and 10 ~,1 of HCV substrate (AnaSpec, Inc., San
Jose CA)
is added. The plate is mixed and read on a Fluostar plate reader. Results are
expressed as relative fluorescence units (RFC per minute.
EXAMPLE 2
Priming of HCV-specific CTLs in Vaccinated Animals
The HCV fusion protein, NS3*NS4NSSaCore, produced as described above is
used to produce an HCV fusion-ISCOM as follows. The fusion-ISCOM formulations
are prepared by mixing the fusion protein with a preformed ISCOMATRIX (empty
ISCOMs) utilizing ionic interactions to maximize association between the
antigen and
the adjuvant. ISCOMATRIX is prepared essentially as described in Coulter et
al.
(1998) Iracci~ze 16:1243.
Rhesus macaques are immunized under anesthesia. Animals are divided into
two groups. The first group is infected with 2 x 108 plaque forming units
(pfu) (1 x
108 intradermally and 1 x 108 by scarification) of rVVC/E1 at month 0. This
group
serves as a positive control for CTL priming. Animals from the second group
are
immunized with 25-100 ~,g of an HCV fusion polypeptide, as described above,
that
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
has been adsorbed to an ISCOM, by intramuscular (IM) injection in the left
quadriceps at months 0, 1, 2 and 6. Cytotoxic activity is assayed in a
standard 5lCr
release assay as described in, e.g., Paliard et al. (2000) AIDS Res. Hufn.
Retroviruses
16:273.
EXAMPLE 3
Immunization With NS3*NS4NSSaCore Polynucleotides
In one immunization protocol, animals are immunized with 50-250 p,g of
plasmid DNA encoding an NS3*NS4NSSaCore fusion protein by intramuscular
injection into the tibialis anterior. A booster injection of 107 pfu of
vaccinia virus
(VV)-NSSa (intraperitoneal) or 50-250 ~,g of plasmid control (intramuscular)
is
provided 6 weeps later.
In another immunization protocol, animals are inj ected intramuscularly in the
tibialis anterior with 1010 adenovirus particles encoding an NS3*NS4NSSaCore
fusion protein. An intraperitoneal booster injection of 107 pfu of VV-NSSa or
an
intramuscular booster injection of 1010 adenovirus particles encoding
NS3*NS4NSSaCore is provided 6 weeles later.
EXAMPLE 4
Activation of HCV-Specific CD8+ T Cells
SI Cr Release Assay. A 51 Cr release assay is used to measure the ability of
HCV-specific T cells to lyse target cells displaying an NSSa epitope. Spleen
cells are
pooled from the immunized animals. These cells are restimulated i~z vitro for
6 days
with the CTL epitopic peptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO:l)
from HCV-NSSa in the presence of IL-2. The spleen cells are then assayed for
cytotoxic activity in a standard 51 Cr release assay against peptide-
sensitized target
cells (L929) expressing class I, but not class II MHC molecules, as described
in Weiss
(1980) J. Biol. Chem. 255:9912-9917. Ratios of effector (T cells) to target (B
cells)
of 60:1, 20:1, and 7:1 are tested. Percent specific lysis is calculated for
each effector
to target ratio.
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EXAMPLE 5
Activation of HCV-Specific CD8+ T Cells Which Express IFN-y
hZtracellular StairaifZg fog Irate~fef°of2-gamma (IFN y). Intracellular
staining for
1FN-y is used to identify the CD8+ T cells that secrete IFN-y after i~2 vitro
stimulation
with the NSSa epitope p214K9. Spleen cells of individual immunized animals are
restimulated in vitYO either with p214K9 or with a non-specific peptide for 6-
12 hours
in the presence of IL-2 and monensin. The cells are then stained for surface
CD8 and
for intracellular IFN-y and analyzed by flow cytometry. The percent of CD8+ T
cells
which are also positive for IFN-y is then calculated.
EXAMPLE 6
Proliferation of HCV-Specific CD4+ T Cells
Lyf~zphopYOlife~atiofz assay. Spleen cells from pooled immunized animals are
depleted of CD8+ T cells using magnetic beads and are cultured in triplicate
with
either p222D, an NSSa-epitopic peptide from HCV-NSSa
(2224-AELIEANLLWRQEMG-2238; SEQ ID N0:2), or in medium alone. After 72
hours, cells are pulsed with 1 ~, Ci per well of 3H-thymidine and harvested 6-
8 hours
later. Incorporation of radioactivity is measured after harvesting. The mean
cpm is
calculated.
EXAMPLE 7
Ability of NS3*45aCore-Encoding DNA Vaccine Formulations to prime
CTLs
Animals are immunized with either 10-250 ~.g of plasmid DNA encoding
NS3*45aCore fusion protein as described in Example 3, with PLG-linl~ed DNA
encoding NS3*45aCore (see below), or with DNA encoding NS3*45aCore, delivered
via electroporation (see, e.g., W ternational Publication No. WO/0045823 for
this
delivery technique). The immunizations are followed by a booster injection 6
weeps
later of plasmid DNA encoding NS3*45aCore.
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PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers are
obtained from Boehringer Ingelheim, U.S.A. The PLG polymer is RG505, which has
a copolymer ratio of 50/50 and a molecular weight of 65 l~Da (manufacturers
data).
Cationic microparticles with adsorbed DNA are prepared using a modified
solvent
evaporation process, essentially as described in Singh et al., Proc. Natl.
Acad. Sci.
