Note: Descriptions are shown in the official language in which they were submitted.
'V'94127153 PCTIUS94/05151
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216 2 2 5
METHODS OF TYPING HEPATITIS C VIRUS
AND REAGENTS FOR USE THEREIN
Description
Technical Field
This invention relates to typing hepatitis C viruses (HCV). In
particular, this invention relates to a method of typing HCV using novel type-
dependent peptides.
Back rg o, und
Viral hepatitis is known to be caused by five different viruses
known as hepatitis A,B,C, D and E. HAV is an RNA virus and does not lead
to long-term clinical symptoms. HBV is a DNA virus. HDV is a dependent
virus that is unable to infect cells in the absence of HBV. HEV is a water-
borne virus. HCV was first identified and characterized as a cause of non-A.
non-B hepatitis (NANBH). Houghton et al., EPO Pub. No. 388,232. This
led to the disclosure of a number of general and specific polypeptides useful
2 5 as immunological reagents in identifying HCV. See, e. g. , Choo et al.
(1989)
Science, 244:359-362; Kuo et al. (1989) Science, 244:362-364; and Houghton
et al. (1991) Hepatology, 14:381-388. HCV is the major cause of blood
transfusion-related hepatitis.
The prototype isolate of HCV was characterized EP Publication
Nos. 318,216 and 388,232. As used herein, the term "HCV" includes newly
isolated NANBH viral species. The term "HCV-1" refers to the virus
described in the above-mentioned publications.
Since the initial identification of HCV, at least six different
viral types have been identified and designated HCV-1 to HCV-6.. Cha et al.
(1992) Proc. Natl. Acad. Sci. USA, 89:7144-7148. Within these types are
numerous subtypes. The type of virus with which a patient is infected may
affect the clinical prognosis and also response to various treatments.
2162250
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Yoshioka et al. (1992) Hepatology, 16:293-299. In light of the fact that the
most serious clinical outcome of HCV infection is hepatocellular carcinoma, it
would
be useful to be able to determine with which type or types of HCV a patient is
infected.
The method currently in use to determine virus type is genotyping; that is,
isolation of viral RNA and determination of the sequence of various segments
by
polymerase chain reaction (PCR). Not only is this method laborious and time
consuming but it is not suitable for use on samples that have been stored
under
conditions that do not allow for preservation of RNA or samples from patients
that do
not have sufficient viral titer. It would be useful to have a method for
typing HCV by
immunoanalysis or serotyping.
The current method for screening blood and diagnosing patients is an
immunoassay. The immunoassay utilizes an antigen from HCV-1 which contains a
sufficient number of common epitopes to detect antibodies to other types of
HCV.
The immunoassay does not distinguish between infections by different types of
HCV.
Disclosure of the Invention 20 The present invention includes compositions and
methods for typing of HCVs
by genotype and serotype. The compositions include type specific epitopes,
type-
cluster specific epitopes nucleic acids encoding the epitopes for use as
probes and
nucleic acids complementary to the regions flanking those encoding the
epitopes for
use as primers.
According to an aspect of the invention, there is provided a method for
detecting one or more types of a hepatitis C virus comprises the steps of:
(a) providing a biological sample from an individual suspected of being
infected by a hepatitis C virus;
(b) contacting the sample with a first reagent comprising hepatitis C virus
epitopes, said epitopes consisting essentially of a combination of type
specific
epitopes specific for a first type of hepatitis C virus, wherein said epitopes
are
selected from the core, NS4 or NS5 regions of the hepatitis C virus and
contacting is
carried out under conditions which allow the formation of first epitope-
antibody
complexes; and
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(c) assaying :for the presence of the first epitope-antibody complexes in the
sample.
According to another aspect of the invention, there is provided a method for
detecting one or more types of a hepatitis C virus comprises the steps of:
(a) providing a biological sample suspected of containing HCV antigens;
(b) contacting the sample with a first reagent comprising a combination of
antibodies specific for at least two different type specific epitopes selected
from the
core, NS4, or NS5 region of a first type of hepatitis C virus, wherein the
contacting is
carried out under conditions which allow the formation of first antigen-
antibody
complexes; and
(c) assaying for the presence at the first antigen-antibody complexes in the
sample.
According to another aspect of the invention, there is provided a polypeptide
having an amino acid sequence corresponding to a type specific epitope within
the
core region of the hepatitis C virus, wherein the epitope is derived from the
amino
acid sequence spanning amino acids 67 to 84 of hepatitis C virus-1 or a
homologous
region thereof from other hepatitis C virus types.
According to another aspect of the invention, there is provided a polypeptide
having an amino acid sequence corresponding to a type specific epitope within
the
NS4 region of the hepatitis C virus, wherein the epitope is derived from the
amino
acid sequence spanning amino acids 1689 to 1718 of hepatitis C virus-1 or a
homologous region thereof from other hepatitis C virus types.
According to another aspect of the invention, there is provided nucleic acid
molecule comprises a nucleotide sequence encoding by a hepatitis C virus
genome
corresponding to amino acid residues 67 to 84 of hepatitis C virus-1 or a
homologous
region thereof obtained from other hepatitis C virus types.
According to another aspect of the invention, there is provided a nucleic acid
molecule comprises a nucleotide sequence encoding by a hepatitis C virus
genome
corresponding to amino acid residues 1689 to 1718 of hepatitis C virus-1 or a
homologous region thereof obtained from other hepatitis C virus types.
