U.S. patent application number 12/231351 was filed with the patent office on 2009-04-16 for activation of hcv-specific t cells.
Invention is credited to Steve Coates, Michael Houghton, Xavier Paliard, Mark Selby.
Application Number | 20090098153 12/231351 |
Document ID | / |
Family ID | 22582387 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090098153 |
Kind Code |
A1 |
Houghton; Michael ; et
al. |
April 16, 2009 |
Activation of HCV-specific T cells
Abstract
The invention provides a method of activating hepatitis C virus
(HCV)-specific T cells, including CD4.sup.+ and CD8.sup.+ T cells.
HCV-specific T cells are activated using fusion proteins comprising
HCV NS3, NS4, NS5a, and NS5b polypeptides, polynucleotides encoding
such fusion proteins, or polypeptide or polynucleotide compositions
containing the individual components of these fusions. The method
can be used in model systems to develop HCV-specific immunogenic
compositions, as well as to immunize a mammal against HCV.
Inventors: |
Houghton; Michael;
(Danville, CA) ; Coates; Steve; (Emeryville,
CA) ; Selby; Mark; (San Francisco, CA) ;
Paliard; Xavier; (San Francisco, CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS INC.
INTELLECTUAL PROPERTY R338, P.O. BOX 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
22582387 |
Appl. No.: |
12/231351 |
Filed: |
September 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10281341 |
Oct 25, 2002 |
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12231351 |
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09698874 |
Oct 27, 2000 |
6562346 |
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10281341 |
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60161713 |
Oct 27, 1999 |
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Current U.S.
Class: |
424/189.1 |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 43/00 20180101; A61K 2039/53 20130101; A61P 1/16 20180101;
A61P 37/04 20180101; A61K 48/00 20130101; C07K 2319/00 20130101;
C12N 2770/24222 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/189.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 2000 |
US |
PCT/US00/29594 |
Claims
1. A composition comprising: (a) a fusion protein comprising HCV
polypeptides, wherein the HCV polypeptides consist of an NS3, an
NS4, an NS5a, and NS5b polypeptide of a hepatitis C virus (HCV) and
optionally a core polypeptide; and (b) a polynucleotide encoding an
HCV E1E2 complex, wherein the composition stimulates an immune
response to HCV.
2. The composition of claim 1, wherein at least one of the HCV
polypeptides is derived from a different strain of HCV than the
other HCV polypeptides.
3. The composition of claim 1, further comprising a
pharmaceutically acceptable excipient.
4. The composition of claim 3, further comprising an adjuvant.
5. The composition of claim 3, further comprising a CpG
oligonucleotide.
6. The composition of claim 3, wherein said fusion protein is
adsorbed to or entrapped within a microparticle or ISCOM.
7. A composition comprising a hepatitis C virus (HCV)
polynucleotide encoding an HCV E1E2 complex, HCV polypeptides, and
a pharmaceutically acceptable excipient, wherein the HCV
polypeptides consist of: (a) an isolated and purified NS3
polypeptide; (b) an isolated and purified NS4; (c) an isolated and
purified NS5a polypeptide; (d) an isolated and purified NS5b
polypeptide; and optionally, (e) an isolated and purified core
polypeptide, wherein the composition stimulates an immune response
to HCV.
8. The composition of claim 7, further comprising an adjuvant.
9. The composition of claim 7, further comprising a CpG
oligonucleotide.
10. The composition of claim 7, wherein one or more of said HCV
polypeptides is adsorbed to or entrapped within a microparticle or
ISCOM.
11. A method of activating T cells of a vertebrate subject which
recognize an epitope of an HCV polypeptide, comprising the step of:
administering the composition of claim 1 to said vertebrate
subject, whereby a population of activated T cells recognizes an
epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
12. A method of activating T cells of a vertebrate subject which
recognize an epitope of an HCV polypeptide, comprising the step of:
administering the composition of claim 4 to said vertebrate
subject, whereby a population of activated T cells recognizes an
epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
13. A method of activating T cells of a vertebrate subject which
recognize an epitope of an HCV polypeptide, comprising the step of:
administering the composition of claim 5 to said vertebrate
subject, whereby a population of activated T cells recognizes an
epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
14. A method of activating T cells of a vertebrate subject which
recognize an epitope of an HCV polypeptide, comprising the step of:
administering the composition of claim 6 to said vertebrate
subject, whereby a population of activated T cells recognizes an
epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
15. A method of activating T cells of a vertebrate subject which
recognize an epitope of an HCV polypeptide, comprising the step of:
administering the composition of claim 7 to said vertebrate
subject, whereby a population of activated T cells recognizes an
epitope of the NS3, NS4, NS5a, NS5b and/or core polypeptides.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 10/281,341, filed Oct. 25, 2002, which is a
continuation-in-part of U.S. application Ser. No. 09/698,874, filed
Oct. 27, 2000, from which applications priority is claimed under 35
USC .sctn.120. U.S. application Ser. No. 09/698,874 claims the
benefit of provisional patent application Ser. No. 60/161,713,
filed Oct. 27, 1999 under 35 USC .sctn.119(e)(1). The foregoing
applications are incorporated herein by reference in their
entireties.
TECHNICAL AREA OF THE INVENTION
[0002] The invention relates to the activation of hepatitis C
virus(HCV)-specific T cells. More particularly, the invention
relates to the use of multiple HCV polypeptides, either alone or as
fusions, to stimulate cell-mediated immune responses, such as to
activate HCV-specific T cells.
BACKGROUND OF THE INVENTION
[0003] 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 chronic carrier state that can result in cirrhosis,
liver failure, and hepatocellular carcinoma. See Alter et al.
(1992) N. Engl. J. Med. 327:1899-1905; Resnick 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.
[0004] 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 particular,
generation of a strong cytotoxic T lymphocyte (CTL) response 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 inducing strong CTL responses against HCV.
SUMMARY OF THE INVENTION
[0005] It is an object of the invention to provide reagents and
methods for stimulating immune responses, such as activating T
cells which recognize epitopes of HCV polypeptides. This and other
objects of the invention are provided by one or more of the
embodiments described below.
[0006] The invention provides HCV proteins useful for stimulating
immune responses, such as activating HCV-specific T cells. One
embodiment provides a fusion protein that comprises HCV
polypeptides, wherein the HCV polypeptides consist essentially of
an NS3, an NS4, an NS5a polypeptide, and optionally a core
polypeptide. In certain embodiments, the fusion protein includes an
NS5b polypeptide.
[0007] In certain embodiments, at least one of the HCV polypeptides
is derived from a different strain of HCV than the other
polypeptides.
[0008] The invention also provides compositions comprising any of
these fusion proteins and a pharmaceutically acceptable excipient.
In certain embodiments, the compositions further comprise an
adjuvant, a CpG polynucleotide and/or the fusion protein is
adsorbed to or entrapped within a microparticle or ISCOM. The
compositions can further comprise a polynucleotide encoding an E1E2
complex. The E1E2 polynucleotide can also be adsorbed to or
entrapped withing a microparticle.
[0009] Another embodiment provides a composition comprising HCV
polypeptides and a pharmaceutically acceptable excipient. The HCV
polypeptides consist essentially of an NS3, an NS4, an NS5a
polypeptide, and optionally a core polypeptide. In certain
embodiments, the composition includes an NS5b polypeptide. In other
embodiments, the compositions further comprise an adjuvant, a CpG
polynucleotide and/or one or more of the HCV polypeptides is
adsorbed to or entrapped within a microparticle or ISCOM. The
compositions can further comprise a polynucleotide encoding an E1E2
complex. The E1E2 polynucleotide can also be adsorbed to or
entrapped withing a microparticle. Moreover, one of the HCV
polypeptides may be derived from a different strain of HCV than the
others.
[0010] Even another embodiment of the invention provides an
isolated and purified polynucleotide which encodes a fusion protein
as described above. In additional embodiments, the fusion proteins
further include a polynucleotide encoding an E1E2 complex.
[0011] Yet another embodiment of the invention provides a
composition comprising the polynucleotides described above and a
pharmaceutically acceptable excipient. In certain embodiments, the
compositions further comprise an adjuvant and/or the polynucleotide
may be adsorbed to or entrapped within a microparticle. The
compositions can further comprise a polynucleotide encoding an E1E2
complex. The E1E2 polynucleotide can also be adsorbed to or
entrapped withing a microparticle.
[0012] In a further embodiment, the invention provides a
composition comprising HCV polynucleotides and a pharmaceutically
acceptable excipient, wherein the HCV polynucleotides consist
essentially of polynucleotides encoding an NS3, an NS4, an NS5a
polypeptide, and optionally a core polypeptide. In certain
embodiments, the composition also includes a polynucleotide
encoding an NS5b polypeptide. The compositions may further comprise
an adjuvant and/or one or more of the polynucleotides may be
adsorbed to or entrapped within a microparticle. The compositions
can further comprise a polynucleotide encoding an E1E2 complex. The
E1E2 polynucleotide can also be adsorbed to or entrapped withing a
microparticle. Additionally, one or more of the polynucleotides may
be derived from a different strain of HCV than the others.
[0013] In another embodiment, the invention provides a method of
activating T cells which recognize an epitope of an HCV
polypeptide. T cells are contacted with any of the fusions,
polynucleotides or compositions described above. A population of
activated T cells recognizes an epitope of the NS3, NS4, NS5a,
NS5b, core and/or E1E2 polypeptide.
[0014] In the proteins and polynucleotides above, the regions in
the fusions need not be in the order in which they naturally occur
in the native HCV polyprotein. Thus, for example, the NS5b
polypeptide, if present, may be at the N- and/or C-terminus of the
fusion, or may be located internally. Similarly, the E1 polypeptide
may precede or follow the E2 polypeptide. The E1E2 polypeptide may
also be part of the nonstructural fusion protein or may be provided
separately, as an E1E2 complex, or as individual polypeptides.
[0015] Moreover, the NS3 polypeptide may include a modification to
inhibit protease activity, such that cleavage of the fusion is
inhibited. Such modifications are described more fully below.
Additionally, the compositions can comprise more than one HCV
nonstructural fusion protein, such as a fusion protein with NS3,
NS4 and NS5a, and a fusion protein with NS3, NS4, NS5a, NS5b and
E1E2. The E1E2 complexes, whether present separately or as part of
the fusion, can have varying E1E2 polypeptides (described more
fully below).
[0016] In certain embodiments, the nonstructural fusion protein
consists of, from the amino terminus to the carboxyl terminus, an
NS3, an NS4, an NS5a and, optionally, an NS5b polypeptide and the
E1E2 complex consists of, from amino terminus to the carboxyl
terminus, an E1 polypeptide and an E2 polypeptide.
[0017] The various polypeptides (and polynucleotides encoding
therefor) are derived from the same HCV isolate, 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.
[0018] Yet another embodiment of the invention provides a method of
stimulating an immune response, such as a cellular immune response,
in a vertebrate subject by administering a composition as described
herein. In certain embodiments, the composition activates T cells
which recognize an epitope of an HCV polypeptide. T cells are
contacted with a composition as described above. A population of
activated T cells recognizes an epitope of one or more of the HCV
polypeptide(s).
[0019] The invention thus provides methods and reagents for
stimulating immune responses to HCV, such as for 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
[0020] FIG. 1 is a diagrammatic representation of the HCV genome,
depicting the various regions of the HCV polyprotein.
[0021] FIG. 2 depicts the DNA and corresponding amino acid sequence
of a representative native NS3 protease domain.
[0022] FIGS. 3A-3C (SEQ ID NOS:3 and 4) shows the nucleotide and
corresponding amino acid sequence for the HCV-1 E1/E2/p7 region.
The numbers shown in the figure are relative to the full-length
HCV-1 polyprotein. The E1, E2 and p7 regions are shown.
[0023] FIG. 4 is a diagram of plasmid pMHE1E2-809, encoding
E1E2.sub.809, a representative E1E2 protein for use with the
present invention.
[0024] FIGS. 5A-5J (SEQ ID NOS:7 and 8) depict the DNA and
corresponding amino acid sequence of a representative NS345Core
fusion protein. The depicted sequence includes amino acids
1242-3011 of the HCV polyprotein (representing polypeptides from
NS3, NS4, NS5a and NS5b) with amino acids. 1-121 of the HCV
polyprotein (representing a polypeptide from the core region) fused
to the C-terminus of NS5b. This numbering is relative to the HCV-1
polyprotein.
[0025] FIG. 6 shows a side-by-side comparison of IFN-.gamma.
expression generated in animals in response to delivery of
alphavirus constructs encoding NS3NS4NS5a.
[0026] FIG. 7 shows IFN-.gamma. expression generated in animals in
response to delivery of plasmid DNA encoding NS3NS4NS5a ("naked"),
PLG-linked DNA encoding NS3NS4NS5a ("PLG), separate DNA plasmids
encoding NS5a, NS34a, and NS4ab ("naked"), and PLG-linked DNA
encoding NS5a, NS34a, and NS4ab ("PLG").
[0027] FIG. 8 shows HCV-specific CD8+ and CD4+ responses in
vaccinated chimpanzees.
[0028] FIG. 9 depicts the specificity of T cell responses primed by
electroporation of plasmid DNA two weeks subsequent to the third
immunization.
[0029] FIG. 10 shows the specificity of T cell responses primed by
vaccinating chimpanzees with NS345Core.sub.121-ISCOMS two weeks
subsequent to the third immunization.
DETAILED DESCRIPTION OF THE INVENTION
[0030] 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., Molecular Cloning: A
Laboratory Manual (2nd Edition); Methods In Enzymology (S. Colowick
and N. Kaplan eds., Academic Press, Inc.); DNA Cloning, Vols. I and
II (D. N. Glover ed.); Oligonucleotide Synthesis M. J. Gait ed.);
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.);
Animal Cell Culture (R. K. Freshney ed.); Perbal, B., A Practical
Guide to Molecular Cloning.
[0031] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0032] 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 antigens, and the like.
[0033] The following amino acid abbreviations are used throughout
the text: [0034] Alanine: Ala (A) Arginine: Arg (R) [0035]
Asparagine: Asn (N) Aspartic acid: Asp (D) [0036] Cysteine: Cys (C)
Glutamine: Gln (Q) [0037] Glutamic acid: Glu (E) Glycine: Gly (G)
[0038] Histidine: His (H) Isoleucine: Ile (I) [0039] Leucine: Leu
(L) Lysine: Lys (K) [0040] Methionine: Met (M) Phenylalanine: Phe
(F) [0041] Proline: Pro (P) Serine: Ser (S) [0042] Threonine: Thr
(T) Tryptophan: Trp (W) [0043] Tyrosine: Tyr (Y) Valine: Val
(V)
I. DEFINITIONS
[0044] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0045] 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.
[0046] 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. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.),
as well as newly identified isolates, and subtypes of these
isolates, such as HCV1a, HCV1b, 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.
[0047] By an "E1 polypeptide" is meant a molecule derived from an
HCV E1 region. The mature E1 region of HCV-1 begins at
approximately amino acid 192 of the polyprotein and continues to
approximately amino acid 383, numbered relative to the full-length
HCV-1 polyprotein. (See, FIGS. 1 and 3A-3C. Amino acids 192-383 of
FIGS. 3A-3C correspond to amino acid positions 20-211 of SEQ ID
NO:4.) Amino acids at around 173 through approximately 191 (amino
acids 1-19 of SEQ ID NO: 4) serve as a signal sequence for E1.
Thus, by an "E1 polypeptide" is meant either a precursor E1
protein, including the signal sequence, or a mature E1 polypeptide
which lacks this sequence, or even an E1 polypeptide with a
heterologous signal sequence. The E1 polypeptide includes a
C-terminal membrane anchor sequence which occurs at approximately
amino acid positions 360-383 (see, International Publication No. WO
96/04301, published Feb. 15, 1996). An E1 polypeptide, as defined
herein, may or may not include the C-terminal anchor sequence or
portions thereof.
[0048] By an "E2 polypeptide" is meant a molecule derived from an
HCV E2 region. The mature E2 region of HCV-1 begins at
approximately amino acid 383-385, numbered relative to the
full-length HCV-1 polyprotein. (See, FIGS. 1 and 3A-3C. Amino acids
383-385 of FIGS. 3A-3C correspond to amino acid-positions 211-213
of SEQ ID NO:4.) A signal peptide begins at approximately amino
acid 364 of the polyprotein. Thus, by an "E2 polypeptide" is meant
either a precursor E2 protein, including the signal sequence, or a
mature E2 polypeptide which lacks this sequence, or even an E2
polypeptide with a heterologous signal sequence. The E2 polypeptide
includes a C-terminal membrane anchor sequence which occurs at
approximately amino acid positions 715-730 and may extend as far as
approximately amino acid residue 746 (see, Lin et al., J. Virol.
(1994) 68:5063-5073). An E2 polypeptide, as defined herein, may or
may not include the C-terminal anchor sequence or portions thereof.
Moreover, an E2 polypeptide may also include all or a portion of
the p7 region which occurs immediately adjacent to the C-terminus
of E2. As shown in FIGS. 1 and 3A-3C, the p7 region is found at
positions 747-809, numbered relative to the full-length HCV-1
polyprotein (amino acid positions 575-637 of SEQ ID NO:4).
Additionally, it is known that multiple species of HCV E2 exist
(Spaete et al., Virol. (1992) 188:819-830; Selby et al., J. Virol.
(1996) 70:5177-5182; Grakoui et al., J. Virol. (1993) 67:1385-1395;
Tomei et al., J. Virol. (1993) 67:4017-4026). Accordingly, for
purposes of the present invention, the term "E2" encompasses any of
these species of E2 including, without limitation, species that
have deletions of 1-20 or more of the amino acids from the
N-terminus of the E2, such as, e.g, deletions of 1, 2, 3, 4, 5 . .
. 10 . . . 15, 16, 17, 18, 19 . . . etc. amino acids. Such E2
species include those beginning at amino acid 387, amino acid 402,
amino acid 403, etc.
[0049] Representative E1 and E2 regions from HCV-1 are shown in
FIGS. 3A-3C and SEQ ID NO:4. For purposes of the present invention,
the E1 and E2 regions are defined with respect to the amino acid
number of the polyprotein encoded by the genome of HCV-1, with the
initiator methionine being designated position 1. See, e.g., Choo
et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455. However, it
should be noted that the term an "E1 polypeptide" or an "E2
polypeptide" as used herein is not limited to the HCV-1 sequence.
In this regard, the corresponding E1 or E2 regions in other HCV
isolates can be readily determined by aligning sequences from the
isolates in a manner that brings the sequences into maximum
alignment. This can be performed with any of a number of computer
software packages, such as ALIGN 1.0, available from the University
of Virginia, Department of Biochemistry (Attn: Dr. William R.
Pearson). See, Pearson et al., Proc. Natl. Acad. Sci. USA (1988)
85:2444-2448.
[0050] Furthermore, an "E1 polypeptide" or an "E2 polypeptide" as
defined herein is not limited to a polypeptide having the exact
sequence depicted in the Figures. Indeed, the HCV genome is in a
state of constant flux in vivo and contains several variable
domains which exhibit relatively high degrees of variability
between isolates. 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%, more than 60%, and even
more than 80-90% homology, when the two sequences are aligned. It
is readily apparent that the terms encompass E1 and E2 polypeptides
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. Gen. Virol. (1993) 74:2391-2399 (e.g., strains 1, 2, 3, 4 etc.),
as well as newly identified isolates, and subtypes of these
isolates, such as HCV1a, HCV1b etc.
[0051] Thus, for example, the term "E1" or "E2" polypeptide refers
to native E1 or E2 sequences from any of the various HCV strains,
as well as analogs, muteins and immunogenic fragments, as defined
further below. The complete genotypes of many of these strains are
known. See, e.g., U.S. Pat. No. 6,150,087 and GenBank Accession
Nos. AJ238800 and AJ238799.
[0052] Additionally, the terms "E1 polypeptide" and "E2
polypeptide" encompass proteins which include modifications to the
native sequence, such as internal deletions, additions and
substitutions (generally conservative in nature). These
modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through naturally
occurring mutational events. All of these modifications are
encompassed in the present invention so long as the modified E1 and
E2 polypeptides function for their intended purpose. Thus, for
example, if the E1 and/or E2 polypeptides are to be used in vaccine
compositions, the modifications must be such that immunological
activity (i.e., the ability to elicit a humoral or cellular immune
response to the polypeptide) is not lost.