USA (2000) 97:811-816. Briefly, the microparticles are prepared by emulsifying
10
ml of a 5% w/v polymer solution in methylene chloride with 1 ml of PBS at high
speed using an II~A homogenizer. The primary emulsion is then added to SOmI of
distilled water containing cetyl trimethyl ammonium bromide (CTAB) (0.5% w/v).
This results in the formation of a w/o/w emulsion which is stirred at 6000 rpm
for 12
hours at room temperature, allowing the methylene chloride to evaporate. The
resulting microparticles are washed twice in distilled water by centrifugation
at
10,000 g and freeze dried. Following preparation, washing and collection, DNA
is
adsorbed onto the microparticles by incubating 100 mg of cationic
microparticles in a
lmg/ml solution of DNA at 4 C for 6 hours. The microparticles are then
separated by
centrifugation, the pellet washed with TE buffer and the microparticles are
freeze
dried.
CTL activity and IFN-y expression is measured by 5lCr release assay or
intracellular staining as described in the examples above.
EXAMPLE ~
Immunization Routes and Replicon particles SINCR (DC+)
Encoding for NS3*45aCore
Alphavirus replicon particles, for example, SINCR (DC+) are prepared as
described in Polo et al., Proc. Natl. Acad. Sci. USA (1999) 96:4598-4603.
Animals
are injected with 5 x 106 ILJ SINCR (DC+) replicon particles encoding for
NS3*45aCore intramuscularly (IM) as described in Example 3, or subcutaneously
(S/C) at the base of the tail (BoT) and foot pad (FP), or with a combination
of 2/3 of
the DNA delivered via IM administration and 1/3 via a BoT route. The
immunizations are followed by a booster injection of vaccinia virus encoding
NSSa as
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WO 2004/005473 PCT/US2003/020996
described in Example 3. IFN-y expression is measured by intracellular staining
as
described in Example 5.
EXAMPLE 9
Alphavirus Replicon Priming, Followed by Various Boosting Regimes
Alphavirus replicon particles, for example, SINCR (DC+) are prepared as
described in Polo et al., Py°oc. Natl. Acad. Sci. USA (1999) 96:4598-
4603. Animals
are primed with SINCR (DC+), 1.5 x 106 IU replicon particles encoding NS345a,
by
intramuscular injection into the tibialis anterior, followed by a booster of
either
10-100 ~,g of plasmid DNA encoding for NSSa, 1010 adenovirus particles
encoding
NS3*45aCore, 1.5 x 106 IU SINCR (DC+) replicon particles encoding NS3*45aCore,
or l0y pfu vaccinia virus encoding NSSa at 6 weeps. IFN-y expression is
measured
by intracellular staining as described in Example 5.
EXAMPLE 10
Alphaviruses Expressing NS3*45aCore
Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)
are prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA (1999)
96:4598-4603. Animals are immunized with 1 x 102 to 1 x 106 ICT SINCR (DC+)
replicons encoding NS3*45aCore via a combination of delivery routes (2/3 IM
and
1/3 S/C) as well as by S/C alone, or with 1 x 102 to 1 x 106 ICT S1NCR (LP)
replicon
particles encoding NS3~45aCore via a combination of delivery routes (2/3 IM
and 1/3
S/C) as well as by S/C alone. The immunizations are followed by a booster
injection
of 10~ pfu vaccinia virus encoding NSSa at 6 weelcs. IFN-y expression is
measured
by intracellular staining as described in Example 5.
Thus, HCV fusion polypeptides, to stimulate cell-mediated immune responses,
are disclosed. Although preferred embodiments of the subject invention have
been
described in some detail, it is understood that obvious variations can be made
without
departing from the spirit and the scope of the invention as defined herein.
49
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SEQUENCE LISTING
<110> Chiron Corporation
<120> HCV FUSION PROTEINS WITH MODIFIED NS3 DOMAINS
<130> PP19545.004 (2300-19545.40)
<150> 60/394,510
<151> 2002-07-08
<150> 60/393,694
<l5l> 2002-07-02
<150> 09/721,479
<151> 2000-11-22
<150> 60/167,502
<151> 1999-11-24
<160> 8
<170> PatentIn version 3.2
<210> 1
<211> 9
<212> PRT
<213> Artificial
<220>
<223> epitope recognized by a Tcell receptor
<400> 1
His Glu Tyr Pro Val Gly Ser G1n Leu
1 5
<210> 2
<211> 15
<212> PRT
<213> Artificial
<220>
<223> epitope recognized by a Tcell receptor
<400> 2
Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly
l 5 10 15
<210> 3
<211> 546
<212> DNA
<213> Artificial
<220>
<223> DNA sequence of a representative native, unmodified NS3 protease
domain
1
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
<400> 3
atggcg cccatcacg gcgtacgcc cagcagaca aggggcctc ctaggg 48
MetAla ProIleThr AlaTyrAla GlnGlnThr ArgGlyLeu LeuGly
1 5 10 15
tgcata atcaccagc ctaactggc cgggacaaa aaccaagtg gagggt 96
CysIle TleThrSer LeuThrGly ArgAspLys AsnGlnVal GluGly
20 25 30
gaggtc cagattgtg tcaactget gcccaaacc ttcctggca acgtgc 144
GluVal GlnIleVal SerThrAla AlaGlnThr PheLeuAla ThrCys
35 40 45
atcaat ggggtgtgc tggactgtc taccacggg gccggaacg aggacc 192