7
CA 02162250 2003-09-16
2b
According to a further aspect of the invention, there is provided a reagent
useful for typing
one or more types of a hepatitis C virus, comprising a combination of type
specific epitopes from
the hepatitis C virus, wherein the combination includes at least one epitope
from the NS4 region
of the hepatitis C virus.
One aspect of the invention is a method for typing HCV comprising the steps of
providing an antibody-containing sample from an individual; contacting the
sample with a type
specific epitope or type-cluster specific epitopes under conditions which
permit antigen-antibody
binding; and determining whether antibodies in the sample bind the epitope.
According to one aspect of the invention, there is provided a method of typing
a hepatitis
C virus comprising the steps of:
(a) providing a biological sample from an individual suspected of being
infected by a
hepatitis C virus;
(b) contacting the sample with a first reagent comprising a combination of
polypeptides containing type-specific epitopes specific for a first type of
hepatitis C virus
wherein the epitopes are from core, NS4 or NS5 regions of the hepatitis C
virus and at least one
epitope is derived from the amino acid sequences found between amino acid
residues 67 and 84,
amino acids residues 1689 and 1718, amino acid residues 2281 and 2313, said
amino acid
residues numbered relative to a mature HCV-1 polyprotein, or epitope
corresponding to these
regions of other hepatitis C virus types and wherein said epitopes are from 5
to 15 amino acids in
length; and
(c) assaying for the presence of first epitope-antibody complexes in the
sample.
According to a further aspect of the invention, there is provided a method for
detecting
one or more types of hepatitis C virus comprising the steps of:
(a) providing a biological sample suspected of containing HCV antigens;
(b) contacting the sample with a first reagent comprising a combination of
antibodies
specific epitopes selected from the core, NS4, or NS5 region of a first type
of hepatitis C virus,
wherein at least one epitope is derived from the amino acid sequences found
between amino acid
residues 67 and 84, amino acids residues 1689 and 1718, amino acid residues
2281 and 2313 and
wherein said epitopes are 5 to 15 amino acids in length, under conditions
which allow the
formation of first antigen-antibody complexes; and
(c) assaying for the presence of said first antigen-antibody complexes in the
sample.
CA 02162250 2005-01-17
- 2c -
According to a further aspect of the invention, there is provided a
polypeptide having an
amino acid sequence corresponding to a type specific epitope within NS4 region
of the hepatitis
C virus, wherein said epitope is derived from the amino acid sequence spanning
amino acids
1689 to 1718 of hepatitis C virus-1 or a homologous region thereof from other
hepatitis C virus
types, and consists of a sequence selected from the group consisting of
CASHPLY, CASRAAL,
CASKAAL, ASRAAL, and ASKAAL.
According to a further aspect of the invention, there is provided a reagent
comprising a
combination of polypeptides containing type-specific epitopes wherein the
epitopes are selected
from epitopes located between amino acid residues 67 and 84, 1689 and 1718,
and 2281 and 2313
of HCV-1 or epitopes from the region corresponding to this region in other
hepatitis C virus
types, and the epitopes are from 5 to 15 amino acids in length.
Another aspect of the invention relates to a method of typing HCV comprising
the steps
of providing an antibody-containing sample from an individual; contacting the
sample with a first
type specific epitope or type-cluster specific epitope under conditions which
permit antigen-
antibody binding; contacting the sample with a second type specific epitope or
type-
Wr94/27153 PCTIUS94/05151
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cluster specific epitope under conditions which permit antigen-antibody
binding; and determining whether antibodies in the sample bind to either the
' first or second epitope.
Another aspect of the invention relates to polypeptides
containing type specific epitopes or type-cluster specific epitopes. The
polypeptides are derived from three different regions of the HCV genome.
One set of polypeptides includes a type specific epitope or type-cluster
specific'
1O epitopes obtained from the HCV core region. This first set is found between
amino acid residues sixty-seven and eighty-four of HCV-1 and homologous
regions of other types of HCV. As used herein, the amino acid residue
abbreviaitons are as follows: A, alanine; I, isoleucine; L, leucine; M.
atn.ethionine; F, phenylalanine; P, proline; W, tryptophan; V, valine; N,
asparagine; C, cysteine; Q, glutamine;; G, glycine; S, serine; T, threonine;
Y, tyrosine; R, arginine; H, histidine; K, lysine; D, aspartic acid; and E,
glutamic acid.
The particular amino acid residue sequences derived from the
core region and subtypes from which they are derived are as follows:
1. PEGRTWAQ, subtype la or lb.
2. STGKSWGK, subtype 2a or 2b.
3. SEGRSWAQ, subtype 3a or 4.
Another set of polypeptides includes a type specific epitope
obtained from the HCV non-structural region 4 (NS4). This second set is
found between amino acid residues 1689-1718 of HCV-1 and homologous
regions of other types of HCV.
The particular amino acid residue sequences and types or
subtypes from which they are derived are as follows:
1. CSQHLPY, subtype la.
2. CASHLPY, subtype lb.
3. CASRAAL, subtype 2a or 2b.
Another set of polypeptides includes a type specifc.epitope or
type-cluster specific epitopes obtained from the non-structural region 5 (NS5)
of a hepatitis C virus. This set is found between amino acid residues 2281-
2313 of HCV-1 and homologous regions of other types of hepatitis C vinis.