[0053] By "E1E2" complex is meant a protein containing at least one
E1 polypeptide and at least one E2 polypeptide, as described above.
Such a complex may also include all or a portion of the p7 region
which occurs immediately adjacent to the C-terminus of E2. As shown
in FIGS. 1 and 3A-3C, the p7 region is found at positions 747-809,
numbered relative to the full-length HCV-1 polyprotein (amino acid
positions 575-637 of SEQ ID NO:4). A representative E1E2 complex
which includes the p7 protein is termed "E1E2.sub.809" herein.
[0054] The mode of association of E1 and E2 in an E1E2 complex is
immaterial. The E1 and E2 polypeptides may be associated through
non-covalent interactions such as through electrostatic forces, or
by covalent bonds. For example, the E1E2 polypeptides of the
present invention may be in the form of a fusion protein which
includes an immunogenic E1 polypeptide and an immunogenic E2
polypeptide, as defined above. The fusion may be expressed from a
polynucleotide encoding an E1E2 chimera. Alternatively, E1E2
complexes may form spontaneously simply by mixing E1 and E2
proteins which have been produced individually. Similarly, when
co-expressed and secreted into media, the E1 and E2 proteins can
form a complex spontaneously. Thus, the term encompasses E1E2
complexes (also called aggregates) that spontaneously form upon
purification of E1 and/or E2. Such aggregates may include one or
more E1 monomers in association with one or more E2 monomers. The
number of E1 and E2 monomers present need not be equal so long as
at least one E1 monomer and one E2 monomer are present. Detection
of the presence of an E1E2 complex is readily determined using
standard protein detection techniques such as polyacrylamide gel
electrophoresis and immunological techniques such as
immunoprecipitation.
[0055] The terms "analog" and "mutein" refer to biologically active
derivatives of the reference molecule, or fragments of such
derivatives, that retain desired activity, such as the ability to
stimulate a cell-mediated immune response, as defined below. 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) 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 known in the art and are
described further below.
[0056] 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, arginine, 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.
[0057] 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.
[0058] 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 immunogenic activity, as measured by the assays described
herein.
[0059] 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).
[0060] 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 Mapping Protocols in Methods
in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana
Press, Totowa, N.J. 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 known in the art and described in, e.g., U.S. Pat. No.
4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA
81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all
incorporated herein by reference in their entireties. 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 Mapping Protocols, 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 from the Oxford Molecular
Group. This computer program employs the Hopp/Woods method, Hopp et
al., Proc. 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.
[0061] For a description of various HCV epitopes, see, e.g., Chien
et al., Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et
al., J. Gastroent. Hepatol. (1993) 8:S33-39; Chien et al.,
International Publication No. WO 93/00365; Chien, D. Y.,
International Publication No. WO 94/01778; and U.S. Pat. Nos.
6,280,927 and 6,150,087, incorporated herein by reference in their
entireties.
[0062] As used herein, the term "conformational epitope" refers to
a portion of a full-length protein, or an analog or mutein thereof,
having structural features native to the amino acid sequence
encoding the epitope within the full-length natural protein. Native
structural features include, but are not limited to, glycosylation
and three dimensional structure. Preferably, a conformational
epitope is produced recombinantly and is expressed in a cell from
which it is extractable under conditions which preserve its desired
structural features, e.g. without denaturation of the epitope. Such
cells include bacteria, yeast, insect, and mammalian cells.
Expression and isolation of recombinant conformational epitopes
from the HCV polyprotein are described in e.g., International
Publication Nos. WO 96/04301, WO 94/01778, WO 95/33053, WO
92/08734, which applications are herein incorporated by reference
in their entirety.
[0063] 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, (Unanue 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 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., Computer Prediction of T-cell
Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G. C.
Woodrow et al., (1990) pp. 109-116) and further that the
amphipathic structures have an .alpha.-helical configuration (see,
e.g., Spouge et al., J. Immunol. (1987) 138:204-212; Berkower et
al., J. Immunol. (1986) 136:2498-2503).
[0064] Hence, segments of proteins that include T-cell epitopes can
be readily predicted using numerous computer programs. (See e.g.,
Margalit et al., Computer Prediction 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 which are believed
to be required for a T-cell epitope.
[0065] An "immunological response" to an 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. Another 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 cells against cells
displaying peptide antigens in association with MHC molecules on
their surface. A "cellular immune response" also refers to the
production of cytokines, chemokines and other such molecules
produced by activated T-cells and/or other white blood cells,
including those derived from CD4+ and CD8+ T-cells.
[0066] 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.
[0067] 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. Immunol.
(1993) 151:4189-4199; Doe et al., Eur. J. Immunol. (1994)
24:2369-2376; and the examples below.
[0068] Thus, an immunological 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 immune 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 .gamma..delta. 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.
[0069] By "equivalent antigenic 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 are
not necessarily identical due to sequence variation, but which
occur in equivalent positions in the HCV sequence in question. In
general 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.
[0070] 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 in 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.
[0071] 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.
[0072] An "HCV polynucleotide" is a polynucleotide that encodes an
HCV polypeptide, as defined above.
[0073] "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.
[0074] "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 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.
[0075] A "control element" refers to a polynucleotide sequence
which aids in the expression of a coding sequence to which it is
linked. The term 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.
[0076] A "promoter" as used herein is a DNA regulatory region
capable of binding RNA polymerase in a host cell and initiating
transcription of a downstream (3' direction) coding sequence
operably linked thereto. For purposes of the present invention, a
promoter sequence includes the minimum 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
polymerase. Eucaryotic promoters will often, but not always,
contain "TATA" boxes and "CAT" boxes.
[0077] A control sequence "directs the transcription" of a coding
sequence in a cell when RNA polymerase will bind the promoter
sequence and transcribe the coding sequence into mRNA, which is
then translated into the polypeptide encoded by the coding
sequence.
[0078] "Expression cassette" or "expression construct" refers to an
assembly which is capable of directing the expression of the
sequence(s) or gene(s) 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
sequence(s) or gene(s) 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 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).
[0079] "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
uptake, transfection, infection, and the like. 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.
[0080] A "host cell" is a cell which has been transformed, or is
capable of transformation, by an exogenous DNA sequence.
[0081] 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 macro-molecules 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.
[0082] The term "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.
[0083] "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.
[0084] 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 Protein Sequence and Structure M. O.
Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research
Foundation, Washington, D.C., which adapts the local homology
algorithm of Smith and Waterman Advances in 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, Wis.) for example, the BESTFIT, FASTA and GAP programs,
which also rely on the Smith and Waterman algorithm. These programs
are readily utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package 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.
[0085] 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, Calif.). 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;
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.nln.gov/cgi-bin/BLAST.
[0086] 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., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0087] By "nucleic acid immunization" is meant the introduction of
a nucleic acid molecule encoding one or more selected antigens into
a host cell, for the in vivo expression of the antigen or antigens.
The nucleic acid molecule can be introduced directly into the
recipient subject, such as by injection, inhalation, oral,
intranasal and mucosal administration, or the like, 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.
[0088] 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).
[0089] 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 chickens, turkeys
and other gallinaceous birds, ducks, geese, and the like. 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 immune systems of all of these vertebrates operate
similarly.
II. MODES OF CARRYING OUT THE INVENTION
[0090] 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.
[0091] 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.
[0092] It is a discovery of the present invention that fusion
proteins, combinations of the individual components of these
fusions, and polynucleotides encoding the same, comprising an NS3,
an NS4, and an NS5a polypeptide with or without a core polypeptide,
or an NS3, an NS4, an NS5a, and an NS5b polypeptide, with or
without a core polypeptide, of an HCV virus can be used to activate
HCV-specific T cells, i.e., T cells which recognize epitopes of
these polypeptides.
[0093] The present invention also pertains to compositions
comprising HCV nonstructural fusion proteins and HCV E1E2
complexes, as well as compositions comprising polynucleotides
encoding the same or combinations of polypeptides and
polynucleotides.
[0094] The proteins, polynucleotides, compositions and combinations
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. Activation of
HCV-specific T cells provides both in vitro and in vivo model
systems for the development of HCV vaccines, particularly for
identifying HCV polypeptide epitopes associated with a response.
The compositions can also be used to generate an immune response
against HCV in a mammal, particularly a CTL response for either
therapeutic or prophylactic purposes.
Fusion Proteins
[0095] 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. As shown in FIG. 1 and the table
below, an HCV polyprotein, upon cleavage, produces at least ten
distinct products, in the order of
NH.sub.2-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The core
polypeptide occurs at positions 1-191, numbered relative to HCV-1
(see, Choo et al. (1991) Proc. 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 NS5a found at about positions 1973-2420),
and an RNA-dependent RNA polymerase (NS5b 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-US-00001 Domain Approximate Boundaries* C (core) 1-191 E1
192-383 E2 384-746 P7 747-809 NS2 810-1026 NS3 1027-1657 NS4a
1658-1711 NS4b 1712-1972 NS5a 1973-2420 NS5b 2421-3011 *Numbered
relative to HCV-1. See, Choo et al. (1991) Proc. Natl. Acad. Sci.
USA 88: 2451-2455.
[0096] Fusion proteins for use in the compositions and methods, and
polynucleotides encoding therefor, include or encode an NS3
polypeptide, an NS4 (NS4a and/or NS4b) polypeptide, an NS5a
polypeptide and, optionally, an NS5b polypeptide. The fusion
proteins may or may not include all or part of the core region. In
certain embodiments, none of the core region is present in the
compositions. The nonstructural regions need not be in the order in
which they naturally occur in the native HCV polyprotein. Thus, for
example, the NS5b polypeptide may be at the N- and/or C-terminus of
the fusion or may be found internally. These polypeptides may be
derived from the same HCV isolate, 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 clades endemic in specific geographic 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.
[0097] In one embodiment, the fusion protein of the present
invention includes an NS3 polypeptide that has been 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.
[0098] 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 FIG. 3. The
structure of the NS3 protease and active site are known. See, e.g.,
De Francesco et al., Antivir. Ther. (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 occurring at positions 1- or
2-182, preferably 1- or 2-170, or 1- or 2-155 of FIG. 3. Preferred
modifications are to the catalytic triad at the active site of the
protease, i.e., H, D 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 FIG. 3). 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.
[0099] For example, protease activity or lack thereof may be
determined using assays well known in the art. See, e.g., Takeshita
et al., Anal. Biochem. (1997) 247:242-246; Kakiuchi et al., J.
Biochem. (1997) 122:749-755; SalI et al., Biochemistry (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. Biochem. (1999) 270:268-275; Kakiuchi et al., J. Virol. Meth.
(1999) 80:77-84; Fowler et al., J. Biomol. Screen. (2000)
5:153-158; and Kim et al., Anal. Biochem. (2000) 284:42-48.
[0100] The NS3; NS4; NS5a, and NS5b polypeptides present in the
various fusions described above can either be full-length
polypeptides or portions of NS3, NS4 (NS4a and/or NS4b), NS5a, and
NS5b polypeptides. The portions of NS3, NS4, NS5a, and NS5b
polypeptides making up the fusion protein preferably 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 NO:2). Epitopes of NS3, NS4 (NS4a
and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can be
identified by several methods. For example, NS3, NS4, NS5a, NS5b
polypeptides or fusion proteins comprising any combination of the
above, can be isolated, for example, by 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.
[0101] Epitopes recognized by a T-cell receptor on an HCV-activated
T cell can be identified by, for example, .sup.51Cr release assay
or by lymphoproliferation assay (see the examples). In a .sup.51Cr
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 into the target cells. HCV-specific CD8.sup.+ T cells will
lyse target cells displaying, for example, an NS3, NS4, NS5a, NS5b,
NS3NS4NS5a, or NS3NS4NS5aNS5b epitope and will not lyse cells that
do not display such an epitope. In a lymphoproliferation assay,
HCV-activated CD4.sup.+ T cells will proliferate when cultured
with, for example, an NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or
NS3NS4NS5aNS5b epitopic peptide, but not in the absence of an HCV
epitopic peptide.
[0102] NS3, NS4, NS5a, and NS5b 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 NS3, NS4,
NS5a, and NS5b polypeptides of any of these strains can be used in
a fusion protein. A representative fusion protein for use in the
present invention is shown if FIGS. 5A-5J. The depicted sequence
includes amino acids 1242-3011 of the HCV polyprotein (representing
polypeptides from NS3, NS4, NS5a and NS5b) with amino acids 1-121
of the HCV polyprotein (representing a polypeptide from the core
region) fused to the C-terminus of NS5b. This numbering is relative
to the HCV-1 polyprotein.
[0103] Nucleic acid and amino acid sequences of a number of HCV
strains and isolates, including nucleic acid and amino acid
sequences of NS3, NS4, NS5a, NS5b genes and polypeptides have been
determined. For example, isolate HCV J1.1 is described in Kubo et
al. (1989) Japan. Nucl. Acids Res. 17:10367-10372; Takeuchi et al.
(1990) Gene 91:287-291; Takeuchi et al. (1990) J. Gen. Virol.
71:3027-3033; and Takeuchi et al. (1990) Nucl. Acids Res. 18:4626.
The complete coding sequences of two independent isolates, HCV-J
and BK, are described by Kato et al., (1990) Proc. Natl. Acad. Sci.
USA 87:9524-9528 and Takamizawa et al., (1991) J. Virol.
65:1105-1113 respectively.
[0104] 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.about., HCT 23, Th, HCT 27, EC1 and EC10 are
described in Weiner et al. (1991) Virol. 180:842-848. HCV isolates
Pt-1, HCV-K1 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 Tsukiyama-Kohara et al. (1991) Virus Genes
5:243-254.
[0105] Each of the NS3, NS4, NS5a, and NS5b 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 NS4, and NS5a polypeptides can
be derived from a second strain of HCV. Alternatively, the NS4
polypeptide can be derived from a first strain of HCV, and the NS3
and NS5a polypeptides can be derived from a second strain of HCV.
Optionally, the NS5a polypeptide can be derived from a first strain
of HCV, and the NS3 and NS4 polypeptides can be derived from a
second strain of HCV. NS3, NS4 and NS5a polypeptides that are each
derived from different HCV strains can also be used in an HCV
fusion protein. Similarly, in a fusion protein comprising NS5b, at
least one of the NS3, NS4, NS5a, and NS5b polypeptides can be
derived from a different HCV strain than the other polypeptides.
Optionally, NS3, NS4, NS5a, and NS5b polypeptides that are each
derived from different HCV strains can also be used in an
NS3NS4NS5aNS5b fusion protein.
[0106] In addition to NS3, NS4a, NS4b, NS5a and NS5b, the fusion
proteins can contain other polypeptides derived from the HCV
polyprotein. For example, it may be desirable to include
polypeptides derived from the core region of the HCV polyprotein.
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 1-150, e.g., amino
acids 1-130, 1-120, for example, amino acids 1-121, 1-122, 1-123,
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. Pat. No. 5,350,671; Chien et al.,
Proc. Natl. Acad. Sci. USA (1992) 89:10011-10015; Chien et al., J.
Gastroent. Hepatol. (1993) 8:S33-39; Chien et al., International
Publication No. WO 93/00365; Chien, D. Y., International
Publication No. WO 94/01778; and U.S. Pat. Nos. 6,280,927 and
6,150,087, the disclosures of which are incorporated herein by
reference in their entireties. 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. The
fusions may also contain polynucleotides encoding E1E2
polypeptides, as described further below.
[0107] 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.
[0108] If desired, the fusion proteins, or the individual
components of these proteins, also can contain other non-HCV 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.
E1E2 Polypeptides
[0109] As explained above, the compositions of the present
invention may also include E1 and E2 polypeptides, complexes of
these polypeptides or polynucleotides encoding the same. The E1 and
E2 polypeptides and complexes thereof can be provided independent
of the nonstructural fusion protein or can be incorporated into the
same fusion. Moreover, E1E2 complexes can be provided as proteins,
or as polynucleotides encoding the same.
[0110] In this regard, E1, E2 and p7 are known to contain human
T-cell epitopes (both CD4+ and 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 E1E2 complexes, such as a composite of epitopes
from different genotypes.
[0111] As explained above, the E1 and E2 polypeptides that make up
the E1E2 complexes can be associated either through non-covalent or
covalent interactions. Such complexes may be made up of immunogenic
fragments of E1 and E2 which comprise epitopes. For example,
fragments of E1 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. The E1 and E2 polypeptides may be from
the same or different HCV strains. 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 E2
polypeptide. A particularly effective E2 epitope to incorporate
into the E2 sequence or E1E2 complexes is one which includes a
consensus sequence derived from this region, such as the consensus
sequence
Gly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-L-
ys-Gln-Asn (SEQ ID NO:5), which represents a consensus sequence for
amino acids 390-410 of the HCV type 1 genome. Additional epitopes
of E1 and E2 are known and described in, e.g., Chien et al.,
International Publication No. WO 93/00365, incorporated by
reference herein in its entirety.
[0112] Moreover, the E1 and E2 polypeptides may lack all or a
portion of the membrane spanning domain. The membrane anchor
sequence functions to associate the polypeptide to the endoplasmic
reticulum. Normally, such polypeptides are capable of secretion
into growth medium in which an organism expressing the protein is
cultured. However, as described in International Publication No. WO
98/50556, such polypeptides may also be recovered intracellularly.
Secretion into growth medium is readily determined using a number
of detection techniques, including, e.g., polyacrylamide gel
electrophoresis and the like, and immunological techniques such as
immunoprecipitation assays as described in, e.g., International
Publication No. WO 96/04301, published Feb. 15, 1996. With E1,
generally polypeptides terminating with about amino acid position
370 and higher (based on the numbering of HCV1 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 HCV1 E2 sequence) will
be retained by the ER and not secreted. (See, e.g., International
Publication No. WO 96/04301, published Feb. 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
E1 and E2 polypeptides which retain the transmembrane binding
domain, as well as polypeptides which lack 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, are intended to be
captured by the present invention. 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. If truncated molecules are used, it is
preferable to use E1 and E2 molecules that are both truncated.
[0113] E2 exists as multiple species (Spaete et al., Virol. (1992)
188:819-830; Selby et al., J. Virol. (1996) 70:5177-5182; Grakoui
et al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol.
(1993) 67:4017-4026) and clipping and proteolysis may occur at the
N- and C-termini of the E1 and E2 polypeptides. Thus, an E2
polypeptide for use herein may comprise at least amino acids
405-661, e.g., 400,401, 402 . . . to 661, such as 384-661, 384-715,
384-746, 384-749 or 384-809, or 384 to any C-terminus between
661-809, of an HCV polyprotein, numbered relative to the
full-length HCV-1 polyprotein. Similarly, preferable 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.
[0114] The E1 and E2 polypeptides and complexes thereof may also be
present as asialoglycoproteins. Such asialoglycoproteins are
produced by methods known in the art, such as by using cells in
which terminal glycosylation is blocked. When these proteins are
expressed in such cells and isolated by GNA lectin affinity
chromatography, the E1 and E2 proteins aggregate spontaneously.
Detailed methods for producing these E1E2 aggregates are described
in, e.g., U.S. Pat. No. 6,074,852, incorporated herein by reference
in its entirety. For example, E1E2 complexes are readily produced
recombinantly, either as fusion proteins or by e.g.,
co-transfecting host cells with constructs encoding for the E1 and
E2 polypeptides of interest. Co-transfection can be accomplished
either in trans or cis, i.e., by using separate vectors or by using
a single vector which bears both of the E1 and E2 genes. If done
using a single vector, both genes can be driven by a single set of
control elements or, alternatively, the genes can be present on the
vector in individual expression cassettes, driven by individual
control elements. Following expression, the E1 and E2 proteins will
spontaneously associate. Alternatively, the complexes can be formed
by mixing the individual proteins together which have been produced
separately, either in purified or semi-purified form, or even by
mixing culture media in which host cells expressing the proteins,
have been cultured, if the proteins are secreted. Finally, the E1E2
complexes of the present invention may be expressed as a fusion
protein wherein the desired portion of E1 is fused to the desired
portion of E2.
[0115] Moreover, the E1E2 complexes may be present as a
heterogeneous mixture of molecules, due to clipping and proteolytic
cleavage, as described above. Thus, a composition including E1E2
complexes may include multiple species of E1E2, such as E1E2
terminating at amino acid 746 (E1E2.sub.746), E1E2 terminating at
amino acid 809 (E1E2.sub.809), or any of the other various E1 and
E2 molecules described above, such as E2 molecules with N-terminal
truncations of from 1-20 amino acids, such as E2 species beginning
at amino acid 387, amino acid 402, amino acid 403, etc.