IleAsn GlyValCys TrpThrVal TyrHisGly AlaGlyThr ArgThr
50 55 60
atcgcg tcacccaag ggtcctgtc atccagatg tataccaat gtagac 240
IleAla SerProLys GlyProVal IleGlnMet TyrThrAsn ValAsp
65 70 75 80
caagac cttgtgggc tggcccget ccgcaaggt agccgatca ttgaca 288
GlnAsp LeuValG1y TrpProAla ProGlnG1y SerArgSer LeuThr
85 90 95
ccctgc acttgcggc tcctcggac ctttacctg gtcacgagg cacgcc 336
ProCys ThrCysGly SerSerAsp LeuTyrLeu Va1ThrArg HisAla
100 105 110
gatgtc attcccgtg cgccggcgg ggtgatagc aggggcagc ctgctg 384
AspVal I1eProVal ArgArgArg G1yAspSer ArgGlySer LeuLeu
115 120 125
tcgccc cggcccatt tcctacttg aaaggctcc tcggggggt ccgctg 432
SerPro ArgProIle SerTyrLeu LysGlySer SerGlyGly ProLeu
130 135 140
ttgtgc cccgcgggg cacgccgtg ggcatattt agggccgcg gtgtgc 480
LeuCys ProAlaGly HisAlaVal GlyIlePhe ArgAlaAla ValCys
145 150 155 160
acccgt ggagtgget aaggcggtg gactttatc cctgtggag aaccta 528
ThrArg GlyValAla LysAlaVal AspPheTle ProValGlu AsnLeu
165 170 175
gagaca accatgagg tcc 546
GluThr ThrMetArg Ser
180
<210> 4
<211> 182
<212> PRT
<213> Artificial
<220>
<223> amino acid sequence of a representative native unmodified NS3
protease domain
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
<400> 4
Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly
1 5 10 15
Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly
20 25 30
Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys
35 40 45
Ile Asn G1y Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr
50 55 60
Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp
65 70 75 80
Gln Asp Leu Val Gly Trp Pro Ala Pro G1n Gly Ser Arg Ser Leu Thr
85 90 95
Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala
100 105 1l0
Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu
115 120 125
Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly G1y Pro Leu
130 135 140
Leu Cys Pro Ala Gly His Ala Val Gly Tle Phe Arg Ala Ala Val Cys
145 150 155 160
Thr Arg Gly Val Ala Lys Ala Val Asp Phe I1e Pro Val Glu Asn Leu
165 170 175
Glu Thr Thr Met Arg Ser
180
<210> 5
<211> 5676
<212> DNA
<213> Artificial
<220>
G223> DNA sequence of a representative modified fusion protein, with
the NS3 protease domain deleted from the N-terminus and including
amino acids l-l21 of Core on the C-terminus
<400> 5
atg get gca tat gca get cag ggc tat aag gtg cta gta ctc aac ccc 48
Met Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro
1 5 10 15
tct gtt get gca aca ctg ggc ttt ggt get tac atg tcc aag get cat 96
Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His
20 25 30
ggg atc gat cct aac atc agg acc ggg gtg aga aca att acc act ggc 144
Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly
3
CA 02491508 2004-12-31
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35 40 45
agc ccc atc acg tac tcc acc tac ggc aag ttc ctt gcc gac ggc ggg 192
Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly
50 55 60
tgc tcg ggg ggc get tat gac ata ata att tgt gac gag tgc cac tcc 240
Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser
65 70 75 80
acg gat gcc aca tcc atc ttg ggc att ggc act gtc ctt gac caa gca 288
Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala
85 90 95
gag act gcg ggg gcg aga ctg gtt gtg ctc gcc acc gcc acc cct ccg 336
Glu Thr A1a Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro
100 105 110
ggc tcc gtc act gtg ccc cat ccc aac atc gag gag gtt get ctg tcc 384
Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser
115 7,20 125
acc acc gga gag atc cct ttt tac ggc aag get atc ccc ctc gaa gta 432
Thr Thr Gly G1u Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val
130 135 140
atc aag ggg ggg aga cat ctc atc ttc tgt cat tca aag aag aag tgc 480
Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys
145 150 155 160
gac gaa ctc gcc gca aag ctg gtc gca ttg ggc atc aat gcc gtg gcc 528
Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala
165 170 175
tac tac cgc ggt ctt gac gtg tcc gtc atc ccg acc agc ggc gat gtt 576
Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val
180 185 190
gtc gtc gtg gca acc gat gcc ctc atg acc ggc tat acc ggc gac ttc 624
Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe
195 200 205
gac tcg gtg ata gac tgc aat acg tgt gtc acc cag aca gtc gat ttc 672
Asp Ser Va1 Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe
210 215 220
agc ctt gac cct acc ttc acc att gag aca atc acg ctc ccc caa gat 720
Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro Gln Asp
225 230 235 240
get gtc tcc cgc act caa cgt cgg ggc agg act ggc agg ggg aag cca 768
Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg G1y Lys Pro
245 250 255
ggc atc tac aga ttt gtg gca ccg ggg gag cgc ccc tcc ggc atg ttc 816
Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu Arg Pro Ser Gly Met Phe
260 265 270
gac tcg tcc gtc ctc tgt gag tgc tat gac gca ggc tgt get tgg tat 864
Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr
275 ~ 280 285
4
CA 02491508 2004-12-31