VF,,k,94127153 PCT1US94105151
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The particular amino acid residue sequences and types or
subtypes from which they are derived are as follows:
1. PDYEPPVVHG, subtypes 1 a.
2. PDYVPPVVHG, subtype lb.
3. PDYQPATVAG, subtype 2a.
4. PGYEPPTVLG, subtype 2b.
5. FAQASPVW, subtype Ia.
6. FPPQALPIW, subtype lb.
7. FPQALPAW, subtype 2a.
8. FPPQALPPW, subtype 2b.
Another aspect of the invention includes nucleic acid molecules
encoding the amino acid residue sequences of the type specific and type-
cluster epitopes described. These nucleic acid molecules are useful as probes
for instance in Southern blots or other DNA recognition assays such as the
capture assay described in US Patent Nos. 4,868;105; and 5,124,246.
Another aspect of the invention includes nucleic acid molecules
complementary to the nucleic acid sequences flanking regions encoding the
type specific and type-cluster specific epitopes. Such nucleic acid molecules
are useful in performing PCR to determine the genotype of a particular HCV.
Brief Description of the Drawings
Figure I is a flow diagram of the serotyping experimental strategy.
Figure 2 is a flow diagram of the comprehensive epitope mapping strategy.
Figure 3 is a compilation of graphs depicting the results of epitope mapping
of
HCV la (Rodney).
Figure 4 is a compilation of graphs depicting the results of epitope inappins!
of
HCV 2b (Nomoto).
1~~94/27153 PCT/US94/05151
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Definitions
"Hepatitis C virus" or "HCV" refers to the viral species of
which pathogenic types cause NANBH, and attenuated types or defective
interfering particles derived therefrom. See generally, publications cited in
the section entitled "Background." The'HCV genome is comprised of RNA.
RNA containing viruses have relatively high rates of spontaneous mutation
reportedly on the order of 1Y3 to 104 per incorporated nucleotide.
Fields & Knipe (1986) "Fundamental Virology" (Raven Press, NY). Since
heterogeneity and fluidity of genotype are inherent in RNA viruses, there are
multiple types/subtypes, within the HCV species which may be virulent or
avirulent. The propagation, identification, detection, and isolation of
various
HCV types or isolates is documented in the literature. As depicted herein, all
nucleotide and amino acid residue sequences are from the HCV types noted.
the number of the HCV=-1 genome and amino acid residues sequences is as
descibed in Choo et al. (1990) Brit. Med. Bull., 46:423-441. The disclosure
herein allows the diagnosis of the various types.
As used herein, "type" refers to HCVs that differ genotypically
by more than about 30%; "subtype" refers to HCVs that differ genotypically
by about 10-20% and "isolate" refers to HCVs that differ genotypically by
about less than 10%. "'Typing" refers to distinguishing one type of HCV
from another type.
Information on several different HCV types/ subtypes is
disclosed in International Publication No. WO 93/00365 particularly type or
subtype CDC/HCV 1(also called HCV- l). Information from one type or
subtype, such as a partial genomic or amino acid sequence, is sufficient to
allow those skilled in the art using standard techniques to isolate new types
of
HCV. For example, several different types of HCV were screened as
described below. These types, which were obtained from a number of human
~ sera (and from different geographical areas), were typed utilizing the
method
and reagents described herein.
The genomic structure and the nucleotide sequence of HCV-1
genomic RNA has been deduced. The genome appears to be single-stranded
RNA containinc, 10.000 nucleotides. The genome is positive-stranded. and
V~"",94/27153 PCTNS94l05151
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possesses a continuous, translational open reading frame (ORF) that encodes a
polyprotein of about 3,000 amino acids. In the ORF, the structural protein(s)
appear to be encoded in approximately the first quarter of the amino-terminus
=
region, with the majority of the polyprotein responsible for non-structural
(NS) proteins. When compared with all known viral sequences, small but
significant co-linear homologies are observed with the non-structural (NS)
proteins of the flavivirus family, and with the pestiviruses (which are now
also
considered to be part of the Flavivirus family).
Based upon the putative amino acid residues encoded in the
nucleotide sequence of HCV-1 and other evidence, possible protein domains
of the encoded HCV polyprotein, as well as the approximate boundaries, are
presented in Table 1.
Table I
Putative Domain Approximate Boundarv
(amino acid nos.)
C(nucleocapsid protein) 1-191
E, (virion envelope protein) 192-383
E,/NS 1 (envelope) 384-800
NS2 (unknown function) 800-1050
NS3 (protease) 1050-1650
NS4 (unknown function) 1651-2100
NS5 (polymerase) 2100-3011(end)
These dornains are tentative. For example, the E1-NS2 border
is probably in the 750-810 region, and NS3-NS4 border is about 1640-1650.
There is also evidence that the 191 amino acid (aa) version of C is a
precursor
that is further processed to about 170 aa in length, and that the NS2. NS4 and
NS5 proteins are each further processed into two mature proteins. =
Different types of HCV are defined according to various criteria
sucli as, for example, an ORF of approximately 9,000 nucleotides to
approximately 12,000 nucleotides, encoding a polyprotein similar in size to
that of HCV-l. an encoded polyprotein of similar hydrophobic and/or
W,A94127153 PCT/13S94/05151
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antigenic character to that of HCV-1, and the presence of co-linear
polypeptide sequences that are conserved with HCV-1.
= The following parameters of nucleic acid homology and amino
acid homology are applicable, either alone or in combination, in identifying
HCV types. Generally, as described above, different types of HCV are about
70% homologous whereas subtypes are about 80-90% homologous and isolates
are about 90% homologous.