[0116] E1E2 complexes are readily produced recombinantly, either as
fusion proteins or by e.g., co-transfecting host cells with
constructs encoding for the E1 and E2 polypeptides of interest.
Co-transfection can be accomplished either in trans or cis, i.e.,
by using separate vectors or by using a single vector which bears
both of the E1 and E2 genes. If done using a single vector, both
genes can be driven by a single set of control elements or,
alternatively, the genes can be present on the vector in individual
expression cassettes, driven by individual control elements.
Following expression, the E1 and E2 proteins will spontaneously
associate. Alternatively, the complexes can be formed by mixing the
individual proteins together which have been produced separately,
either in purified or semi-purified form, or even by mixing culture
media in which host cells expressing the proteins, have been
cultured, if the proteins are secreted. Finally, the E1E2 complexes
of the present invention may be expressed as a fusion protein
wherein the desired portion of E1 is fused to the desired portion
of E2.
[0117] Methods for producing E1E2 complexes from full-length,
truncated E1 and E2 proteins which are secreted into media, as well
as intracellularly produced truncated proteins, are known in the
art. For example, such complexes may be produced recombinantly, as
described in U.S. Pat. No. 6,121,020; Ralston et al., J. Virol.
(1993) 67:6753-6761, Grakoui et al., J Virol. (1993) 67:1385-1395;
and Lanford et al., Virology (1993) 197:225-235.
Polynucleotides Encoding the Fusion Proteins and E1E2 Complexes
[0118] Polynucleotides contain less than an entire HCV genome and
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, E1 and E2 polypeptides and complexes thereof, described
above, and thus comprise coding sequences thereof. Polynucleotides
of the invention can also comprise other non-HCV 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.
[0119] 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.
[0120] Polynucleotides can comprise coding sequences for these
polypeptides which occur naturally or can include artificial
sequences which do not occur in nature. These polynucleotides can
be ligated to form a coding sequence for the fusion proteins and
E1E2 complexes 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.
[0121] 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 an immunological
response, such as a cellular immune response, using standard gene
delivery protocols. Methods for gene delivery are known in the art.
See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466,
incorporated by reference herein in their entireties. 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 ColE1
[0122] Additionally, the expression constructs can be packaged 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
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., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.
[0123] 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-triethylammonium (DOTMA) liposomes
are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island, N.Y. (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.,
Proc. 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; Szoka et al., Proc. Natl.
Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al.,
Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979)
17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629;
Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley
et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and
Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et
al., J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos,
Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et
al., Science (1982) 215:166.
[0124] The DNA can also be delivered in cochleate lipid
compositions similar to those described by Papahadjopoulos et al.,
Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat.
Nos. 4,663,161 and 4,871,488.
[0125] A number of viral based systems have been developed for gene
transfer into mammalian cells. For example, retroviruses provide a
convenient platform for gene 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 packaged in retroviral particles using techniques
known in the art. The recombinant virus can then be isolated and
delivered to cells of the subject either in vivo or ex vivo. A
number of retroviral systems have been described (U.S. Pat. No.
5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990;
Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al.,
Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci.
USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin.
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 and 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.
[0126] A number of adenovirus vectors have also been described,
such as adenovirus Type 2 and Type 5 vectors. Unlike retroviruses
which integrate into the host genome, adenoviruses persist
extrachromosomally thus minimizing the risks associated with
insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986)
57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder
et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol.
(1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58;
Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al.,
Human Gene Therapy (1993) 4:461-476).
[0127] 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., Proc. Natl. Acad. Sci. USA (1992)
89:6099-6103, can also be used for gene delivery.
[0128] 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 useful
for the practice of the instant methods, see, Dubensky et al., J.
Virol. (1996) 70:508-519; and International Publication Nos. WO
95/07995 and WO 96/17072.
[0129] Other vectors can be used, including but not limited to
simian virus 40 and cytomegalovirus. Bacterial vectors, such as
Salmonella ssp. Yersinia enterocolitica, Shigella spp., Vibrio
cholerae, Mycobacterium strain BCG, and Listeria monocytogenes can
be used. Minichromosomes 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.
[0130] 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 cytokine release. Examples of
particulate carriers 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., Pharm. Res. (1993) 10:362-368; and McGee et
al., J. Microencap. (1996).
[0131] One preferred method for adsorbing macromolecules onto
prepared microparticles is described in International Publication
No. WO 00/050006, incorporated herein by reference in its entirety.
Briefly, microparticles are rehydrated and dispersed to an
essentially monomeric suspension of microparticles using dialyzable
anionic or cationic detergents. Useful detergents include, but are
not limited to, any of the various N-methylglucamides (known as
MEGAs), such as heptanoyl-N-methylglucamide (MEGA-7),
octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide
(MEGA-9), and decanoyl-N-methyl-glucamide (MEGA-10); cholic acid;
sodium cholate; deoxycholic acid; sodium deoxycholate; taurocholic
acid; sodium taurocholate; taurodeoxycholic acid; sodium
taurodeoxycholate;
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS);
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate
(CHAPSO); -dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate
(ZWITTERGENT 3-12); N,N-bis-(3-D-gluconeamidopropyl)-deoxycholamide
(DEOXY-BIGCHAP); -octylglucoside; sucrose monolaurate; glycocholic
acid/sodium glycocholate; laurosarcosine (sodium salt);
glycodeoxycholic acid/sodium glycodeoxycholate; sodium dodceyl
sulfate (SDS); 3-(trimethylsilyl)-1-propanesulfonic acid (DSS);
cetrimide (CTAB, the principal component of which is
hexadecyltrimethylammonium bromide); hexadecyltrimethylammonium
bromide; dodecyltrimethylammonium bromide;
hexadecyltrimethyl-ammonium bromide; tetradecyltrimethylammonium
bromide; benzyl dimethyldodecylammonium bromide; benzyl
dimethyl-hexadecylammonium chloride; and benzyl
dimethyltetra-decylammonium bromide. The above detergents are
commercially available from e.g., Sigma Chemical Co., St. Louis,
Mo. Various cationic lipids known in the art can also be used as
detergents. See Balasubramaniam et al., 1996, Gene Ther., 3:163-72
and Gao, X., and L. Huang. 1995, Gene Ther., 2:7110-722.
[0132] A wide variety of other methods can be used to deliver the
expression constructs to cells. Such methods include DEAE
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., supra, for a discussion
of techniques for transforming cells of interest; and Felgner, P.
L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review
of delivery systems useful for gene transfer. Methods of delivering
DNA using electroporation are described in, e.g., U.S. Pat. Nos.
6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication
No. 2002/0146831; and International Publication No. WO/0045823, all
of which are incorporated herein by reference in their
entireties.
[0133] Moreover, the HCV polynucleotides 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, the HCV molecules
(usually with a hydrophobic region) are solubilized in detergent
and added to the reaction mixture, whereby ISCOMs are formed with
the HCV molecule 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. Drug Delivery
Reviews 32:247-271 (1998); U.S. Pat. Nos. 4,981,684, 5,178,860,
5,679,354 and 6,027,732, incorporated herein by reference in their
entireties; European Publ. Nos. EPA 109,942; 180,564 and 231,039;
and Coulter et al. (1998) Vaccine 16:1243.
[0134] 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. Pat. Nos. 4,945,050; 5,036,006;
5,100,792; 5,179,022; 5,371,015; and 5,478,744.
Compositions Comprising Fusion Proteins or Polynucleotides
[0135] The invention also provides compositions comprising the
fusion proteins or polynucleotides, as well as compositions
including the individual components of these fusion proteins or
polynucleotides. Compositions of the invention preferably 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.
[0136] 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. Liposomes can also be used as a carrier for
a composition of the invention, such liposomes are described
above.
[0137] If desired, co-stimulatory molecules which improve immunogen
presentation to lymphocytes, such as B7-1 or B7-2, or cytokines
such as GM-CSF, IL-2, and IL-12, can be included in a composition
of the invention. 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 (U.S.
Pat. No. 6,299,884, incorporated herein by reference in its
entirety; Chapter 10 in Vaccine design: the subunit and adjuvant
approach, eds. Powell & Newman, Plenum Press 1995), containing
5% Squalene, 0.5% TWEEN 80.TM., and 0.5% SPAN 85.TM. (optionally
containing various amounts of MTP-PE (see below), although not
required) formulated into submicron particles using a
microfluidizer such as Model 110Y microfluidizer (Microfluidics,
Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% TWEEN
80.TM., 5% pluronic-blocked polymer L121, and thr-MDP either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion, and (c) RIBI.TM. adjuvant system
(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,
0.2% TWEEN 80.TM., 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
(DETOX.TM.); (3) saponin adjuvants, such as QS21 or STIMULON.TM.
(Cambridge Bioscience, Worcester, Mass.) may be used or particles
generated therefrom such as ISCOMs (immunostimulating complexes),
which ISCOMs may be devoid of additional detergent, see, e.g.,
International Publication No. WO 00/07621; (4) Complete Freund's
Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5)
cytokines, such as interleukins (IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12 (International Publication No. WO 99/44636), etc.),
interferons (e.g., 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. 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-S109
(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) MPL or 3-O-deacylated MPL (3dMPL) (see, e.g.,
GB 2220221), EP-A-0689454, optionally in the substantial absence of
alum when used with pneumococcal saccharides (see, e.g.,
International Publication No. WO 00/56358); (8) combinations of
3dMPL with, for example, QS21 and/or oil-in-water emulsions (see,
e.g., EP-A-0835318, EP-A-0735898, EP-A-0761231; (9)
oligonucleotides comprising CpG motifs (see, e.g., Roman et al.
(1997) Nat. Med. 3:849-854; Weiner et al. (1997) Proc. Natl. Acad.
Sci. USA 94:10833-10837; Davis et al. (1998) J. Immunol.
160:870-876; Chu et al. (1997) J. Exp. Med. 186:1623-1631; Lipford
et al. (1997) Eur. J. Immunol. 27:2340-2344; Moldoveanu et al.
(1988) Vaccine 16:1216-1224; Krieg et al. (1995) Nature
374:546-549; Klinman et al. (1996) Proc. Natl. Acad. Sci. USA
93:2879-2883; Ballas et al. (1996) J. Immunol. 157:1840-1845;
Cowdery et al. (1996) J. Immunol. 156:4570-4575; Halpern et al.
(1996) Cell Immunol. 167:72-78; Yamamoto et al. (1988) Jpn. J.
Cancer Res. 79:866-873; Stacey et al. (1996) J. Immunol.
157:2116-2122; Messina et al. (1991) J. Immunol. 147:1759-1764; Yi
et al. (1996) J. Immunol. 157:4918-4925; Yi et al. (1996) J.
Immunol. 157:5394-5402; Yi et al. (1998) J. Immunol. 160:4755-4761;
Yi et al. (1998) J. Immunol. 160:5898-5906; International
Publication Nos. WO 96/02555, WO 98/16247, WO 98/18810, WO
98/40100, WO 98/55495, WO 98/37919 and WO 98/52581), such as those
containing at least on CG dinucleotide, with cytosine optionally
replaced with 5-methylcytosine; (10) a polyoxyethylene ether or a
polyoxyethylene ester (see, e.g., International Publication No. WO
99/52549); (11) a polyoxyethylene sorbitan ester surfactant in
combination with an octoxynol (see, e.g., International Publication
No. WO 01/21207) or a polyoxyethylene alkyl ether or ester
surfactant in combination with at least one additional non-ionic
surfactant such as an octoxynol (see, e.g., International
Publication No. WO 01/21152); (12) a saponin and an
immunostimulatory oligonucleotide such as a CpG oligonucleotide
(see, e.g., International Publication No. WO 00/62800); (13) an
immunostimulant and a particle of metal salt (see, e.g.,
International Publication No. WO 00/23105); and (14) other
substances that act as immunostimulating agents to enhance the
effectiveness of the composition.
[0138] Muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),
-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-
-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0139] Moreover, the HCV proteins can be adsorbed to, or entrapped
within, an ISCOM, as described above. Additionally, ISCOMs with
adsorbed HCV core proteins, either the entire core region or a
fragment of HCV core protein, may be added to the formulations.
Most preferably, the HCV core protein is a fragment comprising a
polypeptide from the region spanning amino acid positions 121-135.
See, e.g., International Publication No. WO 01/37869A, incorporated
herein by reference in its entirety.
[0140] As explained above, the composition may also contain
immunostimulatory molecules, either in addition to or in place of
the antigen delivery system. Immunostimulatory agents for use
herein include, without limitation, monophosphorylipid A (MPL),
trehalose dimycolate (TDM), and cell wall skeleton (CWS),
preferably MPL+CWS (Detox.TM.). MPL may be formulated into an
emulsion to enhance its immunostimulatory affect. See, e.g., Ulrich
et al., "MPLr immunostimulat: adjuvant formulations." in Vaccine
Adjuvants: Prepartion Methods and Research Protocols (O'Hagan D T,
ed.) Human Press Inc., NJ (2000) pp. 273-282. MPL has been shown to
induce the synthesis and release of cytokines, particularly IL-2
and IFN-.gamma.. Other useful immunostimulatory molecules include
LPS and immunostimulatory nucleic acid sequences (ISS), including
but not limited to, unmethylated CpG motifs, such as CpG
oligonucleotides.
[0141] Oligonucleotides containing unmethylated CpG motifs have
been shown to induce activation of B cells, NK cells and
antigen-presenting cells (APCs), such as monocytes and macrophages.
See, e.g., U.S. Pat. No. 6,207,646. Thus, adjuvants derived from
the CpG family of molecules, CpG dinucleotides and synthetic
oligonucleotides which comprise CpG motifs (see, e.g., Krieg et al.
Nature (1995) 374:546 and Davis et al. J. Immunol. (1998)
160:870-876) such as any of the various immunostimulatory CpG
oligonucleotides disclosed in U.S. Pat. No. 6,207,646, may be used
in the subject methods and compositions. Such CpG oligonucleotides
generally comprise at least 8 up to about 100 basepairs, preferably
8 to 40 basepairs, more preferably 15-35 basepairs, preferably
15-25 basepairs, and any number of basepairs between these values.
For example, oligonucleotides comprising the consensus CpG motif,
represented by the formula 5'-X.sub.1CGX.sub.2-3', where X.sub.1
and X.sub.2 are nucleotides and C is unmethylated, will find use as
immunostimulatory CpG molecules. Generally, X.sub.1 is A, G or T,
and X.sub.2 is C or T. Other useful CpG molecules include those
captured by the formula 5'-X.sub.1X.sub.2CGX.sub.3X.sub.4, where
X.sub.1 and X.sub.2 are a sequence such as GpT, GpG, GpA, ApA, ApT,
ApG, CpT, CpA, CpG, TpA, TpT or TpG, and X.sub.3 and X.sub.4 are
TpT, CpT, ApT, ApG, CpG, TpC, ApC, CpC, TpA, ApA, GpT, CpA, or TpG,
wherein "p" signifies a phosphate bond. Preferably, the
oligonucleotides do not include a GCG sequence at or near the 5'-
and/or 3' terminus. Additionally, the CpG is preferably flanked on
its 5'-end with two purines (preferably a GpA dinucleotide) or with
a purine and a pyrimidine (preferably, GpT), and flanked on its
3'-end with two pyrimidines, preferably a TpT or TpC dinucleotide.
Thus, preferred molecules will comprise the sequence GACGTT,
GACGTC, GTCGTT or GTCGCT, and these sequences will be flanked by
several additional nucleotides. The nucleotides outside of this
central core area appear to be extremely amendable to change.
[0142] Moreover, the CpG oligonucleotides for use herein may be
double- or single-stranded. Double-stranded molecules are more
stable in vivo while single-stranded molecules display enhanced
immune activity. Additionally, the phosphate backbone may be
modified, such as phosphorodithioate-modified, in order to enhance
the immunostimulatory activity of the CpG molecule. As described in
U.S. Pat. No. 6,207,646, CpG molecules with phosphorothioate
backbones preferentially activate B-cells, while those having
phosphodiester backbones preferentially activate monocytic
(macrophages, dendritic cells and monocytes) and NK cells.
[0143] One exemplary CpG oligonucleotide for use in the present
compositions has the sequence 5'-TCCATGACGTTCCTGACGTT-3' (SEQ ID
NO:6).
[0144] CpG molecules can readily be tested for their ability to
stimulate an immune response using standard techniques, well known
in the art. For example, the ability of the molecule to stimulate a
humoral and/or cellular immune response is readily determined using
the immunoassays described above. Moreover, the antigen and
adjuvant compositions can be administered with and without the CpG
molecule to determine whether an immune response is enhanced.
[0145] The HCV proteins may also be encapsulated, adsorbed to, or
associated with, particulate carriers, as described above with
reference to the HCV polynucleotides. As explained above, examples
of particulate carriers 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., Pharm. Res. (1993) 10:362-368; and McGee et
al., J. Microencap. (1996). One preferred method for adsorbing
macromolecules onto prepared microparticles is described above and
in International Publication No. WO 00/050006, incorporated herein
by reference in its entirety.
Methods of Producing HCV-Specific Antibodies
[0146] The HCV proteins can be used to produce HCV-specific
polyclonal and monoclonal antibodies. 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.
[0147] Monoclonal antibodies directed against HCV-specific epitopes
present in the proteins can also be readily produced. Normal B
cells from a mammal, such as a mouse, immunized with an HCV
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.
[0148] 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 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.
[0149] 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 linkage 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.
HCV-Specific T Cells
[0150] HCV-specific T cells that are activated by the
above-described fusions and E1E2 complexes, including the
NS3NS4NS5a fusion protein or NS3NS4NS5aNS5b fusion protein, and the
E1E2 complexes, expressed in vivo or in vitro, preferably recognize
an epitope of an HCV polypeptide such as an E1, E2, NS3, NS4, NS5a,
NS5b polypeptide, including an epitope of an NS3NS4NS5a fusion
protein or an NS3NS4NS5aNS5b fusion protein, or an E1E2 complex.
HCV-specific T cells can be CD8.sup.+ or CD4.sup.+.
[0151] HCV-specific CD8.sup.+ T cells preferably are cytotoxic T
lymphocytes (CTL) which can kill HCV-infected cells that display
E1, E2, NS3, NS4, NS5a, NS5b epitopes complexed with an MHC class I
molecule. HCV-specific CD8.sup.+ T cells may also express
interferon-.gamma. (IFN-.gamma.). HCV-specific CD8.sup.+ T cells
can be detected by, for example, .sup.51Cr release assays (see the
examples). .sup.51Cr release assays measure the ability of
HCV-specific CD8.sup.+ T cells to lyse target cells displaying an
E1, E2, E1E2, NS3, NS4, NS5a, NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b
epitope. HCV-specific CD8.sup.+ T cells which express IFN-.gamma.
can also be detected by immunological methods, preferably by
intracellular staining for IFN-.gamma. after in vitro stimulation
with an E1, E2, NS3, an NS4, an NS5a, or an NS5b polypeptide (see
the examples).
[0152] HCV-specific CD4.sup.+ cells activated by the
above-described E1E2 complexes and fusions, such as an E1
polypeptide, an E2 polypeptide, an E1E2 complex, NS3NS4NS5a or
NS3NS4NS5aNS5b fusion protein, expressed in vivo or in vitro,
preferably recognize an epitope of an E1, E2, NS3, NS4, NS5a, or
NS5b polypeptide, including an epitope of an E1E2 complex,
NS3NS4NS5a or NS3NS4NS5aNS5b fusion protein, that is bound to an
MHC class II molecule on an HCV-infected cell and proliferate in
response to stimulating E1E2 complexes with NS3NS4NS5a or
NS3NS4NS5aNS5b peptides, with or without a core polypeptide.
[0153] HCV-specific CD4.sup.+ T cells can be detected by a
lymphoproliferation assay (see examples). Lymphoproliferation
assays measure the ability of HCV-specific CD4.sup.+ T cells to
proliferate in response to an E1, E2, NS3, an NS4, an NS5a, or an
NS5b epitope.
Methods of Activating HCV-Specific T Cells.
[0154] The HCV proteins or polynucleotides can be used to stimulate
an immune response, such as to activate HCV-specific T cells either
in vitro or in vivo. Activation of HCV-specific T cells can be
used, inter alia, to provide model systems to optimize CTL
responses to HCV and to provide prophylactic or therapeutic
treatment against HCV infection. For in vitro activation, proteins
are preferably supplied to T cells via a plasmid or a viral vector,
such as an adenovirus vector, as described above.