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gag ctc acg ccc gcc gag act aca gtt agg cta cga gcg tac atg aac 912
Glu Leu Thr Pro Ala Glu Thr Thr Va1 Arg Leu Arg Ala Tyr Met Asn
290 295 300
acc ccg ggg ctt ccc gtg tgc cag gac cat ctt gaa ttt tgg gag ggc 960
Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly
305 310 315 320
gtc ttt aca ggc ctc act cat ata gat gcc cac ttt cta tcc cag aca 1008
Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr
325 330 335
aag cag agt ggg gag aac ctt cct tac ctg gta gcg tac caa gcc acc 1056
Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Va1 Ala Tyr Gln Ala Thr-
340 345 350
gtg tgc get agg get caa gcc cct ccc cca tcg tgg gac cag atg tgg 1104
Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp
355 360 365
aag tgt ttg att cgc ctc aag ccc acc ctc cat ggg cca aca ccc ctg 1152
Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu
370 375 380
cta tac aga ctg ggc get gtt cag aat gaa atc acc ctg acg cac cca 1200
Leu Tyr Arg Leu Gly Ala Val G1n Asn Glu Ile Thr Leu Thr His Pro
385 390 395 400
gtc acc aaa tac atc atg aca tgc atg tcg gcc gac ctg gag gtc gtc 1248
Vai Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val
405 410 415
acg agc acc tgg gtg ctc gtt ggc ggc gtc ctg get get ttg gcc gcg 1296
Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu A1a Ala
420 425 430
tat tgc ctg tca aca ggc tgc gtg gtc ata gtg ggc agg gtc gtc ttg 1344
Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu
435 440 445
tcc ggg aag ccg gca atc ata cct gac agg gaa gtc ctc tac cga gag 1392
Ser G1y Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu
450 455 460
ttc gat gag atg gaa gag tgc tct cag cac tta ccg tac atc gag caa 1440
Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln
465 470 475 480
ggg atg atg ctc gcc gag cag ttc aag cag aag gcc ctc ggc ctc ctg 1488
Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu
485 490 495
cag acc gcg tcc cgt cag gca gag gtt atc gcc cct get gtc cag acc 1536
Gln Thr Ala Ser Arg Gln Ala Glu Va1 Ile Ala Pro Ala Val Gln Thr
500 505 510
aac tgg caa aaa ctc gag acc ttc tgg gcg aag cat atg tgg aac ttc 1584
Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe
515 520 525
CA 02491508 2004-12-31
WO PCT/US2003/020996
2004/005473
atcagt gggatacaa tacttg gcgggcttgtca acgctgcct ggtaac 1632
I1eSer GlyIleGln TyrLeu AlaGlyLeuSer ThrLeuPro G1yAsn
530 535 540
cccgcc attgettca ttgatg gettttacaget getgtcacc agccca 1680
ProAla IleAlaSer LeuMet AlaPheThrAla AlaValThr SerPro
545 550 555 560
ctaacc actagccaa accctc ctcttcaacata ttggggggg tgggtg 1728
LeuThr ThrSerGln ThrLeu LeuPheAsnIle LeuGlyGly TrpVal
565 570 575
getgcc cagctcgcc gccccc ggtgccgetact gcctttgtg ggcget 1776
AlaAla GlnLeuAla AlaPro GlyAlaAlaThr AlaPheVal GlyAla
580 585 590
ggctta getggcgcc gccatc ggcagtgttgga ctggggaag gtcctc 1824
GlyLeu AlaGlyAla AlaIle GlySerValGly LeuGlyLys ValLeu
595 600 605
atagac atccttgca gggtat ggcgcgggcgtg gcgggaget cttgtg 1872
IleAsp IleLeuAla GlyTyr GlyAlaGlyVal AlaGlyAla LeuVa1
610 615 620
gcattc aagatcatg agcggt gaggtcccctcc acggaggac ctggtc 1920
AlaPhe LysTleMet SerGly GluValProSer ThrGluAsp LeuVal
625 630 635 640
aatcta ctgcccgcc atcctc tcgcccggagcc ctcgtagtc ggcgtg 1968
AsnLeu LeuProAla IleLeu SerProGlyAla LeuValVal GlyVal
645 650 655
gtctgt gcagcaata ctgcgc cggcacgttggc ccgggcgag ggggca 2016
ValCys AlaAlaIle LeuArg ArgHisVa1Gly ProGlyGlu GlyA1a
660 665 670
gtgcag tggatgaac cggctg atagccttcgcc tcccggggg aaccat 2064
ValGln TrpMetAsn ArgLeu IleA1aPheA1a SerArgGly AsnHis
675 680 685
gtttcc cccacgcac tacgtg ccggagagcgat gcagetgcc cgcgtc 2112
Va1Ser ProThrHis TyrVal ProGluSerAsp AlaAlaAla ArgVal
690 695 700
actgcc atactcagc agcctc actgtaacccag ctcctgagg cgactg 2160
ThrA1a IleLeuSer SerLeu ThrValThrGln LeuLeuArg ArgLeu
705 710 715 720
caccag tggataagc tcggag tgtaccactcca tgctccggt tcctgg 2208
HisGln TrpIleSer SerGlu CysThrThrPro CysSerGly SerTrp
725 730 735
ctaagg gacatctgg gactgg atatgcgaggtg ttgagcgac tttaag 2256
LeuArg AspIleTrp AspTrp IleCysGluVal LeuSerAsp PheLys
740 745 750
acctgg ctaaaaget aagctc atgccacagctg cctgggatc cccttt 2304
ThrTrp LeuLysAla LysLeu MetProGlnLeu ProGlyIle ProPhe
755 760 765
gtgtcc tgccagcgc gggtat aagggggtctgg cgaggggac ggcatc 2352
6
CA 02491508 2004-12-31
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Val Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile
770 775 780
atg cac act cgc tgc cac tgt gga get gag atc act gga cat gtc aaa 2400
Met His Thr Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys
785 790 795 800
aac ggg acg atg agg atc gtc ggt cct agg acc tgc agg aac atg tgg 2448
Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp
805 810 815
agt ggg acc ttc ccc att aat gcc tac acc acg ggc ccc tgt acc ccc 2496
Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro
820 825 830
ctt cct gcg ccg aac tac acg ttc gcg cta tgg agg gtg tct gca gag 2544
Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser A1a Glu