As used herein, a polynucleotide "derived from" a designated
sequence refers to a polynucleotide sequence which is comprised of a
sequence of at least about 6 nucleotides, preferably at least about 8
nucleotides, more preferably at least about 10-12 nucleotides, and even more
preferably at least about 15-20 nucleotides corresponding to a region of the
.
designated nucleotide sequence. "Corresponding" means homologous to or
complementary to the designated sequence. Preferably, the sequence of the
region from which the polynucleotide is derived is homologous to or
complementary to a sequence which is unique to an HCV genome.
Hybridization techniques for determining the complementarity of nucleic acid
sequences are known in the art. See, for example, Maniatis et al. (1982). In
addition, mismatches of duplex polynucleotides formed by hybridization can
be determined by known techniques, including for example, digestion with a
nuclease such as Sl that specifically digests single-stranded areas in duplex
polynucleotides. Regions from which typical DNA sequences may be
"derived" include but are not limited to, for example, regions encoding type
specific epitopes, as well as non-transcribed and/or non-translated regions.
The derived polynucleotide is not necessarily physically derived
form the nucleotide sequence shown, but may be generated .in any manner,
including for example, chemical synthesis or DNA replication or reverse
transcription or transcription. In addition, combinations of regions
. corresponding to that of the designated sequence may be modified in ways
known in the art to be consistent with an intended use.
Similarly, a polypeptide or amino acid sequence "derived froni"
a designated amino acid or nucleic acid sequence refers to a polypeptide
having an amino acid sequence identical to that of a polypeptide encoded in
.~.
V'~14/27153 PCTlUS94/05151
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216225 0
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the sequence, or a portion thereof wherein the portion consists of at least 3-
5
amino acids, and more preferably at least 8-10 amino acids, and even more
preferably at least 11-15 amino acids, or which is immunologically
identifiable
with a polypeptide encoded in the sequence. This terminology also includes a
polypeptide expressed from a designated nucleic acid sequence.
A recombinant or derived polypeptide is not necessarily
translated from a designated nucleic acid sequence; it may be generated in any
manner, including for example, chemical synthesis, or expression of a
recombinant expression system, or isolation from HCV, including mutated
HCV. The polypeptides described herein are generally relatively short and
are thus most easily chemically synthesized.
A recombinant or derived polypeptide may include one or inore
analogs of amino acids or unnatural amino acids in its sequence. Methods of
inserting analogs of amirio acids into a sequence are known in the art. It
also
may include one or more labels, which are known to those of skill in the art.
A detailed description of analogs and "mimotopes" is found in U.S. Patent
No. 5,194,392.
Peptide analogs include deletions, additions, substitutions or
modifications thereof which retain the HCV typing capability. Preferred
"substitutions" are those which are conservative, i.e., wherein a residue is
replaced by another of the same general type. As is well understood,
naturally-occurring amino acids can be subclassified as acidic, basic, neutral
and polar, or neutral and nonpolar. Furthermore, three of the encoded amino
acids are aromatic. It is generally preferred that encoded polypeptides
differing from the natural epitope contain substituted codons for amino acids
which are from the same group as that of the amino acid replaced. Thus, in
general, the basic amino acids Lys, Arg, and His are interchangeable; the
acidic amino acids aspartic and glutamic are interchangeable; the neutral
polar
amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; the nonpolar
aliphatic amino acids Gly, Ala, Val, Ile, and Leu are conservative.with
respect to each other (but because of size, Gly and Ala are more closely
related and Val, Ile and Leu are more closely related), and the aromatic amino
acids Phe. Trp. and Tyr are interchangeable. While proline is a nonpolar
~ 94/27153 PCTIUS94/05151
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216226A
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neutral amino acid, it represents difficulties because of its effects on
conformation, and substitutions by or for proline are not preferred, except
when the same or similar conformational results can be obtained. Polar amino
acids which represent conservative changes include Ser, Thr, Gin, Asn; and to
a lesser extent, Met. In addition, although classified in different
categories,
Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into
this group, or may be classified with the polar neutral amino acids.
It should further be noted that if the polypeptides are made
synthetically, substitutions by amino acids which are not encoded by the gene
may also be made. Alternative residues include, for example, the omega
amino acids of the formula HZN(CHZ),COOH wherein n is 2-6. These are
neutral, nonpolar amino acids, as are sarcosine (Sar), t-butyl alanine (t-
BuA),
t-butyl glycine (t-BuG), N-methyl Ile (N-MeIle), and norleucine (Nie).
Phenyl glycine, for example, can be substituted for Trp, Tyr or Phe an
aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO)
are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar,
cysteic
acid (Cya) is acidic, and ornithine (Orn) is basic. The conformation
conferring properties of the proline residues may be retained if one or more
of
these is substituted by hydroxyproline (Hyp).
The term "recombinant polynucleotide" as used herein intends a
polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which,
by virtue of its origin or manipulatiozi: (1) is not associated with all or a
portion of a polynucleotide with which it is associated in nature, (2) is
linked
to a polynucleotide other than that to which it is linked in nature, or (3)
does
not occur in nature.