[0155] 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 activated
HCV-specific T cells in the mammal. The HCV-specific T cells
derived from the mammal can then be restimulated in vitro by
adding, e.g., HCV E1E2 and NS3NS4NS5a or NS3NS4NS5aNS5b epitopic
peptides, with or without a core polypeptide, to the T cells. The
HCV-specific T cells can then be tested for, inter alia,
proliferation, the production of IFN-.gamma., and the ability to
lyse target cells displaying E1E2, NS3NS4NS5a or NS3NS4NS5aNS5b
epitopes in vitro.
[0156] In a lymphoproliferation assay (see examples), HCV-activated
CD4.sup.+ T cells proliferate when cultured with an NS3, NS4, NS5a,
NS5b, NS3NS4NS5a, or NS3NS4NS5aNS5b epitopic peptide, but not in
the absence of an epitopic peptide. Thus, particular E1, E2, NS3,
NS4, NS5a, NS5b, NS3NS4NS5a and NS3NS4NS5aNS5b epitopes that are
recognized by HCV-specific CD4.sup.+ T cells can be identified
using a lymphoproliferation assay.
[0157] Similarly, detection of IFN-.gamma. in HCV-specific
CD8.sup.+ T cells after in vitro stimulation with the
above-described HCV proteins, can be used to identify E1, E2, E1E2,
NS3, NS4, NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b epitopes that
particularly effective at stimulating CD8.sup.+ T cells to produce
IFN-.gamma. (see examples).
[0158] Further, .sup.51Cr release assays are useful for determining
the level of CTL response to HCV. See Cooper et al. Immunity
10:439-449. For example, HCV-specific CD8.sup.+ T cells can be
derived from the liver of an HCV infected mammal. These T cells can
be tested in .sup.51Cr release assays against target cells
displaying, e.g., E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes.
Several target cell populations expressing different E1E2,
NS3NS4NS5a and/or NS3NS4NS5aNS5b epitopes can be constructed so
that each target cell population displays different epitopes of
E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b. The HCV-specific CD8.sup.+
cells can be assayed against each of these target cell populations.
The results of the .sup.51Cr release assays can be used to
determine which epitopes of E1E2, NS3NS4NS5a and/or NS3NS4NS5aNS5b
are responsible for the strongest CTL response to HCV. E1E2
complexes, NS3NS4NS5a fusion proteins or NS3NS4NS5aNS5b 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 .sup.51Cr
release assays.
[0159] HCV proteins as described above, or polynucleotides encoding
such proteins, can be administered to a mammal, such as a mouse,
baboon, chimpanzee, or human, to stimulate an immune response, such
as to activate HCV-specific T cells in 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.
[0160] 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 mg/kg.
[0161] A composition of the invention comprising the HCV proteins
or polynucleotides is administered in a manner compatible with the
particular composition used and in an amount which is effective to
stimulate an immune response, such as to activate HCV-specific T
cells as measured by, inter alia, a .sup.51Cr release assay, a
lymphoproliferation assay, or by intracellular staining for
IFN-.gamma.. 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 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
optimize the activation of HCV-specific T cells either in vivo or
in vitro. Generally, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg of
an HCV fusion and E1 and E2 polypeptides, such as an E1E2 complex,
an NS3NS4NS5a or NS3NS4NS5aNS5b 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.
[0162] 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 reenforce an
immune response, for example, at 1-4 months for a second dose, and
if needed, a subsequent dose or doses after several months.
Deposits of Strains Useful in Practicing the Invention
[0163] A deposit of biologically pure cultures of the following
strains was made with the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. The accession number indicated
was assigned after successful viability testing, and the requisite
fees were paid. made under the provisions of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for
the Purpose of Patent Procedure and the Regulations thereunder
(Budapest Treaty). This assures maintenance of viable cultures for
a period of thirty (30) years from the date of deposit. The
organisms will be made available by the ATCC under the terms of the
Budapest Treaty, which assures permanent and unrestricted
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 U.S.C. .sctn.122 and the Commissioner's rules
pursuant thereto (including 37 C.F.R. .sctn.1.12 with particular
reference to 886 OG 638). Upon the granting of a patent, all
restrictions on the availability to the public of the deposited
cultures will be irrevocably removed.
[0164] These deposits are provided merely as convenience to those
of skill in the art, and are not an admission that a deposit is
required under 35 U.S.C. .sctn.112. The nucleic acid sequences of
these genes, as well as the amino acid sequences of the molecules
encoded thereby, are incorporated herein by reference and are
controlling in the event of any conflict with the description
herein. A license may be required to make, use, or sell the
deposited materials, and no such license is hereby granted.
TABLE-US-00002 Plasmid Deposit Date ATCC No. E1E2-809 Aug. 16, 2001
PTA-3643
III. EXPERIMENTAL
[0165] 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 skill in the art will
readily appreciate that the invention may be practiced in a variety
of ways given the teaching of this disclosure.
[0166] 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 NS3NS4NS5a Polynucleotides
[0167] A polynucleotide encoding NS3NS4NS5a (approximately amino
acids 1027 to 2399, numbered relative to HCV-1) (also termed
"NS345a" herein) or NS5a (approximately amino acids 1973 to 2399,
numbered relative to HCV-1) was isolated from an HCV.
Polynucleotides encoding a methionine residue were ligated to the
5' end of these polynucleotides and the polynucleotides were cloned
into plasmid, vaccinia virus, and adenovirus vectors.
[0168] Immunization Protocols. In one immunization protocol, mice
were immunized with 50 .mu.g of plasmid DNA encoding either NS5a or
encoding an NS3NS4NS5a fusion protein by intramuscular injection
into the tibialis anterior. A booster injection of 10.sup.7 pfu of
vaccinia virus (VV)-NS5a (intraperitoneal) or 50 .mu.g of plasmid
control (intramuscular) was provided 6 weeks later.
[0169] In another immunization protocol, mice were injected
intramuscularly in the tibialis anterior with 10.sup.10 adenovirus
particles encoding an NS3NS4NS5a fusion protein. An intraperitoneal
booster injection of 10.sup.7 pfu of VV-NS5a or an intramuscular
booster injection of 10.sup.10 adenovirus particles encoding
NS3NS4NS5a was provided 6 weeks later.
Example 2
Immunization with DNA Encoding an NS3NS4NS5a Fusion Protein
Activates HCV-Specific CD8.sup.+ T Cells
[0170] .sup.51Cr Release Assay. A .sup.51Cr release assay was used
to measure the ability of HCV-specific T cells to lyse target cells
displaying an NS5a epitope. Spleen cells were pooled from the
immunized animals. These cells were restimulated in vitro for 6
days with the CTL epitopic peptide p214K9 (2152-HEYPVGSQL-2160; SEQ
ID NO:1) from HCV-NS5a in the presence of IL-2. The spleen cells
were then assayed for cytotoxic activity in a standard .sup.51Cr
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 were tested.
Percent specific lysis was calculated for each effector to target
ratio.
[0171] The results of the assays are shown in Tables 1 and 2. Table
1 demonstrates that immunization with plasmid DNA encoding an
NS3NS4NS5a fusion protein activates CD8.sup.+ T cells which
recognize and lyse target cells displaying an NS5a epitope.
Surprisingly the NS5a polypeptide of the NS3NS4NS5a fusion protein
was able to activate T cells even though the NS5a polypeptide was
present in a fusion protein.
[0172] Similarly, Table 2 demonstrates that delivery of the
NS3NS4NS5a fusion protein to mice by means of an adenovirus vector
also activates CD8.sup.+ T cells which recognize and lyse target
cells displaying an HCV NS5a epitope. Thus, immunization with
either "naked" (plasmid) DNA encoding an NS3NS4NS5a fusion protein
or adenovirus vector-encoded fusion protein can be used to activate
HCV-specific T cells.
Example 3
Immunization with DNA Encoding an NS3NS4NS5a Fusion Protein
Activates HCV-Specific CD8.sup.+ T-Cells which Express
IFN-.gamma.
[0173] Intracellular Staining for Interferon-gamma (IFN-.gamma.).
Intracellular staining for IFN-.gamma. was used to identify the
CD8.sup.+ T cells that secrete IFN-.gamma. after in vitro
stimulation with the NS5a epitope p214K9. Spleen cells of
individual immunized mice were restimulated in vitro either with
p214K9 or with a non-specific peptide for 6-12 hours in the
presence of IL-2 and monensin. The cells were then stained for
surface CD8 and for intracellular IFN-.gamma. and analyzed by flow
cytometry. The percent of CD8.sup.+ T cells which were also
positive for IFN-.gamma. was then calculated. The results of these
assays are shown in Tables 1 and 2. Table 1 demonstrates that
CD8.sup.+ T cells activated in response to immunization with
plasmid DNA encoding an NS3NS4NS5a fusion protein also express
IFN-.gamma.. Immunization with an NS3NS4NS5a fusion protein encoded
in an adenovirus also results in CD8.sup.+ HCV-specific T cells
which express IFN-.gamma., although to a lesser extent than
immunization with a plasmid-encoded NS3NS4NS5a fusion protein
(Table 2).
TABLE-US-00003 TABLE 1 HCV-NS5a-Specific CD8+ T Cells in Mice
Immunized with NS5a or NS345a DNA Intracellular .sup.51Cr Release
Assay Staining for IFN-.gamma. Percent Specific Percent of CD8+ T
Lysis of Targets* Cells Positive for IFN-g** E:T NS5a DNA NS345a
DNA NS5a DNA NS345a DNA ratio p214K9 -- p214K9 -- p214K9 -- p214K9
-- 60:1 77 5 66 6 20:1 61 4 49 2 1.74 0.26 1.18 0.40 7:1 29 1 29 4
*Target cells (L929) were pulsed with p214K9 or media alone and
labeled with .sup.51Cr. **Spleen cells were cultured with p214K9 or
media alone for 12 hours in the presence of monensin. p214K9 is a
CTL epitopic peptide (2152-HEYPVGSQL-2160, SEQ ID NO: 1) from
HCV-NS5a `--` refers to the absence of peptide
TABLE-US-00004 TABLE 2 HCV-NS5a-Specific CD8+ T Cells Primed by
Adenovirus or DNA Encoding for NS345a Intracellular .sup.51Cr
Release Assay Staining for IFN-.gamma. Percent Specific Percent of
CD8+ T Lysis of Targets* Cells Positive for IFN-g** NS345a NS345a
NS345a NS345a E:T Adeno DNA Adeno DNA ratio p214K9 -- p214K9 --
p214K9 p214J p214K9 p214J 60:1 76 2 55 5 20:1 85 2 22 3 3.24 0.13
0.25 0.09 7:1 62 <1 10 3 *Target cells (L929) were pulsed with
p214K9 or p214J and labeled with .sup.51Cr. **Spleen cells were
cultured with p214K9 or p214J for 12 hours in the presence of
monensin. p214K9 is a CTL epitopic peptide (2152-HEYPVGSQL-2160,
SEQ ID NO: 1) from HCV-NS5a P214J is a control peptide (10 mer)
from HCV-NS5a
Example 4
Immunization with DNA Encoding an NS3NS4NS5a Fusion Protein
Stimulates Proliferation of HCV-Specific CD4.sup.+ T Cells
[0174] Lymphoproliferation assay. Spleen cells from pooled
immunized mice were depleted of CD8.sup.+ T cells using magnetic
beads and were cultured in triplicate with either p222D, an
NS5a-epitopic peptide from HCV-NS5a (2224-AELIEANLLWRQEMG-2238; SEQ
ID NO:2), or in medium alone. After 72 hours, cells were pulsed
with 1.mu. Ci per well of .sup.3H-thymidine and harvested 6-8 hours
later. Incorporation of radioactivity was measured after
harvesting. The mean cpm was calculated.
[0175] As shown in Table 3, immunization with a plasmid-encoded
NS3NS4NS5a fusion protein stimulates proliferation of CD4.sup.+
HCV-specific T cells. Immunization with an adenovirus vector
encoding the fusion protein also resulted in stimulated
proliferation of CD4.sup.+ HCV-specific T cells (Table 4).
TABLE-US-00005 TABLE 3 HCV-NS5a-Specific CD4+ T Cells in Mice
Immunized with NS5a or NS345a DNA Mean CPM NS5a DNA NS345a DNA
p222D media p222D media 4523 740 4562 861 (x6.1) (x5.3) p222D is a
CD4+ epitopic peptide (aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO: 2)
from HCV-NS5a
TABLE-US-00006 TABLE 4 HCV-NS5-Specific CD4+ T Cells Primed by
Adenovirus or DNA Encoding for NS345a Mean CPM NS345a Adeno NS345a
DNA p222D media p222D media 896 357 1510 385 (x2.5) (x3.9) p222D is
a CD4+ epitopic peptide (aa: 2224-AELIEANLLWRQEMG-2238, SEQ ID NO:
2) from HCV-NS5a
Example 5
Efficiency of NS345a-Encoding DNA Vaccine Formulations to Prime
CTLs in Mice
[0176] Mice were immunized with either 10-100 .mu.g of plasmid DNA
encoding NS345a fusion protein as described in Example 1, with
PLG-linked DNA encoding NS345a, described below, or with DNA
encoding NS345a, delivered via electroporation (see, e.g., U.S.
Pat. Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent
Publication No. 2002/0146831; and International Publication No.
WO/0045823, all of which are incorporated herein by reference in
their entireties, for this delivery technique). The immunizations
were followed by a booster injection 6 weeks later of
1.times.10.sup.7 pfu vaccinia virus encoding NS5a, plasmid DNA
encoding NS345a or plasmid DNA encoding NS5a each as described in
Example 1.
[0177] PLG-delivered DNA. The polylactide-co-glycolide (PLG)
polymers were obtained from Boehringer Ingelheim, U.S.A. The PLG
polymer used in this study was RG505, which has a copolymer ratio
of 50/50 and a molecular weight of 65 kDa (manufacturers data).
Cationic microparticles with adsorbed DNA were 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 were 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 IKA homogenizer. The primary emulsion was then
added to 50 ml of distilled water containing cetyl trimethyl
ammonium bromide (CTAB) (0.5% w/v). This resulted in the formation
of a w/o/w emulsion which was stirred at 6000 rpm for 12 hours at
room temperature, allowing the methylene chloride to evaporate. The
resulting microparticles were washed twice in distilled water by
centrifugation at 10,000 g and freeze dried. Following preparation,
washing and collection, DNA was adsorbed onto the microparticles by
incubating 100 mg of cationic microparticles in a 1 mg/ml solution
of DNA at 4 C for 6 hours. The microparticles were then separated
by centrifugation, the pellet washed with TE buffer and the
microparticles were freeze dried.
[0178] CTL activity and IFN-.gamma. expression were measured by
.sup.51Cr release assay or intracellular staining as described in
examples 2 and 3 respectively. The results are shown in Table
5.
[0179] Results demonstrate that immunization using plasmid DNA
encoding for NS345a to prime mice results in activation of
CD8.sup.+ HCV specific T cells.
TABLE-US-00007 TABLE 5 Efficiency of NS345a-Encoding DNA Vaccine
Formulations to Prime CTLs in Mice ICS for IFN-gamma (% CD8+ cells
that are fold IFN-g+) increase NS345a # of vs. DNA mice % # of
`naked` CTL Vaccines Boost Mean Sdtdev P tested responding expts
DNA activity? NS345a VVNS5a 1.02 1.70 41 68% 10 N/A YES DNA NS345a
NS345a 0.02 0.04 22 5% 5 N/A YES DNA DNA NS345a NS5a 0.22 0.21 24
63% 5 N/A YES DNA DNA NS345a VVNS5a 5.00 4.36 7 100% 2 4.90 YES DNA
eV (electro- poration) PLGNS345a VVNS5a 2.65 2.54 6 100% 2 2.60 YES
DNA PLGNS345a NS5a 0.33 0.24 15 80% 3 1.50 YES DNA DNA
Example 6
Immunization Routes and Replicon Particles SINCR (DC+) Encoding for
NS345a
[0180] Alphavirus replicon particles, for example, SINCR (DC+) were
prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA
(1999) 96:4598-4603. Mice were injected with 5.times.10.sup.6 IU
SINCR (DC+) replicon particles encoding for NS345a intramuscularly
(IM) as described in Example 1, 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 were followed by a booster injection of vaccinia
virus encoding NS5a as described in Example 1.
[0181] IPN-.gamma. expression was measured by intracellular
staining as described in Example 3. The results are shown in Table
6. The results demonstrate that immunization via SINCR (DC+)
replicon particles encoding for NS345a by a variety of routes
results in CD8+ HCV specific T cells which express IFN-.gamma..
TABLE-US-00008 TABLE 6 Immunization Routes and SINCR (DC+) Replicon
Particles Encoding NS345a (all mice VVNS5a challenged) ICS for
IFN-gamma (% CD8+ cells that are IFN-g+) # of mice # of %
responding Vaccines Immunization Route Mean Sdtdev P tested expts
mice SINCR (DC+) 100% IM (ta) 1.11 0.63 3 1 100% 5 .times. 10.sup.6
SINCR (DC+) 100% S/C (BoT + 0.62 0.29 3 1 100% 5 .times. 10.sup.6
FP) SINCR (DC+) 2/3 IM (ta) + 1/3 2.43 2.00 3 1 100% 5 .times.
10.sup.6 S/C (BoT)
Example 7
SINCR (DC+) Vs SINDC (LP) Replicon Particles Encoding for
NS345a
[0182] Alphavirus replicon particles, for example, SINCR (DC+) and
SINCR (LP) were prepared as described in Polo et al., Proc. Natl.
Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with
1.times.10.sup.3 to 1.times.10.sup.7 IU of SINCR (DC+) or SINCR
(LP) replicon particles encoding for NS345a, by intramuscular
injection into the tibialis anterior, followed by a booster
injection of 10.sup.7 pfu vaccinia virus encoding NS5a at 6
weeks.
[0183] IFN-.gamma. expression was measured by intracellular
staining as described in Example 3. Administration of an increase
in the number of SINCR (DC+) replicon particles encoding NS345a
resulted in an increase in % of CD8+ T cells expressing
IFN-.gamma..
Example 8
Alphavirus Replicon Priming, Followed by Various Boosting
Regimes
[0184] Alphavirus replicon particles, for example, SINCR (DC+) were
prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA
(1999) 96:4598-4603. Mice were primed with SINCR (DC+),
1.5.times.10.sup.6 IU replicon particles encoding NS345a, by
intramuscular injection into the tibialis anterior, followed by a
booster of either 10-100 .mu.g of plasmid DNA encoding for NS5a,
10.sup.10 adenovirus particles encoding NS345a, 1.5.times.10.sup.6
IU SINCR (DC+) replicon particles encoding NS345a, or 10.sup.7 pfu
vaccinia virus encoding NS5a at 6 weeks.
[0185] IFN-.gamma. expression was measured by intracellular
staining as described in Example 3. The results are shown in Table
7. The results demonstrate that boosting with vaccinia virus
encoding NS5a DNA results in the strongest generation of CD8+ HCV
specific T cells which express IFN-.gamma.. Boosting with plasmid
encoding NS5a DNA also results in a good response, while lesser
responses are noted with adenovirus NS345a or SINCR DC+boosted
animals.
TABLE-US-00009 TABLE 7 Alphavirus Replicon Particle Priming,
Followed by Various Boosting Regimens ICS for IFN-gamma (% CD8+
cells that are IFN-g+) # of mice # of % responding Vaccines Boost
Mean Sdtdev P tested expts mice SINCR (DC+) NS5a DNA 0.46 0.36 4 1
75% 1.5 .times. 10.sup.6 SINCR (DC+) Adeno NS345a 0.04 0.04 4 1 25%
1.5 .times. 10.sup.6 (10 .times. 10.sup.10) SINCR (DC+) SINCR (DC+)
0.06 0.06 8 2 25% 1.5 .times. 10.sup.6 1.5 .times. 10.sup.6 SINCR
(DC+) VVNS5a (1 .times. 10.sup.7) 2.43 2.45 4 1 100% 1.5 .times.
10.sup.6
Example 9
Alphaviruses Expressing NS345a
[0186] Alphavirus replicon particles, for example, SINCR (DC+) and
SINCR (LP) were prepared as described in Polo et al., Proc. Natl.