835 840 845
gaa tac gtg gag ata agg cag gtg ggg gac ttc cac tac gtg acg ggt 2592
Glu Tyr Val Glu Ile Arg Gln Val Gly Asp Phe His Tyr Val Thr Gly
850 ~ 855 860
atg act act gac aat ctt aaa tgc ccg tgc cag gtc cca tcg ccc gaa 2640
Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser Pro Glu
865 870 875 880
ttt ttc aca gaa ttg gac ggg gtg cgc cta cat agg ttt gcg ccc ccc 2688
Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro
885 890 895
tgc aag ccc ttg ctg cgg gag gag gta tca ttc aga gta gga ctc cac 2736
Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His
900 905 910
gaa tac ccg gta ggg tcg caa tta cct tgc gag ccc gaa ccg gac gtg 2784
Glu Tyr Pro Va1 Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val
915 920 925
gcc gtg ttg acg tcc atg ctc act gat ccc tcc cat ata aca gca gag 2832
Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Tle Thr Ala Glu
930 935 940
gcg gcc ggg cga agg ttg gcg agg gga tca ccc ccc tct gtg gcc agc 2880
Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser
945 950 955 960
tcc tcg get agc cag cta tcc get cca tct ctc aag gca act tgc acc 2928
Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr
965 970 975
get aac cat gac tcc cct gat get gag ctc ata gag gcc aac ctc cta 2976
Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu
980 985 990
tgg agg cag gag atg ggc ggc aac atc acc agg gtt gag tca gaa aac 3024
Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn
995 1000 1005
aaa gtg gtg att ctg gac tcc ttc gat ccg ctt gtg gcg gag gag gac 3072
Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp
7
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1010 1015 1020
gagcgggag atctccgtaccc gcagaa atcctgcgg aagtctcgg aga 3120
GluArgGlu IleSerValPro AlaGlu IleLeuArg LysSerArg Arg
1025 1030 1035 1040
ttcgcccag gccctgcccgtt tgggcg cggccggac tataacccc ccg 3168
PheAlaGln AlaLeuProVal TrpAla ArgProAsp TyrAsnPro Pro
1045 1050 1055
cta'gtggag acgtggaaaaag cccgac tacgaacca cctgtggtc cat 3216
LeuValGlu ThrTrpLysLys ProAsp TyrGluPro ProValVal His
1060 1065 1070
ggctgcccg cttccacctcca aagtcc cctcctgtg cctccgcct cgg 3264
GlyCysPro LeuProProPro LysSer ProProVal ProProPro Arg
1075 1080 1085
aagaagcgg acggtggtcctc actgaa tcaacccta tctactgcc ttg 3312
LysLysArg ThrValValLeu ThrGlu SerThrLeu SerThrAla Leu
1090 1095 1100
gccgagctc gccaccagaagc tttggc agctcctca acttccggc att 3360
AlaG1uLeu AlaThrArgSer PheGly SerSerSer ThrSerGly Ile
1105 1110 1115 1120
acgggcgac aatacgacaaca tcctct gagcccgcc ccttctggc tgc 3408
ThrGlyAsp AsnThrThrThr SerSer GluProAla ProSerGly Cys
1125 1130 1135
ccccccgac tccgacgetgag tcctat tcctccatg ccccccctg gag 3456
ProProAsp SerAspAlaGlu SerTyr SerSerMet ProProLeu Glu
1140 1145 1150
ggggagcct ggggatccggat cttagc gacgggtca tggtcaacg gtc 3504
GlyGluPro GlyAspProAsp LeuSer AspGlySer TrpSerThr Val
1155 1160 1165
agt agtgag gccaacgcg gaggat gtcgtgtgctgc tcaatgtcttac 3552
Ser SerGlu AlaAsnAla GluAsp Va1ValCysCys SerMetSerTyr
1 170 1175 1180
tct tggaca ggcgcactc gtcacc ccgtgcgccgcg gaagaacagaaa 3600
Ser TrpThr GlyAlaLeu Va1Thr ProCysAlaAla G1uGluGlnLys
1185 1190 1195 1200
ctg cccatc aatgcacta agcaac tcgttgctacgt caccacaatttg 3648
Leu ProIle AsnAlaLeu SerAsn SerLeuLeuArg HisHisAsnLeu
1205 1210 1215
gtg tattcc accacctca cgcagt gettgccaaagg cagaagaaagtc 3696
Val TyrSer ThrThrSer ArgSer AlaCysGlnArg GlnLysLysVal
1220 1225 1230
aca tttgac agactgcaa gttctg gacagccattac caggacgtactc 3744
Thr PheAsp ArgLeuGln ValLeu AspSerHisTyr GlnAspValLeu
1235 1240 1245
aag gaggtt aaagcagcg gcgtca aaagtgaagget aacttgctatcc 3792
Lys GluVal LysAlaAla AlaSer LysValLysAla AsnLeuLeuSer
1 250 1255 1260
8
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gta gag gaa get tgc agc ctg acg ccc cca cac tca gcc aaa tcc aag 3840
Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys
1265 1270 1275 1280
ttt ggt tat ggg gca aaa gac gtc cgt tgc cat gcc aga aag gcc gta 3888
Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val
1285 1290 1295
acc cac atc aac tcc gtg tgg aaa gac ctt ctg gaa gac aat gta aca 3936
Thr His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr
1300 1305 1310
cca ata gac act acc atc atg get aag aac gag gtt ttc tgc gtt cag 3984
Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln
1315 1320 1325
cct gag aag ggg ggt cgt aag cca get cgt ctc atc gtg ttc ccc gat 4032
Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp
1330 1335 1340
ctg ggc gtg ogc gtg tgc gaa aag atg get ttg tac gac gtg gtt aca 4080
Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr
1345 1350 1355 1360
aag ctc ccc ttg gcc gtg atg gga~agc tcc tac gga ttc caa tac tca 4128
Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser
1365 1370 1375
cca gga cag cgg gtt gaa ttc ctc gtg caa gcg tgg aag tcc aag aaa 4176
Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys
1380 1385 1390
acc cca atg ggg ttc tcg tat gat acc cgc tgc ttt gac tcc aca gtc 4224