The term "polynucleotide" as used herein refers to a polymeric
form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. This term refers only to the primary structure of the
= molecule. Thus, this term includes double- and single-stranded DNA and
RNA. It also incudes known types of modifications, for example,. labels
which are known in the art, methylation, "caps", substitution of one or more
naturally occurring nucleotides with an analog. internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
RO" '4/27153 PCT/US94l0{151
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phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with
charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those
containing pendant moieties, such as, for example proteins including but not =
limited to nucleases, toxins, antibodies, signal peptides and poly-L-lysine;
those with intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.),
those containing alkylators, those with modified linkages (e.g., alpha
anomeric
nucleic acids, etc.), as well as unmodified forms of the polynucleotide. The
polynucleotides described herein are relatively short and are thus most easily
chemically synthesized.
A"purifted" polypeptide refers to the polypeptide being in a
state that is substantially free of other polypeptides, i.e., in a composition
that
contains a minimum of about 50% by weight (desired polypeptide/total
polypeptide in composition), preferably a minimum of about 70%, and even
more preferably a minimum of about 90% of the desired polypeptide, without
regard to nonproteinaceous materials in the composition. Techniques for
purifying viral polypeptides are known in the art. Purified antibodies are
similarly defined in the art.
As used herein, "epitope" refers to an antigenic determinant of
a polypeptide. An epitope could comprise 3 or more amino acids that define
the binding site of an antibody. Generally an epitope consists of at least 5
amino acids, and sometimes consists of at least 8 amino acids. Methods of
epitope mapping are known in the art.
As used herein, "type specific epitope" refers to an epitope that
is found on one HCV type. A "type-cluster specific epitope" is found on
more than one but fewer than all HCV types. For instance, a particular
epitope may be recognized by antibodies from a patient infected with HCV 1
but not recognized by or recognized less efficiently by antibodies from a
patient infected with HCV 2. Similarly, a type-cluster specific epitope =
derived from HCV-3 may be recognized by antibodies from a patient infected
wih HCV-3 or HCV-4 but not by antibodies from a patient infected with
HCV-1 or HCV-2. "Conserved epitopes" are those which are recognized by
antibodies specific to all HCV types.
WO 94/27153 PCT/US94/05151
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A polypeptide is "immunologically reactive" with an antibody
which binds to the peptide due to antibody recognition of a specific epitope
contained within the polypeptide. Immunological reactivity may be
determined by antibody binding, more particularly by the kinetics of antibody
binding, and/or by competition in binding using as competitors known
polypeptides containing an epitope against which the antibody is directed. The
techniques for determining whether a polypeptide is immunologically reactive
IQ with an antibody are known in the art.
As used herein, the term "antibody" refers to a polypeptide or
group of polypeptides which are comprised of at least one antibody combining
site. An "antibody combining site" or "binding domain" is formed from the
folding of variable domains of an antibody molecule(s) to form three-
dimensional binding spaces with an internal surface shape and charge
distribution complementary to the features of an epitope of an antigen, which
allows an immunological reaction with the antigen. An antibody combining
site may be formed from a heavy and/or a light chain domain (V,i and VL,
respectively), which form hypervariable loops which contribute to antigen
binding. The term "antibody" includes, for example, vertebrate antibodies,
hybrid antibodies, chimeric antibodies, altered antibodies, univalent
antibodies, the Fab proteins, and single domain antibodies.
Antibodies specific to polypeptides and polyppetides can be
made by any method known in the art. For instance, the polypeptides are
generally suspended in a physiologically acceptable buffer, mixed with a
suitable adjuvant and injected into an animal. Methods of making polyclonal
and monoclonal antibodies are known in the art and will not be described in
detail herein.
The term "polypeptide" refers to a polymer of amino acids and
does not refer to a specific length of the product; thus, polypeptides,
- oligopeptides, and proteins are included within the definition of
polypeptide.
This term also does not refer to or exclude post-expression modifications of
the polypeptide, for example, glycosylation, acetylation, phosphoryiation, and
the like. Included within the definition are, for example, polypeptides
containing one or more analogs of an ainino acid (including, for example.
w; 4n7153 PCT/US94/05151
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unnatural amino acids, etc.), polypeptides with substituted linkages, as weli
as
other modifications known in the art, both naturally occurring and non-
naturally occurring.
"Treatment", as used herein, refers to prophylaxis and/or
therapy.
An "individual", as used herein, refers to vertebrates,
particularly members of the mammalian species, and includes, but is not
limited to, animals (e.g., dogs, cats, cattle, swine, sheep, goat, rabbits,
mice,
rats, guinea pigs, etc.), and primates, including monkeys, chimps, baboons
and humans.
As used herein, the "sense strand" of a nucleic acid contains the
sequence that has sequence homology to that of mRNA. The "anti-sense
strand" contains a sequence which is complementary to that of the "sense
strand".
As used herein, a "positive stranded genome" of a virus is one
in which the genome, whether RNA or DNA, is single-stranded and which
encodes a viral polypeptide(s). Examples of positive stranded RNA viruses
include Togaviridae, Coronaviridae, Retroviridae, Picornaviridae, and
Caliciviridae. Included also, are the Flaviviridae, which were formerly
classified as Togaviradae. See Fields & Knipe (1986).
As used herein, "antibody containing body sample" refers to a
component of an individual's body which is a source of the antibodies of
interest. Antibody containing body components are known in the art, and
include but are not limited to, for example, plasma, serum, spinal fluid.
lymph fluid, the external sections of the respiratory, intestinal, and
genitourinary tracts, tears, saliva, milk, white blood cells, and myelomas.