Acad. Sci. USA (1999) 96:4598-4603. Mice were immunized with
1.times.10.sup.2 to 1.times.10.sup.6 IU SINCR (DC+) replicons
encoding NS345a via a combination of delivery routes (2/3 IM and
1/3 S/C) as well as by S/C alone, or with 1.times.10.sup.2 to
1.times.10.sup.6 IU SINCR (LP) replicon particles encoding NS345a
via a combination of delivery routes (2/3 IM and 1/3 S/C) as well
as by S/C alone. The immunizations were followed by a booster
injection of 10.sup.7 pfu vaccinia virus encoding NS5a at 6
weeks.
[0187] IFN-.gamma. expression was measured by intracellular
staining as described in Example 3. The results are shown in FIG.
6. The results indicate activation of CD8+ HCV specific T
cells.
Example 10
Efficiency of NS5a Encoding DNA Vaccine Formulations to Prime CTLs
in Mice
[0188] Mice were immunized with either 10-100 .mu.g of plasmid DNA
encoding NS5a as described in Example 1 or with PLG-linked DNA
encoding NS5a as described in Example 5. The immunizations were
followed by a booster injection at 6 weeks of either 10-100 .mu.g
of plasmid DNA encoding for NS5a, 10.sup.10 adenovirus particles
encoding NS345a, 1.5.times.10.sup.6 IU SINCR (DC+) replicon
particles encoding NS345a, or 10.sup.7 pfu vaccinia virus encoding
NS5a.
[0189] CTL activity and IFN-.gamma. expression were measured by the
methods described in Examples 2 and 3.
[0190] The results are shown in Table 8. The results demonstrate
that priming with plasmid DNA encoding for NS5a or PLG-linked DNA
encoding NS5a results in activation of CD8+ HCV specific T
cells.
TABLE-US-00010 TABLE 8 Efficiency of NS5a-Encoding DNA Vaccine
Formulations to Prime CTLs in Mice ICS for IFN-gamma (% CD8+ cells
that are fold IFN-g+) increase # of vs. NS5a mice % # of `naked`
CTL Vaccines Boost Mean Sdtdev P tested responding expts DNA
activity? NS5a VVNS5a 1.67 1.49 8 100% 3 N/A YES DNA NS5a NS5a 0.17
0.09 12 83% 3 N/A YES DNA DNA PLGNS5a NS5a 0.22 0.09 9 100% 2 1.29
YES DNA DNA NS5a AdenoNS345a 0.10 0.08 4 50% 1 N/A NO DNA NS5a
SINCRNS345a 0.20 0.17 4 75% 1 N/A YES DNA
Example 11
Efficiency of NS345b-Encoding DNA Vaccine Formulations to Prime
CTLs in Mice
[0191] Mice were immunized with 10-100 .mu.g of plasmid DNA
encoding NS34b by intramuscular injection to the tibialis anterior
or with PLG linked DNA encoding NS5a as described in Example 5. The
immunizations were followed by a booster injection of plasmid DNA
encoding for NS5a as described in Example 1.
[0192] CTL activity and IFN-.gamma. expression were measured by the
methods described in Examples 2 and 3.
[0193] The results are shown in Table 9. The results demonstrate
that priming with plasmid DNA encoding NS345b or PLG-linked NS345b
results in activation of CD8+ HCV specific T cells.
TABLE-US-00011 TABLE 9 Efficiency of NS345b-Encoding DNA Vaccine
Formulations to Prime CTLs in Mice ICS for IFN-gamma (% CD8+ cells
that are fold IFN-g+) increase NS345 # of vs. DNA mice % # of
`naked` CTL Vaccines Boost Mean Sdtdev P tested responding expts
DNA activity? NS345 NS5a 0.18 0.16 15 60% 3 N/A YES DNA DNA
PLGNS345 NS5a 0.30 0.33 14 57% 3 1.67 YES DNA DNA
Example 12
Administration of DNA Via Separate Plasmids
[0194] Mice were immunized with 100 .mu.g plasmid DNA encoding for
NS345a or with 100 .mu.g PLG-linked DNA encoding NS345a.
Additionally, separate DNA plasmids encoding NS5a, NS34a, and NS4ab
(33.3 .mu.g each) were administered concurrently to another group
of mice. Finally, PLG-linked DNA encoding NS5a, NS34a, and NS4ab
(33.3 .mu.g each) were administered concurrently to another group
of mice. The immunizations were followed by a booster injection of
1.times.10.sup.7 pfu vaccinia virus encoding NS5a, 6 weeks post
first immunization.
[0195] IFN-.gamma. expression was measured by the method described
in Example 3. The results are shown in FIG. 7. The results
demonstrate a particularly vigorous response in the activation of
CD8+ HCV specific T cells when the DNA is broken down into smaller
sub units and linked to PLG.
Example 13
Immunogenicity of NS345Core.sub.121-ISCOMS in Mice
[0196] Groups of 10 C57 black mice were immunized IM at 0, 21 and
60 days with the formulations shown in Table 10. The
NS345Core.sub.121-PLGdss group received a vaccine dose of 50 .mu.l
in each leg whereas the other vaccine groups received a vaccine
dose of 50 .mu.l in one leg.
[0197] NS345Core.sub.121-ISCOMS were comprised of amino acids 1242
to 3011 and 1-121 and the HCV polyprotein, numbered relative to
HCV-1 and were adsorbed to ISCOMS with a ratio of protein to QH of
approximately 8:1, using standard techniques. See, e.g.,
International Publication No. WO 01/37869A, incorporated herein by
reference in its entirety.
[0198] Core-ISCOMS including an HCV core protein fragment from the
region spanning amino acid positions 1-191 of the HCV polyprotein,
numbered relative to HCV-1, with a ratio of protein to QH of 1:1,
were produced using standard techniques. See, e.g., International
Publication No. WO 01/37869A, incorporated herein by reference in
its entirety.
[0199] NS345Core.sub.121 was formulated in 0.1% SDS in PBS and
contained DTT. Protein was diluted in PBS and mixed 1:1 with MF59
(see, Ott et al., "MF59--Design and Evaluation of a Safe and Potent
Adjuvant for Human Vaccines" in Vaccine Design: The Subunit and
Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum
Press, New York (1995) pp. 277-296; and U.S. Pat. No. 6,299,884,
incorporated herein by reference in its entirety) prior to
immunization.
[0200] For NS345Core.sub.121-PLGdss, PLG microparticles produced as
described above were treated with
3-(trimethylsilyl)-1-propanesulfonic acid (DSS) to enhance
adsorption of antigen. DSS is commercially available from, e.g.,
Sigma Chemical Co., St. Louis, Mo. NS345Core.sub.121 was adsorbed
thereto using standard techniques (see, International Publication
No. WO 00/050006). The NS345Core.sub.121-PLGdss was mixed with MF59
prior to immunization.
[0201] As shown in Table 10, NS345Core.sub.121-ISCOMS produced
antibody response only to NS5 in immunized C57 black mice. Higher
levels of antibodies to NS5 were produced in mice immunized with
NS345Core.sub.121 adjuvanted with MF59, however no antibody
response to core, NS3 or NS4 was produced with this adjuvant
either.
[0202] Mice immunized with Core-ISCOMS produced antibodies to core.
In contrast, NS345Core.sub.121-PLGdss immunized mice produced
significantly higher antibodies to NS5 than
NS345Core.sub.121-ISCOMS. In addition, NS345Core.sub.121-PLGdss
immunized mice produced antibodies to NS3 and some antibody
response to core, but no antibodies to NS4.
TABLE-US-00012 TABLE 10 Immunogenicity of NS345Core.sub.121-Iscoms
in Mice. Geometric mean EIA antibody titers to core and
nonstructural proteins are shown. The number of responding mice per
group are also listed. Anti-C33C Anti-C100 Anti-Core (NS3) (NS4)
Anti-NS5 IM Protein Dose Antibody Antibody Antibody Antibody
Vaccine (.mu.g).sup.a EIA GMT EIA GMT EIA GMT EIA GMT
NS345Core.sub.121 6.0, 6.0, 6.0.sup.b <10 <10 <10 31
ISCOMS (0/10) (0/10) (0/10) (7/10) Core- 6.0, 6.0, 6.0.sup.c 188
<10 <10 <10 ISCOMS (9/10) (1/10) (0/10) (2/10)
NS345Core.sub.121 6.0, 6.0, 6.0 <10 <10 <10 279 MF59 (2/9)
(1/9) (0/9) (9/) NS345Core.sub.121 10, 10, 10 5 50 <10 419 PLG-
(6/10) (9/10) (2/10) (9/9) dss/MF59 .sup.aGroups of 10 C57 black
mice were immunized IM at 0, 21 and 60 days. Serum was obtained
after the last immunization. The NS345 Core.sub.121-PLGdss group
received vaccine dose of 50 .mu.l in each leg whereas the other
vaccine groups received vaccine dose of 50 .mu.l in one leg.
.sup.bThe ratio of protein to QH was approximately 8:1. .sup.cThe
ratio of protein to QH was approximately 1:1.
Example 14
Immunogenicity of Different Formulations of NS345Core.sub.121 or
NS345 in Mice
[0203] Groups of 10 C57 black mice were immunized IM at 0, 30 and
60 days with the formulations shown in Tables 11 and 12. For the
studies shown in Table 11, the NS345 and NS345Core.sub.121 protein
concentration was 10 .mu.g per dose, and for those in Table 12, the
concentration was 5 .mu.g per dose.
[0204] For PLG-NS345 (amino acids 1242 to 3011 of the HCV
polyprotein) and PLG-NS345Core.sub.121 (amino acids 1242-3011 and
1-121 of the HCV polyprotein), PLG microparticles were prepared and
NS345 or NS345Core.sub.121 were adsorbed thereto using standard
techniques, as described above.
[0205] For PLG-NS345+PLG-CTAB-E1E2 DNA, PLG microparticles were
prepared and NS345 was adsorbed to the microparticles as described
above. E1E2 DNA was produced as follows. Mammalian expression
plasmid pMH-E1E2-809 (FIG. 4, ATCC Deposit No. PTA-3643) encodes an
E1E2 fusion protein which includes amino acids 192-809 of HCV 1a
(see, Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455).
Chinese Hamster Ovary (CHO) cells were used for expression of the
HCV E1E2 sequence from pMH-E1E2-809. In particular, CHO DG44 cells
were used. These cells, described by Uraub et al., Proc. Natl.
Acad. Sci. USA (1980) 77:4216-4220, were derived from CHO K-1 cells
and were made dihydrofolate reductase (dhfr) deficient by virtue of
a double deletion in the dhfr gene. DG44 cells were transfected
with pMH-E1E2-809. The transfected cells were grown in selective
medium such that only those cells expressing the dhfr gene could
grow (Sambrook et al., supra). Isolated CHO colonies were picked
(.about.800 colonies) into individual wells of a 96-well plate.
From the original 96-well plates, replicates were made to perform
expression experiments. The replicate plates were grown until the
cells made a confluent monolayer. The cells were fixed to the wells
of the plate and permeablized using cold methanol. Anti-E1 and
anti-E2 antibodies, 3D5C3 and 3E5-1 respectively, were used to
probe the fixed cells. After adding an anti-mouse HRP conjugate,
followed by substrate, the cell lines with the highest expression
were determined. The highest expressing cell lines were then
expanded to 24-well cluster plates. The assay for expression was
repeated, and again, the highest expressing cell lines were
expanded to wells of greater volume. This was repeated until the
highest expressing cell lines were expanded from 6-well plates into
tissue culture flasks. At this point there was sufficient quantity
of cells to allow accurate count and harvest of the cells, and
quantitative expression assays were done. An ELISA was performed on
the cell extract, to determine high expressors.
[0206] To produce the PLG-CTAB-E1E2 DNA, PLG microparticles were
treated with CTAB as described above (see, International
Publication No. WO 00/050006).
[0207] For PLG-NS345Core.sub.121+E1E2 DNA PLG-NS345Core.sub.121 and
E1E2 DNA were produced as described above.
[0208] For PLG-NS345 or PLG-NS345Core.sub.121+MF59, PLG-NS345 or
PLG-NS345Core.sub.121, was combined with MF59 as described
above.
[0209] For PLG-NS345 or PLG-NS345Core.sub.121+CTAB-CpG, NS345 or
NS345Core.sub.121 was adsorbed to PLG as described above. The CpG
molecule used was 5'-TCCATGACGTTCCTGACGTT-3' and this was treated
with CTAB, as described above.
[0210] For PLG-NS345 or PLG-NS345Core.sub.121+QS21, the saponin
adjuvant QS21 was combined with the PLG-HCV proteins.
[0211] For PLG-NS345 or PLG-NS345Core.sub.121+CTAB-CpG+MF59, the
various components, as described above, were combined.
[0212] The remaining adjuvants used in the studies and shown in the
tables are self-explanatory.
[0213] The results of these studies are shown in Tables 11 and 12.
As can be seen in Table 11, none of the formulations produced
antibody responses to core, NS3 or NS4 antigens. However,
PLG-NS345+CTAB-CPG in MF59 produced the highest antibody titers to
NS5. PLG-NS345Core.sub.121+QS21, PLG-NS345+CTAB-CPG,
PLG-NS345Core.sub.121+CTAB-CPG, and PLG-NS345+QS21 produced
moderate antibody titers to NS5. The other formulations produced
very low antibody titers to NS5.
[0214] As can be seen in Table 12, NS345Core.sub.121/MF59/MPL and
NS345Core.sub.121/MF59/CpG formulations produced very high antibody
titers to NS345Core.sub.121, NS345Core.sub.121/MF59,
NS345/MF59/CpG, and NS345Core.sub.121/MF59/Cho1/QS21 formulations
produced moderate antibody titers to NS345Core.sub.121. The other
formulations produced very low or no antibody titers to
NS345Core.sub.121.
TABLE-US-00013 TABLE 11 Immunogenicity of different formulations of
HCV NS345Core.sub.121 or NS345 in Mice. Geometric mean EIA antibody
titers to core and nonstructural proteins are shown. Anti-Core
Anti-C33C Anti-C100 Anti-NS5 Antibody EIA (NS3) Antibody (NS4)
Antibody Antibody EIA Vaccine.sup.a GMT EIA GMT EIA GMT GMT
PLG-NS345 <10 <10 <10 10 PLG- <10 <10 <10 15
NS345Core.sub.121 PLG- <10 11 <10 23 NS345 + PLG- CTAB-E1E2
DNA PLG- <10 <10 <10 20 NS345Core.sub.121 + E1E2 DNA
PLG-NS345 + <10 <10 <10 70 MF59 PLG- <10 <10 <10
26 NS345Core.sub.121 + MF59 PLG-NS345 + <10 <10 <10 350
CTAB-CPG PLG- <10 <10 <10 271 NS345Core.sub.121 + CTAB-CPG
PLG-NS345 + <10 <10 <10 201 QS21 PLG- <10 <10 <10
505 NS345Core.sub.121 + QS21 PLG-NS345 + <10 <10 <10 1471
CTAB-CPG + MF59 PLG- <10 <10 <10 63 NS345Core.sub.121 +
CTAB + MF59 .sup.aGroups of 10 C57 black mice were immunized IM at
0, 30 and 60 days. Serum was obtained after the last immunization.
The NS345 or NS345Core.sub.121 protein concentration was 10 .mu.g
per dose.
TABLE-US-00014 TABLE 12 Immunogenicity of different formulations of
HCV NS345Core.sub.121 or HCV NS345 in Mice. Geometric mean EIA
antibody titers to NS345Core.sub.121 protein are shown.
Anti-NS345Core.sub.121 Vaccine.sup.a Antibody EIA GMT
NS345Core.sub.121/MF59 328 NS345Core.sub.121/MF59/CpG 7,926 NS34a +
NS5B + Core/MF59 12 NS34a + NS5B + Core/MF59/CpG 5
PLG-NS345Core.sub.121/MF59 <10 PLG-NS345Core.sub.121/MF59/CpG
<10 PLG-NS345Core.sub.121/PLG-CpG 9 NS345Core.sub.121/alum
phosphate 34 NS345Core.sub.121/alum phosphate//CpG 950
NS345/MF59/CpG 511 PLG-NS345/PLG/CpG 117 NS345Core.sub.121/MF59/MPL
10,292 NS345Core.sub.121/MF59/Chol/QS21 698 NS345Core.sub.121/Alum
phosphate/MPL 23 .sup.aGroups of 10 C57 black mice were immunized
IM at 0, 30 and 60 days. Serum was obtained after the last
immunization. The NS345 or NS345Core.sub.121protein concentration
was 5 .mu.g per dose.
Example 15
Lymphoproliferative Response of if Different Formulations of
NS345Core.sub.121 or NS34A+NS35B+Core in Mice
[0215] Groups of 8 C57 black mice were immunized IM at 0, 30 and 60
days with the formulations shown in Table 13 and are as described
above. Spleens were obtained after the last immunization. The
NS345Core.sub.121 protein concentration was 25 .mu.g per dose. The
NS34a, NS5b and core doses were 3 .mu.g each.
[0216] The results of this study are shown in Table 13. As can be
seen, NS345Core.sub.121/Alum/CpG, PLG-NS345Core.sub.121/PLG/CpG,
NS34a+NS5B+Core/MF59/CpG and PLG-NS345Core.sub.121/MF59/CpG
formulations demonstrated strong LPA responses to NS5, NS34 and
core antigens. The NS345Core.sub.121/MF59 formulation also produced
a strong LPA response to NS5 and NS34. Core was not tested.
Moderate LPA responses were observed to NS5, NS34 and Core antigens
with PLG-NS345Core.sub.121/MF59 and NS34a+NS5B+Core/MF59
formulations. The NS345Core.sub.121/MF59/CpG formulation may not
have been administered properly in that no LPA response was
observed in this experiment. In a subsequent experiment as shown in
Table 14, an LPA was observed to this formulation.
[0217] Groups of 8 C57 black mice were immunized once IM with the
formulations shown in Table 14, produced as described above.
Draining lymph nodes were obtained. The NS345Core.sub.121 protein
concentration was 25 .mu.g per dose.
[0218] The results of this study are shown in Table 14. As can be
seen in Table 14, all the formulations tested produced a strong LPA
response to NS5, NS34 and Core as well as the
NS345Core.sub.121.
TABLE-US-00015 TABLE 13 Lymphoproliferative response of different
formulations of HCV NS345Core.sub.121 or NS34A + NS5B + Core in
Mice. LPA responses (cpm) to core and nonstructural proteins are
shown. The number of mice in each group responding is also
indicated in parentheses. HIV-2 env SOD-C200 SOD-C22-3 (background
Vaccine.sup.a SOD-NS5 (NS34) (Core) control) NS345Core.sub.121/MF59
2250 1800 ND 144 (6/8) (4/8) NS345Core.sub.121/MF59/CpG 80 80 ND
138 PLG-NS345Core.sub.121/MF59 560 120 510 93 (2/8) (2/8) (2/8)
PLG- 1600 1500 620 75 NS345Core.sub.121/MF59/CpG (6/8) (6/8) (8/8)
NS34a + NS5B + Core/MF59 564 710 265 76 (8/8) (8/8) (8/8) NS34a +
NS5B + 1523 885 446 67 Core/MF59/CpG (8/8) (8/8) (6/8)
PLG-NS345Core.sub.121/PLG/CpG 3675 2860 370 88 (8/8) (8/8) (8/8)
NS345Core.sub.121/Alum/CpG 8450 7940 1040 82 (8/8) (8/8) (6/8)
.sup.aGroups of 8 C57 black mice were immunized IM at 0, 30 and 60
days. Spleens were obtained after the last immunization. The
NS345Core.sub.121 protein concentration was 25 .mu.g per dose. The
NS34a, NS5B and Core doses were 3 .mu.g each.
TABLE-US-00016 TABLE 14 Lymphoproliferative response of different
formulations of HCV NS345Core.sub.121 in Mice. The LPA responses
(cpm) form an average of three consecutive experiments to core and
nonstructural proteins are shown. HIV-2 env SOD-C200 SOD-C22-3
(background Vaccine.sup.a SOD-NS5 (NS34) (Core) NS345Core.sub.121
control) NS345Core.sub.121/MF59 8900 8300 3000 20900 890
NS345Core.sub.121/MF59/CpG 1890 1628 1200 20100 623
PLG-NS345Core.sub.121/MF59 10700 12900 1800 23700 818 PLG- 3600
4690 1660 27300 911 NS345Core.sub.121/MF59/CpG
PLG-NS345Core.sub.121 4500 4660 760 18150 315
PLG-NS345Core.sub.121/PLG/CpG 7750 5300 1250 22980 450
NS345Core.sub.121/Alum 3050 3725 744 11300 390
NS345Core.sub.121/Alum/CpG 4130 4670 600 20660 480 .sup.aGroups of
8 C57 black mice were immunized once IM. Draining lymph nodes were
obtained. The NS345Core.sub.121 protein concentration was 25 .mu.g
per dose.