Thr Pro Met G1y Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val
1395 1400 1405
act gag agc gac atc cgt acg gag gag gca atc tac caa tgt tgt gac 4272
Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp
1410 1415 1420
ctc gac ccc caa gcc cgc gtg gcc atc aag tco ctc acc gag agg ctt 4320
Leu Asp Pro Gln Ala Arg Val A1a Ile Lys Ser Leu Thr Glu Arg Leu
1425 1430 1435 ' 1440
tat gtt ggg ggc cct ctt acc aat tca agg ggg gag aac tgc ggc tat 4368
Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr
1445 1450 1455
cgc agg tgc cgc gcg agc ggc gta ctg aca act agc tgt ggt aac acc 4416
Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr
1460 1465 1470
ctc act tgc tac atc aag gcc cgg gca gcc tgt cga gcc gca ggg ctc 4464
Leu Thr Cys Tyr Ile Lys Ala Arg Ala Ala Cys Arg A1a Ala Gly Leu
1475 1480 1485
cag gac tgc acc atg ctc gtg tgt ggc gac gac tta gtc gtt atc tgt 4512
Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Va1 Val Ile Cys
1490 1495 1500
9
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gaa agc gcg ggg gtc cag gag gac gcg gcg agc ctg aga gcc ttc acg 4560
Glu Ser Ala Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr
1505 1510 1515 1520
gag get atg acc agg tac tcc gcc ccc cct ggg gac ccc cca caa cca 4608
Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro
1525 1530 1535
gaa tac gac ttg gag ctc ata aca tca tgc tcc tcc aac gtg tca gtc 4656
Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val
1540 1545 1550
gcc cac gac ggc get gga aag agg gtc tac tac ctc acc cgt gac cct 4704
Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro
1555 1560 1565
aca acc ccc ctc gcg aga get gcg tgg gag aca gca aga cac act cca 4752
Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro
1570 1575 1580
gtc aat tcc tgg cta ggc aac ata atc atg ttt gcc ccc aca ctg tgg 4800
Val Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp
1585 1590 1595 1600
gcg agg atg ata ctg atg acc cat ttc ttt agc gtc ctt ata gcc agg 4848
Ala Arg Met I1e Leu Met Thr His Phe Phe Ser Val Leu Ile Ala Arg
1605 1610 1615
gac cag ctt gaa cag gcc ctc gat tgc gag atc tac ggg gcc tgc tac 4896
Asp Gln Leu Glu Gln A1a Leu Asp Cys Glu Ile Tyr Gly Ala Cys Tyr
1620 1625 1630
tcc ata gaa cca ctg gat cta cct cca atc att caa aga ctc cat ggc 4944
Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly
1635 1640 1645
ctc agc gca ttt tca ctc cac agt tac tct cca ggt gaa atc aat agg 4992
Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu I1e Asn Arg
1650 1655 1660
gtg gcc gca tgc ctc aga aaa ctt ggg gta ccg ccc ttg cga get tgg 5040
Val Ala Ala Cys Leu Arg Lys Leu Gly Va1 Pro Pro Leu Arg Ala Trp
1665 1670 1675 1680
aga cac cgg gcc cgg agc gtc cgc get agg ctt ctg gcc aga gga ggc 5088
Arg His Arg A1a Arg Ser Val Arg A1a Arg Leu Leu Ala Arg Gly Gly
1685 1690 1695
agg get gcc ata tgt ggc aag tac ctc ttc aac tgg gca gta aga aca 5136
Arg Ala Ala I1e Cys G1y Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr
1700 1705 1710
aag ctc aaa ctc act cca ata gcg gcc get ggc cag ctg gac ttg tcc 5184
Lys Leu Lys Leu Thr Pro Ile Ala A1a Ala Gly G1n Leu Asp Leu Ser
1715 1720 1725
ggc tgg ttc acg get ggc tac agc ggg gga gac att tat cac agc gtg 5232
Gly Trp Phe Thr Ala Gly Tyr Ser G1y Gly Asp Ile Tyr His Ser Val
1730 1735 1740
tct cat gcc cgg ccc cgc tgg atc tgg ttt tgc cta ctc ctg ctt get 5280
1~
CA 02491508 2004-12-31
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SerHis AlaArgProArg TrpIleTrp PheCys LeuLeuLeuLeu Ala
1745 1750 1755 1760
gcaggg gtaggcatctac ctcctcccc aaccga atgagcacgaat cct 5328
AlaGly ValGlyIleTyr LeuLeuPro AsnArg MetSerThrAsn Pro
1765 1770 1775
aaacct caaagaaagacc aaacgtaac accaac cggcggccgcag gac 5376
LysPro GlnArgLysThr LysArgAsn ThrAsn ArgArgProGln Asp
1780 1785 1790
gtcaag ttcccgggtggc ggtcagatc gttggt ggagtttacttg ttg 5424
ValLys PheProGlyGly GlyGlnIle ValGly GlyValTyrLeu Leu
1 795 1800 1805
ccgcgc aggggccctaga ttgggtgtg cgcgcg acgagaaagact tcc 5472
ProArg ArgGlyProArg LeuGlyVal ArgAla ThrArgLysThr Ser
1 810 1815 1820
gagcgg tcgcaacctcga ggtagacgt cagcct atccccaagget cgt 5520
GluArg SerGlnProArg GlyArgArg GlnPro IleProLysAla Arg
1825 1830 1835 1840
cggccc gagggcaggacc tgggetcag cccggg tacccttggccc ctc 5568
ArgPro GluGlyArgThr TrpAlaGln ProGly,TyrProTrpPro Leu
1845 1850 1855
tatggc aatgagggctgc gggtgggcg ggatgg ctcctgtctccc cgt 5616
TyrGly AsnGluG1yCys GlyTrpAla GlyTrp LeuLeuSerPro Arg
1860 1865 1870
ggctct cggcctagctgg ggccccaca gacccc cggcgtaggtcg cgc 5664
GlySer ArgProSerTrp G1yProThr AspPro ArgArgArgSer Arg
1875 1880 1885
aatttg ggtaag 5676
AsnLeu GlyLys
1 890
<210>
6
<211> 892
1
<212>
PRT
<213>
Artificial
<220>
<223> amino acid sequence of a representative modified fusion protein,
with the NS3 protease domain deleted from the N-terminus and
including amino acids 1-121 of Core on the C-terminus
<400> 6
Met Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro
1 5 10 15
Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His