As used herein, a "biological sample" refers to a sample of
tissue or fluid isolated from an individual, including, but not limited to.
for
example, plasma, serum, spinal fluid, lymph fluid, the external sections of
the
skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk.
biood
cells, tumors, organs. Also included are samples of in vitro cell culture
constituents (including. but not limited to, conditioned medium resulting
froni
----
~
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the growth of cells in c:ulture medium, putatively virally infected cells,
recombinant ceUs, and cell components).
Modes for Ca ing Out the Invention
The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, polypeptide and nucleic acid synthesis, and
immunology, which are within the skiU of the art. Such techniques are
explained fully in the literature. See, e.g., Maniatis, Fitsch & Sambrook,
"Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning,
Volumes I and U" (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J.
Gait ed., 1984); "Nuclei Acid Hybridization" (B.D. Hames & S.J. Higgins
eds. 1984); "Transcription and Translation" (B.D. Hames & S.J. Higgins eds.
1984); "Animal Cell Culture" (R.I. Freshney ed. 1986); "Immobilized Cells
And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To
Molecular Cloning" (1984); the series, "Methods in Enzymology" (Academic
Press, Inc.); "Gene Transfer Vectors For Mammalian Cells" (J.H. Miller and
M.P. Calos eds., 1987, Cold Spring Harbor Laboratory), Meth. Enzymol.,
Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively),
Mayer and Walker, eds. (1987), "Immunochemical Methods In Cell And
Molecular Biology" (Academic Press, London); Scopes, (1987) "Protein
Purification: Principles and Practice", Second Edition (Springer-Verlag,
N.Y.); and "Handbook of Experimental Immunology", Volumes I-IV (D.M.
Weir and C.C. Blackwell eds. 1986).
The invention includes methods for detecting HCV and
identifying infection by different types of HCV. The invention also includes
polypeptides and nucleic acid molecules for use in the methods.
The methods for detecting and typing infection by HCV include
both immunoassays and nucleic acid identification by methods including but
not limited to Southern blot analysis and polymerase chain reaction. In order
to identify infection by HCV, a biological sample is incubated with one of the
~'~'
Wt, :o4/27153 PCT/US94/05151
==
-14- 21622 5
polypeptides described herein under conditions which permit antigen-antibody
binding and a determination is made as to whether antibodies in the sample
bind to the epitope found on the polypeptide.
Immunoassay and Diagnostic Kits
The peptides containing the type specific epitopes and type-
cluster specific epitopes are useful in immunoassays to detect the presence of
HCV antibodies, or the presence of the virus and/or viral antigens, in
biological samples. Design of the immunoassays is subject to a great deal of
variation, and many formats are known in the art. The immunoassay will
utilize at least one type specific epitope or type-cluster specific epitope.
In
one embodiment, the immunoassay uses a combination of type specific
epitopes and/or type-cluster specific epitopes.
The polypeptides are useful for typing HCV by using the
epitopes to determine the presence of type specific or type-cluster specific
antibodies. The polypeptides are also suitable for use in generating type or
type-cluster specific antibodies that can then be used in an immunoassay to
distinguish between various types of HCV.
The polypeptides are derived from three different regions of the
HCV genome. One set of polypeptides includes a type or type-cluster specific
epitope obtained from the HCV core region. Another set of polypeptides
includes a type or type-cluster specific epitope obtained from the HCV non-
structural region 4 (NS4). Another set of polypeptides includes a type or
type-cluster specific epitope obtained from the non-structural region 5 (NS5)
of a hepatitis C virus. This set is found between amino acid residues 2281-
2313 of HCV-l and homologous regions of other types of hepatitis C virus.
The polypeptides are suitable for use in immunoassays for one
or niore HCV types. In order to assay for one type the sample is contacted
with one or more polypeptides containing a type-cluster specific epitope under
conditions which pennit antigen-antibody binding and detennining .whether
antibodies in the sample bind to the epitope.
In an immunoassay to distinguish a particular type of HCV, a
biological sample is obtained from an individual, contacted with a first type
~_
Re""4127153 PCT/US94/05151
-15- 2162'25 0
specific epitope or type-cluster specific epitope under conditions which
permit
antigen-antibody binding; contacted with a second type specific epitope or
type-cluster specific epitope under conditions which permit antigen-antibody
binding and determining whether antibodies in the sample bind to either the
first or second epitope. These steps can be repeated with any number of
polypeptides containing type and/or type-cluster specific epitopes.
Typically, an immunoassay for anti-HCV antibody(s) involves
selecting and preparing the test sample suspected of containing the
antibodies,
such as a biological sample, then incubating it with the type specific epitope
or type-cluster specific epitope under conditions that allow antigen-antibody
complexes to form, and then detecting the formation of such complexes.
Suitable incubation conditions are well known in the art. The immunoassay
may be, without limitations, in a heterogeneous or in a homogeneous format,
and of a standard or competitive type.
In a heterogeneous format, the type specific epitope or type-
cluster specific epitope is typically bound to a solid support to facilitate
separation of the sample from the polypeptide after incubation. Examples of
solid supports that can be used include but are not limited to nitrocellulose
(e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in
sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter
plates, polyvinylidine fluoride (known as Immulon ), diazotized paper, nylon
membranes, activated beads, and Protein A beads. For example, Dynatech
Immulon 1 or Immulon 2 microtiter plates or 0.25 inch polystyrene beads
(Precision Plastic Ball) can be used in the heterogeneous fonnat. The solid
support containing the type specific epitope or type-cluster epitope is
typically
washed after separating it from the test sample, and prior to detection of
bound antibodies. Both standard and competitive formats are known in the
art.