Example 16
Immunogenicity of Recombinant HCV Protein Vaccines Adjuvanted with
ISCOMS in Rhesus Macaques
[0219] The safety and immunogenicity of HCV proteins completed with
the adjuvant, Iscomatrix, was studied in Rhesus macaques. Three
groups made up of four animals each were immunized IM as detailed
below at week 0, 4 and 8 weeks. Vaccines were prepared as described
above. The ISCOMS used lacked QH-A.
TABLE-US-00017 Group Number n Vaccine Delivery 1 4 Core-ISCOM 0.5
ml R Leg (50 .mu.g in 1 ml) 0.5 ml L Leg 2 4
NS345Core.sub.121-ISCOM 0.5 ml R Leg (1 mg in 1 ml) 0.5 ml L Leg 3
4 Core-ISCOM 0.5 ml Core-ISCOM R Leg (25 .mu.g in 0.5 ml) and 0.35
ml NS5b-ISCOM L Leg NS5b-ISCOM (50 .mu.g in 0.35 ml)
Bleeds occurred as follows and immunogenicity was determined by CTL
assays, lymphoproliferation assays, FACS analysis and antibody
response a previously described (Palakos, et al. (2001) J. of
Immunology 166:3589).
TABLE-US-00018 Week Bleed date Immunized -10 -1 X 0 X 2 X 4 X 6 X 8
X 10 X
[0220] The immunogenicity of the different HCV recombinant protein
vaccines is shown in Tables 15-17.
TABLE-US-00019 TABLE 15 The Immunogenicity of HCV Core-ISCOMS
vaccine two weeks post 2.sup.nd immunization and post 3.sup.rd
immunization as assessed by CTL assays, CD8+ FACS analysis, LPA
stimulation index and CD4+ FACS analysis CD8+ ICS (CTL) CD4+ ICS
(LPA SI) Macaque # C NS3 NS4 NS5a NS5b C NS3 NS4 NS5a NS5b 2 weeks
post 3.degree. X020 -(-) +(-) N001 -(-) +(-) N086 -(-) +(-) X010
-(-) +(11) 2 weeks post 2.degree. X020 -(-) +/-(-) N001 -(-) -(8)
N086 -(-) +(-) X010 -(-) +/- (12)
TABLE-US-00020 TABLE 16 The Immunogenicity of HCV
NS345Core.sub.121-ISCOMS vaccine two weeks post 2.sup.nd
immunization and post 3.sup.rd immunization as assessed by CTL
assays, CD8+ FACS analysis, LPA stimulation index and CD4+ FACS
analysis CD8+ ICS (CTL) CD4+ ICS (LPA SI) Macaque # C NS3 NS4 NS5a
NS5b C NS3 NS4 NS5a NS5b 2 weeks post 3.degree. X016 -(-) +(+) -(-)
-(-) -(-) -(-) +(-) -(-) +/-(-) +/-(-) X008 -(-) -(-) -(-) -(-)
-(-) -(-) -(-) -(-) -(5) -(-) X021 -(-) -(-) -(-) -(-) -(-) -(-)
-(-) -(-) -(-) -(-) X023 +/- (-) -(-) -(-) +/-(-) -(-) -(-) +/-(-)
-(-) -(-) -(-) 2 weeks post 2.degree. X016 - (-) +(+) -(-) +(+)
+(+) -(-) +(-) +/-(-) +(-) +(-) X008 +(-) +(+) -(-) +(+) +(+) -(-)
+(-) -(-) +-(-) +(-) X021 -(-) -(-) +/-(-) -(-) +/-(-) -(-) -(-)
+/-(-) -(-) -(-) X023 +/-(+) +(+) -(-) +/-(-) +(-) -(-) +(-) -(-)
-(-) 7
TABLE-US-00021 TABLE 17 The Immunogenicity of HCV Core-ISCOMS +
NS5b-ISCOMS vaccine two weeks post 2.sup.nd immunization and post
3.sup.rd immunization as assessed by CTL assays, CD8+ FACS
analysis, LPA stimulation index and CD4+ FACS analysis CD8+ ICS
(CTL) CD4+ ICS (LPA SI) Macaque # C NS3 NS4 NS5a NS5b C NS3 NS4
NS5a NS5b 2 weeks post 3.degree. X022 +(-) -(-) -(8) +/-(11) X014
-(-) -(-) +(6) +(11) N154 -(-) -(-) -(-) +(-) N173 -(-) -(-) -(-)
+/-(-) 2 weeks post 2.degree. X022 -(-) -(+) -(-) -(-) X014 +(-)
+/-(-) -(-) +(-) N154 -(-) -(-) -(6) +(8) N173 -(-) -(-) +/-(-)
+(6)
[0221] As can be seen in Table 15, the HCV Core-ISCOM vaccine
produced no CTL positive responses in any of the 4 immunized
macaques after the second or third immunizations. No positive CD8
.gamma.-interferon and/or TNF-.alpha. intracellular staining was
also observed, although backgrounds were high in these particular
arrays. At least two of four macaques produced a strong LPA
response after the second immunizations, but only one remained
positive after the third immunization. Two of four macaques
produced positive CD4 intracellular staining after the second
immunization and four of four after the third immunization.
[0222] As shown in Table 16, the HCV NS345Core.sub.121-ISCOM
vaccine after the second immunization produced CTL positive
responses to peptide pools representing two or more HCV proteins in
three of four macaques (two of these macaques had responses to
peptide pools from NS3, NS5a and NS5b, one to peptide pools from
core and NS3). CD8 positive .gamma.-interferon and/or TNF-.alpha.
intracellular staining to peptide pools representing two or more
HCV proteins was positive in at least three of four macaques. One
of four macaques produced a strong LPA response. At least three of
four macaques produced CD4 positive intracellular staining to two
or more HCV proteins. After the third immunization, only one of
four macaques had a positive CTL response, CD8 positive
intracellular staining and C04 positive intracellular staining. One
other macaque had a positive LPA response and weak CD8+CD4
intracellular staining, This decline in immunogenicity was likely
due to instability of the vaccine formulation (see below).
[0223] As shown in Table 17, the HCV Core-ISCOM+NS5b-ISCOM vaccine
produced a CTL positive response to NS5b in one of the 4 immunized
macaques after the second immunization which did not remain
positive after the third immunization. CD8 positive intracellular
positive staining was observed in one of four animals post second.
Two of four macaques produced a strong LPA response after the
second immunization which did not remain positive after the third
immunization. Two other macaques did develop a strong LPA response
after the third immunization. Three or four developed positive CD4
intracellular staining. One developed positive CD8 intracellular
staining.
[0224] Three weeks after the third immunization, it was noted that
the physical appearance of the polyprotein vaccine solution was
visibly turbid. The core vaccine also was turbid but less so. The
Core-NS5 vaccine was also slightly turbid. Analysis of this
turbidity in the polyprotein formulation indicated that the ISCOM
particles had precipitated into large aggregates. These aggregates
could be dispersed by vortexing with 0.1% TWEEN 80 detergent. It is
probable that this change in the formulation of the vaccine
occurred before the last immunization. This observed change in
appearance of the vaccines may have affected their immunogenicity
as cellular immune results declined in all three vaccines.
[0225] The immunogenicity of HCV Core-ISCOMS,
NS345Core.sub.121-ISCOMS and Core-ISCOMS+NS5b-ISCOMS as assessed by
EIA antibody response is shown in Table 18. As can be seen, all
three vaccines produced an antibody response by the third
immunization to their corresponding HCV proteins, except for the
NS345Core.sub.121-ISCOM vaccine. The NS345Core.sub.121-ISCOM
vaccine produced antibody responses to NS3, NS4 and a very strong
antibody response to NS5, but no antibody response to HCV core.
TABLE-US-00022 TABLE 18 The immunogenicity of HCV Core-ISCOMS,
NS345Core.sub.121-ISCOMS, Core- ISOCMS + NS5b-ISCOMS vaccine two
weeks post 2.sup.nd immunization and post 3.sup.rd immunization as
assessed by EIA antibody response to HCV proteins. Anti-Core EIA
Anti-NS3 EIA Anti-NS4 EIA Anti-NS5 EIA Vaccine Antibody Titer
Antibody Titer Antibody Titer Antibody Titer Macaque # Post
2.sup.nd Post 3.sup.rd Post 2.sup.nd Post 3.sup.rd Post 2.sup.nd
Post 3.sup.rd Post 2.sup.nd Post 3.sup.rd Core- ISCOM X020 66 226
N001 87 46 N086 363 396 X010 108 137 NS345 Core121/ ISCOM X016
<10 <10 <10 554 56 68 3,590 3,405 X008 <10 <10 66
995 14 44 2,109 3,213 X021 <10 <10 128 6,330 41 204 7,213
8,083 X023 <10 <10 <10 3,910 64 64 1,243 4,704 Core- ISCOM
+ NS5b- ISCOM X022 <10 18 <10 134 X014 <10 13 <10 693
N154 542 554 <10 272 N173 28 78 <10 258
Example 17
Immunization of Chimpanzees with Recombinant HCV Protein and DNA
Vaccines
[0226] Five groups of five chimps each were immunized IM at 0, 0.7,
2 and 5 months with the formulations presented below. Blood was
collected at week 0, two weeks subsequent to the second
immunization, two weeks following the third immunization and two
weeks after the fourth immunization.
[0227] Formulation 1: 20 .mu.g E1E2 polypeptide+MF59+500 .mu.g CpG
(produced as described above);
[0228] Formulation 2: 1 mg NS345Core.sub.121-ISCOM (produced as
described above);
[0229] Formulation 3: 6 mg each of CTAB-PLG-E1E2 (bp 574-2427,
encoding amino acids 192-809 of the HCV polyprotein, numbered
relative to HCV-1); CTAB-PLG-NS34a (bp 3079-5133, encoding amino
acids 1027-1711 of the HCV polyprotein, numbered relative to
HCV-1); CTAB-PLG-NS34ab (bp 4972-5916, encoding amino acids
1658-1972 of the HCV polyprotein, numbered relative to HCV-1);
CTAB-PLG-NS5a (bp 5917-7260, encoding amino acids 1973-2420 of the
HCV polyprotein, numbered relative to HCV-1);
[0230] Formulation 4: 6 mg each of E1E2 DNA, NS34a DNA, NS34ab DNA
and NS5a DNA, having the same coordinates as described above,
delivered without PLG via electroporation (see, e.g., U.S. Pat.
Nos. 6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent
Publication No. 2002/0146831; and International Publication No.
WO/0045823, all of which are incorporated herein by reference in
their entireties, for this delivery technique). Results are shown
in FIGS. 8-10.
[0231] As can be seen, in FIG. 8, all vaccines were capable of
priming CD4+ and CD8+ cells specific to HCV. Thus, all vaccines
were successful at inducing a T cell response to HCV. Determination
of the results for the PLG-DNA from formulation 3 at two weeks
subsequent to the fourth vaccination is in progress.
[0232] As shown in FIGS. 9 and 10, multiple T cell specificities
were induced by the two vaccines. Both vaccines primed T-cells
specific for multiple T cell epitopes.
[0233] As can be seen in Tables 19 and 20, E1E2 adjuvanted with
MF59 primed anti-E1E2 titers. CpG enhanced anti-E1E2 responses as
well as TH1 responses and the ISCOM and the two DNA vaccines were
capable of priming CD4+ and CD8+ T cell responses to HCV.
TABLE-US-00023 TABLE 19 Anti-E1E2 EIA antibody titers in chimps
immunized with Electroporated DNA E1E2NS345a or PLG DNA E1E2NS345
Vaccine Chimp Pre 1.sup.st Post 2.sup.nd Post 3.sup.rd Post 4th
Electroporated DNA 4X0330 -- -- -- 9 E1E2-NS34A- 4X0335 -- -- -- 10
NS4AB-NS5A.sup.a 4X0348.sup.c 10 457 198 50 4X0354.sup.d 206 1,261
1,197 207 4X0368.sup.c 245 1,426 1,267 358 PLG DNA 4X0238 -- -- 30
10 E1E2-NS34A- 4X0239 -- 104 309 -- NS4AB-NS5A.sup.b 4X0250 -- --
12 -- 4X0278 -- -- 29 -- 4X0288 -- -- 12 -- .sup.aElectroporated IM
with 1.5 mg of each plasmid at 0, 0.7, 2 and 5 months. Bleeds were
taken 14 days after each immunization. .sup.bIM immunization with
1.5 mg of each PLG plasmid at 0, 0.7, 2 and 6 months. Bleeds were
taken 14 days after each immunization. .sup.cPrior E2 immunization
.sup.dPrior E1E2 immunization
TABLE-US-00024 TABLE 20 Immunogenicity in chimps of low dose (20
.mu.g) HCV E1E2 antigen using MF59 or MF59 combined with CpG as
adjuvants (2 wks post 3.sup.rd) E1E2EIA E1E2 EIA CD4+ Vaccine.sup.a
Chimp Ab Titer Ab GMT (ICS) E1E2/ 4 .times. 0419 84 - MF59 4
.times. 0420 101 - 4 .times. 0431 131 261 - 4 .times. 0371 421 - 4
.times. 0372 2,580 +/- P = 0.029.sup.b E1E2/ 4 .times. 0410 8,835 -
MF59/CpG 4 .times. 0426 2,713 - 4 .times. 0365 3,201 2,713 + 4
.times. 0367 510 - 4 .times. 0346 1,238 ++ .sup.aChimps immunized
IM at 0, 1 and 6 mos with 20 .mu.g of E1E2 antigen using MF59 with
or without 500 .mu.g of CpG. Serum samples were obtained 14 days
after last immunization. .sup.bChimps immunized with E1E2 using CpG
combined with MF59 as adjuvant produced significantly higher (P
< 0.05) levels of E1E2 EIA antibody than chimpanzees with E1E2
using MF59 alone.
[0234] Thus, HCV polypeptides and polynucleotides, either alone or
as fusions, 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 by the appended claims.