20 25 30
Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly
35 40 45
11
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Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly
50 55 ~ 60
Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser
65 70 75 8p
Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala
85 90 95
Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro
100 105 110
Gly Ser Val Thr Val Pro His Pro Asn I1e Glu Glu Val Ala Leu Ser
115 120 125
Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Tle Pro Leu Glu Val
130 135 140
Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys
i45 150 155 160
Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala
165 170 175
Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val
180 185 190
Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe
195 200 205
Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe
210 215 220
Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro G1n Asp
225 230 235 240
Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys Pro
245 250 255
Gly Ile Tyr Arg Phe Va1 Ala Pro Gly Glu Arg Pro Ser Gly Met Phe
260 265 270
Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr
275 280 285
Glu Leu Thr Pro Ala G1u Thr Thr Val Arg Leu Arg Ala Tyr Met Asn
290 295 300
Thr Pro G1y Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly
305 310 315 320
Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr
325 330 335
Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr
340 345 350
Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp
355 360 365
Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu
12
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370 375 380
Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu I1e Thr Leu Thr His Pro
385 390 395 400
Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val
405 4l0 415
Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala
420 425 430
Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu
435 440 445
Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu
450 455 460
Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln
465 470 475 480
Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu
485 490 495
Gln Thr Ala Ser Arg G1n Ala Glu Val Ile Ala Pro Ala Val Gln Thr
500 505 510
Asn Trp Gln Lys Leu G1u Thr Phe Trp Ala Lys His Met Trp Asn Phe
515 520 525
Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn
530 535 540
Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro
545 550 555 560
Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val
565 570 575
Ala Ala Gln Leu A1a Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala
580 585 590
Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys Val Leu
595 600 605
Tle Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val
610 615 620
Ala Phe Lys Ile Met Ser Gly Glu Val Pro Ser Thr Glu Asp Leu Val
X25 630 635 640
Asn Leu Leu Pro Ala I1e Leu Ser Pro G1y Ala Leu Va1 Val Gly Val
645 650 655
Val Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala
660 665 670
Val Gln Trp Met Asn Arg Leu Tle Ala Phe Ala Ser Arg G1y Asn His
675 680 685
Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val
690 695 700
13
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Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu
705 710 715 720
His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp
725 730 735
Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys
740 745 750
Thr Trp Leu Lys Ala Lys Leu Met Pro Gln Leu Pro Gly Ile Pro Phe
755 76b 765
Val Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Tle
770 775 780
Met His Thr Arg Cys His Cys Gly Ala G1u Ile Thr Gly His Val Lys
785 790 795 800
Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp
805 810 815
Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro
820 825 830
Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser Ala Glu
835 840 845
Glu Tyr Val Glu Ile Arg Gln Va1 Gly Asp Phe His Tyr Val Thr Gly
850 855 860
Met Thr Thr Asp Asn Leu Lys Cys Pro Cys Gln Val Pro Ser Pro Glu
865 870 875 gg0
Phe Phe Thr Glu Leu Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro
885 890 895
Cys Lys Pro Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His
900 905 910
Glu Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val
915 920 925
Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu
930 935 940
Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala Ser
945 950 955 960
Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys A1a Thr Cys Thr
965 970 975
Ala Asn His Asp Ser Pro Asp Ala Glu Leu Ile Glu Ala Asn Leu Leu
980 985 990
Trp Arg G1n G1u Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn
995 1000 1005
Lys Val Val Ile Leu Asp Ser'Phe Asp Pro Leu Val Ala Glu Glu Asp
1010 1015 1020
14
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Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg
1025 1030 1035 1040
Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro
1045 1050 1055
Leu Val G1u Thr Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His
1060 1065 1070
G1y Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro Pro Pro Arg
1075 1080 1085
Lys Lys Arg Thr Val Val Leu Thr G1u Ser Thr Leu Ser Thr Ala Leu
1090 1095 1100
A1a Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile
1105 1110 1115 1120
Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser Gly Cys
1125 1130 1135
Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu
1140 1145 1150
Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val
1155 1160 1165
Ser Ser Glu Ala Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr
1170 1175 1180
Ser Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys
1185 1190 1195 1200
Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Leu
1205 1210 1215
Va1 Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln Arg Gln Lys Lys Val
1220 1225 1230
Thr Phe Asp Arg Leu Gln Val Leu Asp Ser His Tyr Gln Asp Val Leu
1235 1240 1245
Lys Glu Val Lys Ala Ala Ala Ser Lys Va1 Lys Ala Asn Leu Leu Ser
1250 1255 1260
Val Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys
1265 1270 1275 1280
Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys Ala Val
1285 1290 1295
Thr His Ile Asn Ser Val Trp Lys Asp Leu Leu Glu Asp Asn Val Thr
1300 1305 1310
Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln
1315 1320 1325
Pro G1u Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp
1330 1335 1340
Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr
IS
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
1345 1350 1355 1360
Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe G1n Tyr Ser
1365 1370 1375
Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala Trp Lys Ser Lys Lys
1380 1385 1390
Thr Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val
1395 1400 1405
Thr Glu Ser Asp Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp
1410 1415 1420
Leu Asp Pro Gln Ala Arg Val Ala Tle Lys Ser Leu Thr Glu Arg Leu
1425 1430 1435 1440
Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys Gly Tyr
1445 1450 1455
Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr
1460 1465 1470
Leu Thr Cys Tyr Ile Lys Ala Arg Ala Ala Cys Arg Ala Ala Gly Leu
1475 1480 1485
Gln Asp Cys Thr Met Leu Val Cys Gly Asp Asp Leu Val Va1 Ile Cys
1490 1495 1500
Glu Ser Ala G1y Val Gln Glu Asp Ala Ala Ser Leu Arg A1a Phe Thr
1505 11510 1515 1520
Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro
1525 1530 1535
Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Sex Ser Asn Val Ser Val
1540 1545 1550
Ala His Asp Gly Ala Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro
1555 1560 1565
Thr Thr Pro Leu A1a Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro
1570 1575 1580
Val Asn Ser Trp Leu Gly Asn Ile, Ile Met Phe Ala Pro Thr Leu Trp
1585 1590 1595 1600
Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Va1 Leu I1e Ala Arg
1605 1610 1615
Asp Gln Leu Glu Gln A1a Leu Asp Cys Glu Ile Tyr Gly Ala Cys Tyr
1630 1625 1630
Ser Ile Glu Pro Leu Asp Leu Pro Pro Ile Ile Gln Arg Leu His Gly
1635 1640 1645
Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg
1650 1655 1660
Val Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp
1665 1670 1675 1680
16
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Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu A1a Arg Gly Gly
1685 1690 1695
Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Arg Thr
1700 1705 1710
Lys Leu Lys Leu Thr Pro Ile Ala Ala Ala Gly Gln Leu Asp Leu Ser
1715 1720 1725
Gly Trp Phe Thr A1a Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val
1730 1735 1740
Ser His Ala Arg Pro Arg Trp Tle Trp Phe Cys Leu Leu Leu Leu Ala
1745 1750 1755 1760
Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg Met Ser Thr Asn Pro
1765 1770 1775
Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp
1780 1785 1790
Val Lys Phe Pro Gly Gly Gly Gln Ile Val G1y Gly Val Tyr Leu Leu
1795 1800 1805
Pro Arg Ar,g Gly Pro Arg Leu Gly Va1 Arg Ala Thr Arg Lys Thr Ser
1810 1815 1820
Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro I1e Pro Lys Ala Arg
1825 1830 1835 1840
Arg Pro Glu Gly Arg Thr Trp Ala G1n Pro Gly Tyr Pro Trp Pro Leu
1845 1850 1855
T'yr Gly Asn Glu G1y Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg
1860 1865 1870
Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg
1875 1880 1885
Asn Leu Gly Lys
1890
<210> 7
<211> 21
<212> PRT
<213> Artificial
<220>
<223> E2 epitope consensus sequence
<400> 7
Gly Ser Ala Ala Arg Thr Thr Ser Gly Phe Val Ser Leu Phe Ala Pro
1 5 10 15
Gly Ala Lys Gln Asn
17
CA 02491508 2004-12-31
WO 2004/005473 PCT/US2003/020996
<210> 8
<211> 23
<212> PRT
<213> Artificial
<220>
<223> NS4A peptide
<400> 8
Lys Lys Gly Ser Val Val Ile Val Gly Arg Ile Val Leu Ser Gly Lys
1 5 10 15
Pro Ala Ile Tle Pro Lys Lys
1$