In a homogeneous fonnat, the test sample is incubated with the
type specific epitope or type-cluster specific epitope in solution. For
example.
it may be under conditions that will precipitate any antigen-antibody
complexes which are formed. Both standard and competitive formats for
these assays are known in the an.
VO'"4127153 PCTIUS94/05151
21fi225
-16-
In a standard format, the amount of HCV antibodies forming
the antibody-type or -type-cluster specific epitope complex is directly
monitored. This may be accomplished by determining whether labeled
anti-xenogeneic (e.g., anti-human) antibodies which recognize an epitope on
anti-HCV antibodies will bind due to complex formation. In a competitive
format, the amount of HCV antibodies in the sample is deduced by monitoring
the competitive effect on the binding of a known amount of labeled antibody
(or other competing ligand) in the complex.
In an inhibition assay, the ability of antibodies to bind to
polypeptides containing various different type specific epitopes to type
cluster-
specific epitopes is determined. The antibodies are first exposed to
polypeptides containing epitope(s) from one type or type-cluster of HCV and
then to polypeptides containing epitope(s) from another type or type-cluster
of
HCV. The process may be repeated for additional types or type-clusters of
HCV.
Complexes formed comprising anti-l-:CV antibody (or, in the
case of competitive assays, the amount of competing antibody) are detected by
any of a number of known techniques, depending on the format. For
example, unlabeled HCV antibodies in the complex may be detected using a
conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme
label).
In typical immunoassays, the test sample, typically a biological
sample, is incubated with polypeptides containing one or more type specific
epitopes or type-cluster specific epitopes under conditions that allow the
formation of antigen-antibody complexes. Various formats can be employed.
For example, a "sandwich assay" may be employed, where antibody bound to
a solid support is incubated with the test sample; washed; incubated with a
second, labeled antibody to the analyte. and the support is washed again.
Analyte is detected by determining if the second antibody is bound to the
support. In a competitive format, which can be either heterogeneous or
homogeneous, a test sample is usually incubated with antibody and a labeled.
competing antigen is also incubated. either sequentially or simultaneously.
These and other formats are well loiown in the art.
_- ~.
VV 4/27153 PCT/US94/05151
-
-17 2162250
Antibodies directed against the type specific epitopes or type-
cluster specific epitopes can be used in immunoassays for the detection of
viral antigens in patients with HCV caused NANBH, and in infectious blood
donors. Moreover, these antibodies may be extremely useful in detecting
acute-phase donors and patients.
An immunoassay may use, for example, a monoclonal antibody
directed towards a type specific epitope or type-cluster specific epitopes, a
1 Q combination of monoclonal antibodies directed towards epitopes of one
viral
antigen, monoclonal antibodies directed towards epitopes of different viral
antigens, polyclonal antibodies directed towards the same viral antigen, or
polyclonal antibodies directed towards different viral antigens. Protocols may
be based, for example, upon competition, or direct reaction, or sandwich type
assays. Protocols may also, for example, use solid supports, or may be by
immunoprecipitation. Most assays involve the use of labeled antibody or
polypeptide; the labels may be, but are not limited to enzymatic, fluorescent,
chemiluminescent, radioactive, or dye molecules. Assays which amplify the
signals from the probe are also known; examples of which are assays which
utilize biotin and avidin, and enzyme-labeled and mediated immunoassays,
such as EI.TSA assays.
The invention further includes nucleic acid molecules encoding
the amino acid residue sequences of the type specific epitopes and type-
cluster
specific epitopes described. These nucleic acid molecules are useful as probes
for instance in Southern blots or other DNA recognition assays such as the
capture assay described in US Patent Nos. 4,868,105 and 5,124,246.
The studies on antigenic mapping by expression of HCV
cDNAs showed that a number of clones containing these cDNAs expressed
polypeptides which were immunologically reactive with serum from
individuals exhibiting NANBH. No single polypeptide was immunologicaliy
reactive with all sera. Five of these polypeptides were very immu.nogenic in
that antibodies to the HCV epitopes in these polypeptides were detected in
many different patient sera, although the overlap in detection was not
complete. Thus. the results on the immunogenicity of the polypeptides
Vt ;4127153 PCTlUS94105151
~ -1g 216225 Q -
encoded in the various clones suggest that efficient detection systems for HCV
infection may include the use of panels of epitopes. The epitopes in the panel
may be constructed into one or multiple polypeptides. The assays for the
varying epitopes may be sequential or simultaneous.
Kits suitable for immunodiagnosis and containing the
appropriate labeled reagents are constructed by packaging the appropriate
materials, including the polypeptides of the invention containing type
specific
epitopes and type-cluster specific epitopes or antibodies directed against
type
specific epitopes and type-cluster specific epitopes in suitable containers,
along
with the remaining reagents and materials required for the conduct of the
assay, as well as a suitable set of assay instructions.
The invention further includes nucleic acid molecules
complementary to the nucleic acid sequences flanking regions encoding the
type specific epitopes and type-cluster specific epitopes. Such nucleic acid
molecules are useful in performing PCR to determine the genotype of a
particular HCV.
It should be noted that variable and hypervariable regions within
the HCV genome; therefore, the homology in these regions is expected to be
significantly less than that in the overall genome.