Sequence CWU 1
1
1019PRTArtificial SequenceDescription of Artificial Sequence fusion
protein epitope 1His Glu Tyr Pro Val Gly Ser Gln Leu 1
5215PRTArtificial SequenceDescription of Artificial Sequence fusion
protein epitope 2Ala Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln
Glu Met Gly 1 5 10 1531914DNAArtificial SequenceDescription of
Artificial Sequence HCV-1 E1/E2/p7 region 3tct ttc tct atc ttc ctt
ctg gcc ctg ctc tct tgc ttg act gtg ccc 48Ser Phe Ser Ile Phe Leu
Leu Ala Leu Leu Ser Cys Leu Thr Val Pro 1 5 10 15gct tcg gcc tac
caa gtg cgc aac tcc acg ggg ctc tac cac gtc acc 96Ala Ser Ala Tyr
Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr 20 25 30aat gat tgc
cct aac tcg agt att gtg tac gag gcg gcc gat gcc atc 144Asn Asp Cys
Pro Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile 35 40 45ctg cac
act ccg ggg tgc gtc cct tgc gtt cgc gag ggc aac gcc tcg 192Leu His
Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser 50 55 60agg
tgt tgg gtg gcg atg acc cct acg gtg gcc acc agg gat ggc aaa 240Arg
Cys Trp Val Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly Lys 65 70
75 80ctc ccc gcg acg cag ctt cga cgt cac atc gat ctg ctt gtc ggg
agc 288Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly
Ser 85 90 95gcc acc ctc tgt tcg gcc ctc tac gtg ggg gac ctg tgc ggg
tct gtc 336Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly
Ser Val 100 105 110ttt ctt gtc ggc caa ctg ttt acc ttc tct ccc agg
cgc cac tgg acg 384Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg
Arg His Trp Thr 115 120 125acg caa ggt tgc aat tgc tct atc tat ccc
ggc cat ata acg ggt cac 432Thr Gln Gly Cys Asn Cys Ser Ile Tyr Pro
Gly His Ile Thr Gly His 130 135 140cgc atg gca tgg gat atg atg atg
aac tgg tcc cct acg acg gcg ttg 480Arg Met Ala Trp Asp Met Met Met
Asn Trp Ser Pro Thr Thr Ala Leu145 150 155 160gta atg gct cag ctg
ctc cgg atc cca caa gcc atc ttg gac atg atc 528Val Met Ala Gln Leu
Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile 165 170 175gct ggt gct
cac tgg gga gtc ctg gcg ggc ata gcg tat ttc tcc atg 576Ala Gly Ala
His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met 180 185 190gtg
ggg aac tgg gcg aag gtc ctg gta gtg ctg ctg cta ttt gcc ggc 624Val
Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly 195 200
205gtc gac gcg gaa acc cac gtc acc ggg gga agt gcc ggc cac act gtg
672Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val
210 215 220tct gga ttt gtt agc ctc ctc gca cca ggc gcc aag cag aac
gtc cag 720Ser Gly Phe Val Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn
Val Gln225 230 235 240ctg atc aac acc aac ggc agt tgg cac ctc aat
agc acg gcc ctg aac 768Leu Ile Asn Thr Asn Gly Ser Trp His Leu Asn
Ser Thr Ala Leu Asn 245 250 255tgc aat gat agc ctc aac acc ggc tgg
ttg gca ggg ctt ttc tat cac 816Cys Asn Asp Ser Leu Asn Thr Gly Trp
Leu Ala Gly Leu Phe Tyr His 260 265 270cac aag ttc aac tct tca ggc
tgt cct gag agg cta gcc agc tgc cga 864His Lys Phe Asn Ser Ser Gly
Cys Pro Glu Arg Leu Ala Ser Cys Arg 275 280 285ccc ctt acc gat ttt
gac cag ggc tgg ggc cct atc agt tat gcc aac 912Pro Leu Thr Asp Phe
Asp Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn 290 295 300gga agc ggc
ccc gac cag cgc ccc tac tgc tgg cac tac ccc cca aaa 960Gly Ser Gly
Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys305 310 315
320cct tgc ggt att gtg ccc gcg aag agt gtg tgt ggt ccg gta tat tgc
1008Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys
325 330 335ttc act ccc agc ccc gtg gtg gtg gga acg acc gac agg tcg
ggc gcg 1056Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser
Gly Ala 340 345 350ccc acc tac agc tgg ggt gaa aat gat acg gac gtc
ttc gtc ctt aac 1104Pro Thr Tyr Ser Trp Gly Glu Asn Asp Thr Asp Val
Phe Val Leu Asn 355 360 365aat acc agg cca ccg ctg ggc aat tgg ttc
ggt tgt acc tgg atg aac 1152Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe
Gly Cys Thr Trp Met Asn 370 375 380tca act gga ttc acc aaa gtg tgc
gga gcg cct cct tgt gtc atc gga 1200Ser Thr Gly Phe Thr Lys Val Cys
Gly Ala Pro Pro Cys Val Ile Gly385 390 395 400ggg gcg ggc aac aac
acc ctg cac tgc ccc act gat tgc ttc cgc aag 1248Gly Ala Gly Asn Asn
Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys 405 410 415cat ccg gac
gcc aca tac tct cgg tgc ggc tcc ggt ccc tgg atc aca 1296His Pro Asp
Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr 420 425 430ccc
agg tgc ctg gtc gac tac ccg tat agg ctt tgg cat tat cct tgt 1344Pro
Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys 435 440
445acc atc aac tac act ata ttt aaa atc agg atg tac gtg gga ggg gtc
1392Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val
450 455 460gag cac agg ctg gaa gct gcc tgc aac tgg acg cgg ggc gaa
cgt tgc 1440Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu
Arg Cys465 470 475 480gat ctg gaa gat agg gac agg tcc gag ctc agc
ccg tta ctg ctg acc 1488Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser
Pro Leu Leu Leu Thr 485 490 495act aca cag tgg cag gtc ctc ccg tgt
tcc ttc aca acc ctg cca gcc 1536Thr Thr Gln Trp Gln Val Leu Pro Cys
Ser Phe Thr Thr Leu Pro Ala 500 505 510ttg tcc acc ggc ctc atc cac
ctc cac cag aac att gtg gac gtg cag 1584Leu Ser Thr Gly Leu Ile His
Leu His Gln Asn Ile Val Asp Val Gln 515 520 525tac ttg tac ggg gtg
ggg tca agc atc gcg tcc tgg gcc att aag tgg 1632Tyr Leu Tyr Gly Val
Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp 530 535 540gag tac gtc
gtc ctc ctg ttc ctt ctg ctt gca gac gcg cgc gtc tgc 1680Glu Tyr Val
Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys545 550 555
560tcc tgc ttg tgg atg atg cta ctc ata tcc caa gcg gaa gcg gct ttg
1728Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu
565 570 575gag aac ctc gta ata ctt aat gca gca tcc ctg gcc ggg acg
cac ggt 1776Glu Asn Leu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr
His Gly 580 585 590ctt gta tcc ttc ctc gtg ttc ttc tgc ttt gca tgg
tat ctg aag ggt 1824Leu Val Ser Phe Leu Val Phe Phe Cys Phe Ala Trp
Tyr Leu Lys Gly 595 600 605aag tgg gtg ccc gga gcg gtc tac acc ttc
tac ggg atg tgg cct ctc 1872Lys Trp Val Pro Gly Ala Val Tyr Thr Phe
Tyr Gly Met Trp Pro Leu 610 615 620ctc ctg ctc ctg ttg gcg ttg ccc
cag cgg gcg tac gcg taa 1914Leu Leu Leu Leu Leu Ala Leu Pro Gln Arg
Ala Tyr Ala625 630 635 4637PRTArtificial SequenceDescription of
Artificial Sequence HCV-1 E1/E2/p7 region 4Ser Phe Ser Ile Phe Leu
Leu Ala Leu Leu Ser Cys Leu Thr Val Pro 1 5 10 15Ala Ser Ala Tyr
Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr 20 25 30Asn Asp Cys
Pro Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Ala Ile 35 40 45Leu His
Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Ala Ser 50 55 60Arg
Cys Trp Val Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly Lys 65 70
75 80Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val Gly
Ser 85 90 95Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly
Ser Val 100 105 110Phe Leu Val Gly Gln Leu Phe Thr Phe Ser Pro Arg
Arg His Trp Thr 115 120 125Thr Gln Gly Cys Asn Cys Ser Ile Tyr Pro
Gly His Ile Thr Gly His 130 135 140Arg Met Ala Trp Asp Met Met Met
Asn Trp Ser Pro Thr Thr Ala Leu145 150 155 160Val Met Ala Gln Leu
Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile 165 170 175Ala Gly Ala
His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Met 180 185 190Val
Gly Asn Trp Ala Lys Val Leu Val Val Leu Leu Leu Phe Ala Gly 195 200
205Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val
210 215 220Ser Gly Phe Val Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn
Val Gln225 230 235 240Leu Ile Asn Thr Asn Gly Ser Trp His Leu Asn
Ser Thr Ala Leu Asn 245 250 255Cys Asn Asp Ser Leu Asn Thr Gly Trp
Leu Ala Gly Leu Phe Tyr His 260 265 270His Lys Phe Asn Ser Ser Gly
Cys Pro Glu Arg Leu Ala Ser Cys Arg 275 280 285Pro Leu Thr Asp Phe
Asp Gln Gly Trp Gly Pro Ile Ser Tyr Ala Asn 290 295 300Gly Ser Gly
Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys305 310 315
320Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys
325 330 335Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser
Gly Ala 340 345 350Pro Thr Tyr Ser Trp Gly Glu Asn Asp Thr Asp Val
Phe Val Leu Asn 355 360 365Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe
Gly Cys Thr Trp Met Asn 370 375 380Ser Thr Gly Phe Thr Lys Val Cys
Gly Ala Pro Pro Cys Val Ile Gly385 390 395 400Gly Ala Gly Asn Asn
Thr Leu His Cys Pro Thr Asp Cys Phe Arg Lys 405 410 415His Pro Asp
Ala Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr 420 425 430Pro
Arg Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys 435 440
445Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val
450 455 460Glu His Arg Leu Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu
Arg Cys465 470 475 480Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser
Pro Leu Leu Leu Thr 485 490 495Thr Thr Gln Trp Gln Val Leu Pro Cys
Ser Phe Thr Thr Leu Pro Ala 500 505 510Leu Ser Thr Gly Leu Ile His
Leu His Gln Asn Ile Val Asp Val Gln 515 520 525Tyr Leu Tyr Gly Val
Gly Ser Ser Ile Ala Ser Trp Ala Ile Lys Trp 530 535 540Glu Tyr Val
Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys545 550 555
560Ser Cys Leu Trp Met Met Leu Leu Ile Ser Gln Ala Glu Ala Ala Leu
565 570 575Glu Asn Leu Val Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr
His Gly 580 585 590Leu Val Ser Phe Leu Val Phe Phe Cys Phe Ala Trp
Tyr Leu Lys Gly 595 600 605Lys Trp Val Pro Gly Ala Val Tyr Thr Phe
Tyr Gly Met Trp Pro Leu 610 615 620Leu Leu Leu Leu Leu Ala Leu Pro
Gln Arg Ala Tyr Ala625 630 635521PRTArtificial SequenceDescription
of Artificial Sequence consensus sequence 5Gly Ser Ala Ala Arg Thr
Thr Ser Gly Phe Val Ser Leu Phe Ala Pro 1 5 10 15Gly Ala Lys Gln
Asn 20620DNAArtificial SequenceDescription of Artificial Sequence
exemplary CpG oligonucleotide 6tccatgacgt tcctgacgtt
2075676DNAArtificial SequenceDescription of Artificial Sequence
representative NS345Core fusion protein 7atg gct gca tat gca gct
cag ggc tat aag gtg cta gta ctc aac ccc 48Met Ala Ala Tyr Ala Ala
Gln Gly Tyr Lys Val Leu Val Leu Asn Pro 1 5 10 15tct gtt gct gca
aca ctg ggc ttt ggt gct tac atg tcc aag gct cat 96Ser Val Ala Ala
Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His 20 25 30ggg atc gat
cct aac atc agg acc ggg gtg aga aca att acc act ggc 144Gly Ile Asp
Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly 35 40 45agc ccc
atc acg tac tcc acc tac ggc aag ttc ctt gcc gac ggc ggg 192Ser Pro
Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly 50 55 60tgc
tcg ggg ggc gct tat gac ata ata att tgt gac gag tgc cac tcc 240Cys
Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser 65 70
75 80acg gat gcc aca tcc atc ttg ggc att ggc act gtc ctt gac caa
gca 288Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln
Ala 85 90 95gag act gcg ggg gcg aga ctg gtt gtg ctc gcc acc gcc acc
cct ccg 336Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr
Pro Pro 100 105 110ggc tcc gtc act gtg ccc cat ccc aac atc gag gag
gtt gct ctg tcc 384Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu
Val Ala Leu Ser 115 120 125acc acc gga gag atc cct ttt tac ggc aag
gct atc ccc ctc gaa gta 432Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys
Ala Ile Pro Leu Glu Val 130 135 140atc aag ggg ggg aga cat ctc atc
ttc tgt cat tca aag aag aag tgc 480Ile Lys Gly Gly Arg His Leu Ile
Phe Cys His Ser Lys Lys Lys Cys145 150 155 160gac gaa ctc gcc gca
aag ctg gtc gca ttg ggc atc aat gcc gtg gcc 528Asp Glu Leu Ala Ala
Lys Leu Val Ala Leu Gly Ile Asn Ala Val Ala 165 170 175tac tac cgc
ggt ctt gac gtg tcc gtc atc ccg acc agc ggc gat gtt 576Tyr Tyr Arg
Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val 180 185 190gtc
gtc gtg gca acc gat gcc ctc atg acc ggc tat acc ggc gac ttc 624Val
Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 195 200
205gac tcg gtg ata gac tgc aat acg tgt gtc acc cag aca gtc gat ttc
672Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe
210 215 220agc ctt gac cct acc ttc acc att gag aca atc acg ctc ccc
caa gat 720Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro
Gln Asp225 230 235 240gct gtc tcc cgc act caa cgt cgg ggc agg act
ggc agg ggg aag cca 768Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr
Gly Arg Gly Lys Pro 245 250 255ggc atc tac aga ttt gtg gca ccg ggg
gag cgc ccc tcc ggc atg ttc 816Gly Ile Tyr Arg Phe Val Ala Pro Gly
Glu Arg Pro Ser Gly Met Phe 260 265 270gac tcg tcc gtc ctc tgt gag
tgc tat gac gca ggc tgt gct tgg tat 864Asp Ser Ser Val Leu Cys Glu
Cys Tyr Asp Ala Gly Cys Ala Trp Tyr 275 280 285gag ctc acg ccc gcc
gag act aca gtt agg cta cga gcg tac atg aac 912Glu Leu Thr Pro Ala
Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn 290 295 300acc ccg ggg
ctt ccc gtg tgc cag gac cat ctt gaa ttt tgg gag ggc 960Thr Pro Gly
Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly305 310 315
320gtc ttt aca ggc ctc act cat ata gat gcc cac ttt cta tcc cag aca
1008Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr
325 330 335aag cag agt ggg gag aac ctt cct tac ctg gta gcg tac caa
gcc acc 1056Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln
Ala Thr 340 345 350gtg tgc gct agg gct caa gcc cct ccc cca tcg tgg
gac cag atg tgg 1104Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp
Asp Gln Met Trp 355 360 365aag tgt ttg att cgc ctc aag ccc acc ctc
cat ggg cca aca ccc ctg 1152Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu
His Gly Pro Thr Pro Leu 370 375 380cta tac aga ctg ggc gct gtt cag
aat gaa atc acc ctg acg cac cca 1200Leu Tyr Arg Leu Gly Ala Val Gln
Asn Glu Ile Thr Leu Thr His Pro385 390 395 400gtc acc aaa tac atc
atg aca tgc atg tcg gcc gac ctg gag gtc gtc 1248Val Thr Lys Tyr Ile
Met Thr Cys Met Ser Ala Asp Leu Glu Val Val 405 410 415acg agc acc
tgg gtg
ctc gtt ggc ggc gtc ctg gct gct ttg gcc gcg 1296Thr Ser Thr Trp Val
Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala 420 425 430tat tgc ctg
tca aca ggc tgc gtg gtc ata gtg ggc agg gtc gtc ttg 1344Tyr Cys Leu
Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu 435 440 445tcc
ggg aag ccg gca atc ata cct gac agg gaa gtc ctc tac cga gag 1392Ser
Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu 450 455
460ttc gat gag atg gaa gag tgc tct cag cac tta ccg tac atc gag caa
1440Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu
Gln465 470 475 480ggg atg atg ctc gcc gag cag ttc aag cag aag gcc
ctc ggc ctc ctg 1488Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala
Leu Gly Leu Leu 485 490 495cag acc gcg tcc cgt cag gca gag gtt atc
gcc cct gct gtc cag acc 1536Gln Thr Ala Ser Arg Gln Ala Glu Val Ile
Ala Pro Ala Val Gln Thr 500 505 510aac tgg caa aaa ctc gag acc ttc
tgg gcg aag cat atg tgg aac ttc 1584Asn Trp Gln Lys Leu Glu Thr Phe
Trp Ala Lys His Met Trp Asn Phe 515 520 525atc agt ggg ata caa tac
ttg gcg ggc ttg tca acg ctg cct ggt aac 1632Ile Ser Gly Ile Gln Tyr
Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn 530 535 540ccc gcc att gct
tca ttg atg gct ttt aca gct gct gtc acc agc cca 1680Pro Ala Ile Ala
Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro545 550 555 560cta
acc act agc caa acc ctc ctc ttc aac ata ttg ggg ggg tgg gtg 1728Leu
Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val 565 570
575gct gcc cag ctc gcc gcc ccc ggt gcc gct act gcc ttt gtg ggc gct
1776Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val Gly Ala
580 585 590ggc tta gct ggc gcc gcc atc ggc agt gtt gga ctg ggg aag
gtc ctc 1824Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu Gly Lys
Val Leu 595 600 605ata gac atc ctt gca ggg tat ggc gcg ggc gtg gcg
gga gct ctt gtg 1872Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala
Gly Ala Leu Val 610 615 620gca ttc aag atc atg agc ggt gag gtc ccc
tcc acg gag gac ctg gtc 1920Ala Phe Lys Ile Met Ser Gly Glu Val Pro
Ser Thr Glu Asp Leu Val625 630 635 640aat cta ctg ccc gcc atc ctc
tcg ccc gga gcc ctc gta gtc ggc gtg 1968Asn Leu Leu Pro Ala Ile Leu
Ser Pro Gly Ala Leu Val Val Gly Val 645 650 655gtc tgt gca gca ata
ctg cgc cgg cac gtt ggc ccg ggc gag ggg gca 2016Val Cys Ala Ala Ile
Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 660 665 670gtg cag tgg
atg aac cgg ctg ata gcc ttc gcc tcc cgg ggg aac cat 2064Val Gln Trp
Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His 675 680 685gtt
tcc ccc acg cac tac gtg ccg gag agc gat gca gct gcc cgc gtc 2112Val
Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val 690 695
700act gcc ata ctc agc agc ctc act gta acc cag ctc ctg agg cga ctg
2160Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg
Leu705 710 715 720cac cag tgg ata agc tcg gag tgt acc act cca tgc
tcc ggt tcc tgg 2208His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys
Ser Gly Ser Trp 725 730 735cta agg gac atc tgg gac tgg ata tgc gag
gtg ttg agc gac ttt aag 2256Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu
Val Leu Ser Asp Phe Lys 740 745 750acc tgg cta aaa gct aag ctc atg
cca cag ctg cct ggg atc ccc ttt 2304Thr Trp Leu Lys Ala Lys Leu Met
Pro Gln Leu Pro Gly Ile Pro Phe 755 760 765gtg tcc tgc cag cgc ggg
tat aag ggg gtc tgg cga ggg gac ggc atc 2352Val Ser Cys Gln Arg Gly
Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile 770 775 780atg cac act cgc
tgc cac tgt gga gct gag atc act gga cat gtc aaa 2400Met His Thr Arg
Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys785 790 795 800aac
ggg acg atg agg atc gtc ggt cct agg acc tgc agg aac atg tgg 2448Asn
Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp 805 810
815agt ggg acc ttc ccc att aat gcc tac acc acg ggc ccc tgt acc ccc
2496Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro
820 825 830ctt cct gcg ccg aac tac acg ttc gcg cta tgg agg gtg tct
gca gag 2544Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg Val Ser
Ala Glu 835 840 845gaa tac gtg gag ata agg cag gtg ggg gac ttc cac
tac gtg acg ggt 2592Glu Tyr Val Glu Ile Arg Gln Val Gly Asp Phe His
Tyr Val Thr Gly 850 855 860atg act act gac aat ctt aaa tgc ccg tgc
cag gtc cca tcg ccc gaa 2640Met Thr Thr Asp Asn Leu Lys Cys Pro Cys
Gln Val Pro Ser Pro Glu865 870 875 880ttt ttc aca gaa ttg gac ggg
gtg cgc cta cat agg ttt gcg ccc ccc 2688Phe Phe Thr Glu Leu Asp Gly
Val Arg Leu His Arg Phe Ala Pro Pro 885 890 895tgc aag ccc ttg ctg
cgg gag gag gta tca ttc aga gta gga ctc cac 2736Cys Lys Pro Leu Leu
Arg Glu Glu Val Ser Phe Arg Val Gly Leu His 900 905 910gaa tac ccg
gta ggg tcg caa tta cct tgc gag ccc gaa ccg gac gtg 2784Glu Tyr Pro
Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val 915 920 925gcc
gtg ttg acg tcc atg ctc act gat ccc tcc cat ata aca gca gag 2832Ala
Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu 930 935
940gcg gcc ggg cga agg ttg gcg agg gga tca ccc ccc tct gtg gcc agc
2880Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val Ala
Ser945 950 955 960tcc tcg gct agc cag cta tcc gct cca tct ctc aag
gca act tgc acc 2928Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys
Ala Thr Cys Thr 965 970 975gct aac cat gac tcc cct gat gct gag ctc
ata gag gcc aac ctc cta 2976Ala Asn His Asp Ser Pro Asp Ala Glu Leu
Ile Glu Ala Asn Leu Leu 980 985 990tgg agg cag gag atg ggc ggc aac
atc acc agg gtt gag tca gaa aac 3024Trp Arg Gln Glu Met Gly Gly Asn
Ile Thr Arg Val Glu Ser Glu Asn 995 1000 1005aaa gtg gtg att ctg
gac tcc ttc gat ccg ctt gtg gcg gag gag gac 3072Lys Val Val Ile Leu
Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp 1010 1015 1020gag cgg
gag atc tcc gta ccc gca gaa atc ctg cgg aag tct cgg aga 3120Glu Arg
Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg1025 1030
1035 1040ttc gcc cag gcc ctg ccc gtt tgg gcg cgg ccg gac tat aac
ccc ccg 3168Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn
Pro Pro 1045 1050 1055cta gtg gag acg tgg aaa aag ccc gac tac gaa
cca cct gtg gtc cat 3216Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Glu
Pro Pro Val Val His 1060 1065 1070ggc tgc ccg ctt cca cct cca aag
tcc cct cct gtg cct ccg cct cgg 3264Gly Cys Pro Leu Pro Pro Pro Lys
Ser Pro Pro Val Pro Pro Pro Arg 1075 1080 1085aag aag cgg acg gtg
gtc ctc act gaa tca acc cta tct act gcc ttg 3312Lys Lys Arg Thr Val
Val Leu Thr Glu Ser Thr Leu Ser Thr Ala Leu 1090 1095 1100gcc gag
ctc gcc acc aga agc ttt ggc agc tcc tca act tcc ggc att 3360Ala Glu
Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile1105 1110
1115 1120acg ggc gac aat acg aca aca tcc tct gag ccc gcc cct tct
ggc tgc 3408Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro Ser
Gly Cys 1125 1130 1135ccc ccc gac tcc gac gct gag tcc tat tcc tcc
atg ccc ccc ctg gag 3456Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser Ser
Met Pro Pro Leu Glu 1140 1145 1150ggg gag cct ggg gat ccg gat ctt
agc gac ggg tca tgg tca acg gtc 3504Gly Glu Pro Gly Asp Pro Asp Leu
Ser Asp Gly Ser Trp Ser Thr Val 1155 1160 1165agt agt gag gcc aac
gcg gag gat gtc gtg tgc tgc tca atg tct tac 3552Ser Ser Glu Ala Asn
Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr 1170 1175 1180tct tgg
aca ggc gca ctc gtc acc ccg tgc gcc gcg gaa gaa cag aaa 3600Ser Trp
Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys1185 1190
1195 1200ctg ccc atc aat gca cta agc aac tcg ttg cta cgt cac cac
aat ttg 3648Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His
Asn Leu 1205 1210 1215gtg tat tcc acc acc tca cgc agt gct tgc caa
agg cag aag aaa gtc 3696Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys Gln
Arg Gln Lys Lys Val 1220 1225 1230aca ttt gac aga ctg caa gtt ctg
gac agc cat tac cag gac gta ctc 3744Thr Phe Asp Arg Leu Gln Val Leu
Asp Ser His Tyr Gln Asp Val Leu 1235 1240 1245aag gag gtt aaa gca
gcg gcg tca aaa gtg aag gct aac ttg cta tcc 3792Lys Glu Val Lys Ala
Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser 1250 1255 1260gta gag
gaa gct tgc agc ctg acg ccc cca cac tca gcc aaa tcc aag 3840Val Glu
Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys1265 1270
1275 1280ttt ggt tat ggg gca aaa gac gtc cgt tgc cat gcc aga aag
gcc gta 3888Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg Lys
Ala Val 1285 1290 1295acc cac atc aac tcc gtg tgg aaa gac ctt ctg
gaa gac aat gta aca 3936Thr His Ile Asn Ser Val Trp Lys Asp Leu Leu
Glu Asp Asn Val Thr 1300 1305 1310cca ata gac act acc atc atg gct
aag aac gag gtt ttc tgc gtt cag 3984Pro Ile Asp Thr Thr Ile Met Ala
Lys Asn Glu Val Phe Cys Val Gln 1315 1320 1325cct gag aag ggg ggt
cgt aag cca gct cgt ctc atc gtg ttc ccc gat 4032Pro Glu Lys Gly Gly
Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp 1330 1335 1340ctg ggc
gtg cgc gtg tgc gaa aag atg gct ttg tac gac gtg gtt aca 4080Leu Gly
Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr1345 1350
1355 1360aag ctc ccc ttg gcc gtg atg gga agc tcc tac gga ttc caa
tac tca 4128Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe Gln
Tyr Ser 1365 1370 1375cca gga cag cgg gtt gaa ttc ctc gtg caa gcg
tgg aag tcc aag aaa 4176Pro Gly Gln Arg Val Glu Phe Leu Val Gln Ala
Trp Lys Ser Lys Lys 1380 1385 1390acc cca atg ggg ttc tcg tat gat
acc cgc tgc ttt gac tcc aca gtc 4224Thr Pro Met Gly Phe Ser Tyr Asp
Thr Arg Cys Phe Asp Ser Thr Val 1395 1400 1405act gag agc gac atc
cgt acg gag gag gca atc tac caa tgt tgt gac 4272Thr Glu Ser Asp Ile
Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp 1410 1415 1420ctc gac
ccc caa gcc cgc gtg gcc atc aag tcc ctc acc gag agg ctt 4320Leu Asp
Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu1425 1430
1435 1440tat gtt ggg ggc cct ctt acc aat tca agg ggg gag aac tgc
ggc tat 4368Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn Cys
Gly Tyr 1445 1450 1455cgc agg tgc cgc gcg agc ggc gta ctg aca act
agc tgt ggt aac acc 4416Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr
Ser Cys Gly Asn Thr 1460 1465 1470ctc act tgc tac atc aag gcc cgg
gca gcc tgt cga gcc gca ggg ctc 4464Leu Thr Cys Tyr Ile Lys Ala Arg
Ala Ala Cys Arg Ala Ala Gly Leu 1475 1480 1485cag gac tgc acc atg
ctc gtg tgt ggc gac gac tta gtc gtt atc tgt 4512Gln Asp Cys Thr Met
Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys 1490 1495 1500gaa agc
gcg ggg gtc cag gag gac gcg gcg agc ctg aga gcc ttc acg 4560Glu Ser
Ala Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr1505 1510
1515 1520gag gct atg acc agg tac tcc gcc ccc cct ggg gac ccc cca
caa cca 4608Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro
Gln Pro 1525 1530 1535gaa tac gac ttg gag ctc ata aca tca tgc tcc
tcc aac gtg tca gtc 4656Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser
Ser Asn Val Ser Val 1540 1545 1550gcc cac gac ggc gct gga aag agg
gtc tac tac ctc acc cgt gac cct 4704Ala His Asp Gly Ala Gly Lys Arg
Val Tyr Tyr Leu Thr Arg Asp Pro 1555 1560 1565aca acc ccc ctc gcg
aga gct gcg tgg gag aca gca aga cac act cca 4752Thr Thr Pro Leu Ala
Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro 1570 1575 1580gtc aat
tcc tgg cta ggc aac ata atc atg ttt gcc ccc aca ctg tgg 4800Val Asn
Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp1585 1590
1595 1600gcg agg atg ata ctg atg acc cat ttc ttt agc gtc ctt ata
gcc agg 4848Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu Ile
Ala Arg 1605 1610 1615gac cag ctt gaa cag gcc ctc gat tgc gag atc
tac ggg gcc tgc tac 4896Asp Gln Leu Glu Gln Ala Leu Asp Cys Glu Ile
Tyr Gly Ala Cys Tyr 1620 1625 1630tcc ata gaa cca ctg gat cta cct
cca atc att caa aga ctc cat ggc 4944Ser Ile Glu Pro Leu Asp Leu Pro
Pro Ile Ile Gln Arg Leu His Gly 1635 1640 1645ctc agc gca ttt tca
ctc cac agt tac tct cca ggt gaa atc aat agg 4992Leu Ser Ala Phe Ser
Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg 1650 1655 1660gtg gcc
gca tgc ctc aga aaa ctt ggg gta ccg ccc ttg cga gct tgg 5040Val Ala
Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp1665 1670
1675 1680aga cac cgg gcc cgg agc gtc cgc gct agg ctt ctg gcc aga
gga ggc 5088Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala Arg
Gly Gly 1685 1690 1695agg gct gcc ata tgt ggc aag tac ctc ttc aac
tgg gca gta aga aca 5136Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe Asn
Trp Ala Val Arg Thr 1700 1705 1710aag ctc aaa ctc act cca ata gcg
gcc gct ggc cag ctg gac ttg tcc 5184Lys Leu Lys Leu Thr Pro Ile Ala
Ala Ala Gly Gln Leu Asp Leu Ser 1715 1720 1725ggc tgg ttc acg gct
ggc tac agc ggg gga gac att tat cac agc gtg 5232Gly Trp Phe Thr Ala
Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val 1730 1735 1740tct cat
gcc cgg ccc cgc tgg atc tgg ttt tgc cta ctc ctg ctt gct 5280Ser His
Ala Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala1745 1750
1755 1760gca ggg gta ggc atc tac ctc ctc ccc aac cga atg agc acg
aat cct 5328Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg Met Ser Thr
Asn Pro 1765 1770 1775aaa cct caa aga aag acc aaa cgt aac acc aac
cgg cgg ccg cag gac 5376Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
Arg Arg Pro Gln Asp 1780 1785 1790gtc aag ttc ccg ggt ggc ggt cag
atc gtt ggt gga gtt tac ttg ttg 5424Val Lys Phe Pro Gly Gly Gly Gln
Ile Val Gly Gly Val Tyr Leu Leu 1795 1800 1805ccg cgc agg ggc cct
aga ttg ggt gtg cgc gcg acg aga aag act tcc 5472Pro Arg Arg Gly Pro
Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser 1810 1815 1820gag cgg
tcg caa cct cga ggt aga cgt cag cct atc ccc aag gct cgt 5520Glu Arg
Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg1825 1830
1835 1840cgg ccc gag ggc agg acc tgg gct cag ccc ggg tac cct tgg
ccc ctc 5568Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp
Pro Leu 1845 1850 1855tat ggc aat gag ggc tgc ggg tgg gcg gga tgg
ctc ctg tct ccc cgt 5616Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp
Leu Leu Ser Pro Arg 1860 1865 1870ggc tct cgg cct agc tgg ggc ccc
aca gac ccc cgg cgt agg tcg cgc 5664Gly Ser Arg Pro Ser Trp Gly Pro
Thr Asp Pro Arg Arg Arg Ser Arg 1875 1880 1885aat ttg ggt aag
5676Asn Leu Gly Lys 189081892PRTArtificial SequenceDescription of
Artificial Sequence representative NS345Core fusion protein 8Met
Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro 1 5 10
15Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His
20 25 30Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr
Gly 35 40 45Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp
Gly Gly 50 55 60Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu
Cys His Ser 65 70 75 80Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr
Val Leu Asp Gln Ala 85
90 95Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro
Pro 100 105 110Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val
Ala Leu Ser 115 120 125Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala
Ile Pro Leu Glu Val 130 135 140Ile Lys Gly Gly Arg His Leu Ile Phe
Cys His Ser Lys Lys Lys Cys145 150 155 160Asp Glu Leu Ala Ala Lys
Leu Val Ala Leu Gly Ile Asn Ala Val Ala 165 170 175Tyr Tyr Arg Gly
Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val 180 185 190Val Val
Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe 195 200
205Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe
210 215 220Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro
Gln Asp225 230 235 240Ala Val Ser Arg Thr Gln Arg Arg Gly Arg Thr
Gly Arg Gly Lys Pro 245 250 255Gly Ile Tyr Arg Phe Val Ala Pro Gly
Glu Arg Pro Ser Gly Met Phe 260 265 270Asp Ser Ser Val Leu Cys Glu
Cys Tyr Asp Ala Gly Cys Ala Trp Tyr 275 280 285Glu Leu Thr Pro Ala
Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn 290 295 300Thr Pro Gly
Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly305 310 315
320Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr
325 330 335Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln
Ala Thr 340 345 350Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp
Asp Gln Met Trp 355 360 365Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu
His Gly Pro Thr Pro Leu 370 375 380Leu Tyr Arg Leu Gly Ala Val Gln
Asn Glu Ile Thr Leu Thr His Pro385 390 395 400Val Thr Lys Tyr Ile
Met Thr Cys Met Ser Ala Asp Leu Glu Val Val 405 410 415Thr Ser Thr
Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala 420 425 430Tyr
Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu 435 440
445Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu
450 455 460Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile
Glu Gln465 470 475 480Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys
Ala Leu Gly Leu Leu 485 490 495Gln Thr Ala Ser Arg Gln Ala Glu Val
Ile Ala Pro Ala Val Gln Thr 500 505 510Asn Trp Gln Lys Leu Glu Thr
Phe Trp Ala Lys His Met Trp Asn Phe 515 520 525Ile Ser Gly Ile Gln
Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn 530 535 540Pro Ala Ile
Ala Ser Leu Met Ala Phe Thr Ala Ala Val Thr Ser Pro545 550 555
560Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val
565 570 575Ala Ala Gln Leu Ala Ala Pro Gly Ala Ala Thr Ala Phe Val
Gly Ala 580 585 590Gly Leu Ala Gly Ala Ala Ile Gly Ser Val Gly Leu
Gly Lys Val Leu 595 600 605Ile Asp Ile Leu Ala Gly Tyr Gly Ala Gly
Val Ala Gly Ala Leu Val 610 615 620Ala Phe Lys Ile Met Ser Gly Glu
Val Pro Ser Thr Glu Asp Leu Val625 630 635 640Asn Leu Leu Pro Ala
Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val 645 650 655Val Cys Ala
Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 660 665 670Val
Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His 675 680
685Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val
690 695 700Thr Ala Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg
Arg Leu705 710 715 720His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro
Cys Ser Gly Ser Trp 725 730 735Leu Arg Asp Ile Trp Asp Trp Ile Cys
Glu Val Leu Ser Asp Phe Lys 740 745 750Thr Trp Leu Lys Ala Lys Leu
Met Pro Gln Leu Pro Gly Ile Pro Phe 755 760 765Val Ser Cys Gln Arg
Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile 770 775 780Met His Thr
Arg Cys His Cys Gly Ala Glu Ile Thr Gly His Val Lys785 790 795
800Asn Gly Thr Met Arg Ile Val Gly Pro Arg Thr Cys Arg Asn Met Trp
805 810 815Ser Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys
Thr Pro 820 825 830Leu Pro Ala Pro Asn Tyr Thr Phe Ala Leu Trp Arg
Val Ser Ala Glu 835 840 845Glu Tyr Val Glu Ile Arg Gln Val Gly Asp
Phe His Tyr Val Thr Gly 850 855 860Met Thr Thr Asp Asn Leu Lys Cys
Pro Cys Gln Val Pro Ser Pro Glu865 870 875 880Phe Phe Thr Glu Leu
Asp Gly Val Arg Leu His Arg Phe Ala Pro Pro 885 890 895Cys Lys Pro
Leu Leu Arg Glu Glu Val Ser Phe Arg Val Gly Leu His 900 905 910Glu
Tyr Pro Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro Asp Val 915 920
925Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu
930 935 940Ala Ala Gly Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Val
Ala Ser945 950 955 960Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu
Lys Ala Thr Cys Thr 965 970 975Ala Asn His Asp Ser Pro Asp Ala Glu
Leu Ile Glu Ala Asn Leu Leu 980 985 990Trp Arg Gln Glu Met Gly Gly
Asn Ile Thr Arg Val Glu Ser Glu Asn 995 1000 1005Lys Val Val Ile
Leu Asp Ser Phe Asp Pro Leu Val Ala Glu Glu Asp 1010 1015 1020Glu
Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Arg Arg1025
1030 1035 1040Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr
Asn Pro Pro 1045 1050 1055Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr
Glu Pro Pro Val Val His 1060 1065 1070Gly Cys Pro Leu Pro Pro Pro
Lys Ser Pro Pro Val Pro Pro Pro Arg 1075 1080 1085Lys Lys Arg Thr
Val Val Leu Thr Glu Ser Thr Leu Ser Thr Ala Leu 1090 1095 1100Ala
Glu Leu Ala Thr Arg Ser Phe Gly Ser Ser Ser Thr Ser Gly Ile1105
1110 1115 1120Thr Gly Asp Asn Thr Thr Thr Ser Ser Glu Pro Ala Pro
Ser Gly Cys 1125 1130 1135Pro Pro Asp Ser Asp Ala Glu Ser Tyr Ser
Ser Met Pro Pro Leu Glu 1140 1145 1150Gly Glu Pro Gly Asp Pro Asp
Leu Ser Asp Gly Ser Trp Ser Thr Val 1155 1160 1165Ser Ser Glu Ala
Asn Ala Glu Asp Val Val Cys Cys Ser Met Ser Tyr 1170 1175 1180Ser
Trp Thr Gly Ala Leu Val Thr Pro Cys Ala Ala Glu Glu Gln Lys1185
1190 1195 1200Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His
His Asn Leu 1205 1210 1215Val Tyr Ser Thr Thr Ser Arg Ser Ala Cys
Gln Arg Gln Lys Lys Val 1220 1225 1230Thr Phe Asp Arg Leu Gln Val
Leu Asp Ser His Tyr Gln Asp Val Leu 1235 1240 1245Lys Glu Val Lys
Ala Ala Ala Ser Lys Val Lys Ala Asn Leu Leu Ser 1250 1255 1260Val
Glu Glu Ala Cys Ser Leu Thr Pro Pro His Ser Ala Lys Ser Lys1265
1270 1275 1280Phe Gly Tyr Gly Ala Lys Asp Val Arg Cys His Ala Arg
Lys Ala Val 1285 1290 1295Thr His Ile Asn Ser Val Trp Lys Asp Leu
Leu Glu Asp Asn Val Thr 1300 1305 1310Pro Ile Asp Thr Thr Ile Met
Ala Lys Asn Glu Val Phe Cys Val Gln 1315 1320 1325Pro Glu Lys Gly
Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp 1330 1335 1340Leu
Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Thr1345
1350 1355 1360Lys Leu Pro Leu Ala Val Met Gly Ser Ser Tyr Gly Phe
Gln Tyr Ser 1365 1370 1375Pro Gly Gln Arg Val Glu Phe Leu Val Gln
Ala Trp Lys Ser Lys Lys 1380 1385 1390Thr Pro Met Gly Phe Ser Tyr
Asp Thr Arg Cys Phe Asp Ser Thr Val 1395 1400 1405Thr Glu Ser Asp
Ile Arg Thr Glu Glu Ala Ile Tyr Gln Cys Cys Asp 1410 1415 1420Leu
Asp Pro Gln Ala Arg Val Ala Ile Lys Ser Leu Thr Glu Arg Leu1425
1430 1435 1440Tyr Val Gly Gly Pro Leu Thr Asn Ser Arg Gly Glu Asn
Cys Gly Tyr 1445 1450 1455Arg Arg Cys Arg Ala Ser Gly Val Leu Thr
Thr Ser Cys Gly Asn Thr 1460 1465 1470Leu Thr Cys Tyr Ile Lys Ala
Arg Ala Ala Cys Arg Ala Ala Gly Leu 1475 1480 1485Gln Asp Cys Thr
Met Leu Val Cys Gly Asp Asp Leu Val Val Ile Cys 1490 1495 1500Glu
Ser Ala Gly Val Gln Glu Asp Ala Ala Ser Leu Arg Ala Phe Thr1505
1510 1515 1520Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro
Pro Gln Pro 1525 1530 1535Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys
Ser Ser Asn Val Ser Val 1540 1545 1550Ala His Asp Gly Ala Gly Lys
Arg Val Tyr Tyr Leu Thr Arg Asp Pro 1555 1560 1565Thr Thr Pro Leu
Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro 1570 1575 1580Val
Asn Ser Trp Leu Gly Asn Ile Ile Met Phe Ala Pro Thr Leu Trp1585
1590 1595 1600Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Val Leu
Ile Ala Arg 1605 1610 1615Asp Gln Leu Glu Gln Ala Leu Asp Cys Glu
Ile Tyr Gly Ala Cys Tyr 1620 1625 1630Ser Ile Glu Pro Leu Asp Leu
Pro Pro Ile Ile Gln Arg Leu His Gly 1635 1640 1645Leu Ser Ala Phe
Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg 1650 1655 1660Val
Ala Ala Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Ala Trp1665
1670 1675 1680Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ala
Arg Gly Gly 1685 1690 1695Arg Ala Ala Ile Cys Gly Lys Tyr Leu Phe
Asn Trp Ala Val Arg Thr 1700 1705 1710Lys Leu Lys Leu Thr Pro Ile
Ala Ala Ala Gly Gln Leu Asp Leu Ser 1715 1720 1725Gly Trp Phe Thr
Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Val 1730 1735 1740Ser
His Ala Arg Pro Arg Trp Ile Trp Phe Cys Leu Leu Leu Leu Ala1745
1750 1755 1760Ala Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg Met Ser
Thr Asn Pro 1765 1770 1775Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr
Asn Arg Arg Pro Gln Asp 1780 1785 1790Val Lys Phe Pro Gly Gly Gly
Gln Ile Val Gly Gly Val Tyr Leu Leu 1795 1800 1805Pro Arg Arg Gly
Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser 1810 1815 1820Glu
Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg1825
1830 1835 1840Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro
Trp Pro Leu 1845 1850 1855Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly
Trp Leu Leu Ser Pro Arg 1860 1865 1870Gly Ser Arg Pro Ser Trp Gly
Pro Thr Asp Pro Arg Arg Arg Ser Arg 1875 1880 1885Asn Leu Gly Lys
18909546DNAArtificial SequenceDescription of Artificial Sequence
representative native NS3 protease domain 9atg gcg ccc atc acg gcg
tac gcc cag cag aca agg ggc ctc cta ggg 48Met Ala Pro Ile Thr Ala
Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 15tgc ata atc acc
agc cta act ggc cgg gac aaa aac caa gtg gag ggt 96Cys Ile Ile Thr
Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly 20 25 30gag gtc cag
att gtg tca act gct gcc caa acc ttc ctg gca acg tgc 144Glu Val Gln
Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys 35 40 45atc aat
ggg gtg tgc tgg act gtc tac cac ggg gcc gga acg agg acc 192Ile Asn
Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr 50 55 60atc
gcg tca ccc aag ggt cct gtc atc cag atg tat acc aat gta gac 240Ile
Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn Val Asp 65 70
75 80caa gac ctt gtg ggc tgg ccc gct ccg caa ggt agc cga tca ttg
aca 288Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu
Thr 85 90 95ccc tgc act tgc ggc tcc tcg gac ctt tac ctg gtc acg agg
cac gcc 336Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg
His Ala 100 105 110gat gtc att ccc gtg cgc cgg cgg ggt gat agc agg
ggc agc ctg ctg 384Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg
Gly Ser Leu Leu 115 120 125tcg ccc cgg ccc att tcc tac ttg aaa ggc
tcc tcg ggg ggt ccg ctg 432Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly
Ser Ser Gly Gly Pro Leu 130 135 140ttg tgc ccc gcg ggg cac gcc gtg
ggc ata ttt agg gcc gcg gtg tgc 480Leu Cys Pro Ala Gly His Ala Val
Gly Ile Phe Arg Ala Ala Val Cys145 150 155 160acc cgt gga gtg gct
aag gcg gtg gac ttt atc cct gtg gag aac cta 528Thr Arg Gly Val Ala
Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu 165 170 175gag aca acc
atg agg tcc 546Glu Thr Thr Met Arg Ser 18010182PRTArtificial
SequenceDescription of Artificial Sequence representative native
NS3 protease domain 10Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr
Arg Gly Leu Leu Gly 1 5 10 15Cys Ile Ile Thr Ser Leu Thr Gly Arg
Asp Lys Asn Gln Val Glu Gly 20 25 30Glu Val Gln Ile Val Ser Thr Ala
Ala Gln Thr Phe Leu Ala Thr Cys 35 40 45Ile Asn Gly Val Cys Trp Thr
Val Tyr His Gly Ala Gly Thr Arg Thr 50 55 60Ile Ala Ser Pro Lys Gly
Pro Val Ile Gln Met Tyr Thr Asn Val Asp 65 70 75 80Gln Asp Leu Val
Gly Trp Pro Ala Pro Gln Gly Ser Arg Ser Leu Thr 85 90 95Pro Cys Thr
Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala 100 105 110Asp
Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu 115 120
125Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu
130 135 140Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala
Val Cys145 150 155 160Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile
Pro Val Glu Asn Leu 165 170 175Glu Thr Thr Met Arg Ser 180
* * * * *
References