The techniques for determining nucleic acid and amino acid
sequence homology are known in the art. For example, the amino acid
sequence may be detemiined directly and compared to the sequences provided
herein. Alternatively the nucleotide sequence of the genomic material of the
putative HCV may be determined (usually via a cDNA intermediate), the
amino acid sequence encoded therein can be detennined, and the
corresponding regions compared.
The foregoing discussion and examples only illustrate the
invention, persons of ordinary skill in the art will appreciate that the
invention
can be implemented in other ways, and the invention is defined solely by .
reference to the claims.
V4',~4/27153 PCT/US94/05151
- , -19- 216225 p
Example 1
Comparison of Ma,Lor EQitopes of Various
Different Types of HCV
The amino acid residue homology between different types and
' subtypes of HCV was compared for various regions. The subtype of HCV is
as described by Simmonds phylogenetic analysis. The amino acid sequence
numbering corresponds to that described for the prototype HCV-1 sequence.
10. Choo et al. Table 2 shows the percent amino acid residue homology for NS4
region type specific epitopes and type-cluster specific epitopes and the
conserved major epitope. Table 3 shows the amino acid residue homology
between two type specific epitopes or type-cluster specific epitopes of the
NS5
region. Table 4 shows the percent amino acid residue homology for core
region conserved major epitopes and type specific epitopes.
30
94127153 PCTIUS94/05151
-20- 216225 0
Table 2. Amino Acid Homologies (%) Between Different HCV Subtypes
NS4 region type NS4 region
specific major conserved major
HCV Example types epitopes epitope
subtype abbreviation (1689-1718 aa)* (1910-1936 aa)*
la HCV-1 (la) vs (la) 100% 100%
lb HCV-J (1a) vs (lb) 83% 100%
2a HCV-J6 (la) vs (2a) 47% 93%
2b HCV-J8 (la) vs (2b) 43% 93%
N 94127133 PCTlUS94/05151
-21- 2162250
Table 3. Amino Acid.Homologies (%) Between Different HCV
Subtypes
NS5 region type NS5 region type
= specific major specific major
8CV Example types epitopes epitope
subtype abbreviation (2281-2313 aa)* (2673-2707 aa)*
la HCV-1 (la) vs (la) 100% 100%
lb HCV-J (la) vs (ib) 76% 89%
2a HCV-J6 (la) vs (2a) 70% 83%
2b HCV-J8 (la) vs (2b) 73% 83%
3a HCV-E-bl (la) -vs (3a) 77%
3b HCV-Tb (1a) =vs (3b) 83%
'V. 94/27153 PCT/US94105151
-22- 216 2 2 5 p
Table 4. Amino Acid Residue Homology (%) Between Different HCV
Subtypes
Core region
Core region type
conserved major specific major
Example types epitopes (10-45 epitopes
HCV subtype abbreviation aa)= (67-84 aa)=
la HCV-1 (la) vs (la) 100% 100%
lb HCV-J (la) vs (lb) 98% 100%
2a HCV-J6 (la) vs (2a) 98% 61%
2b HCV-J8 (ia) vs (2b) 98% 61%
3a HCV-E-bl (la) vs (3a) 93% 89%
4 HCV-EG-21 (la) vs (4) 98% 83%
V4'' 4l27153 PCT/US94/05151
-23- 216225
Example 2
PeDtide Synthesis
Two sets of polypeptides were synthesized. The first set was
designed to perform epitope mapping of HCV-1 and the second set was
designed to determine which epitopes identified in the epitope mapping studies
contained type specific epitopes. In the first set of polypeptides, sixty-four
sets (in duplicate) of overlapping octapeptides were synthesized by Mimotopes
across the entire HCV-1 polyprotein (3011 amino acid residues).
The second set of polypeptides were made according to the
method described by Geysen (1990) J. Trop. Med. Pub. Health, 21:523-533;
and Merrifield (1963) J. Am. Chem. Soc., 85:2149-2154.
In the second set of polypeptides, four antigenic regions which
represent the major epitopes of non-conservative sequences in HCV-l from
core, NS4, NS5 and their corresponding sequences from HCV subtypes lb,
2a, 3a and type 4 were selected for type specific epitope synthesis. The
sequence from core was selected from the less conserved region of ainino acid
residues 67-88. The sequence from the NS4 region was selected from the
amino acid residue region 1689-1718. The sequences selected from the NS5
region were from the amino acid residue regions 2281-2313 and 2673-2707.
Examale 3
Biological Samples =
In order to determine the effectiveness of the polypeptides in
distinguishing between antibodies specific to different types of HCV, antisera
were obtained from twenty-four chronic NANBH patients from different areas
of the world including the United States east coast and west coast. Japan.
western European countries, southern European countries and South Africa.
The viral RNA isolation, cDNA synthesis, PCR amplification, DNA
sequencing and oligonucleotide probe hybridization were performed as
described by Cha et al. (1992) Proc. Natl. Acad. Sci. USA, 89:7144-7148.
W 4/27153 PCT/US94/05151
-24- 6 2 2
~ 215 ~
Example 4
;Epitope Mappina Procedures
In order to determine which regions of HCV contain epitopes,
whether group specific or conserved, the entire HCV-1 polyprotein was
subject to epitope mapping. The method used is essentially as outlined in
Figure 2.
Sixty-four sets (in duplicate) of overlapping octapeptides were
synthesized by Mimotopes across the entire HCV-1 polyprotein (3011 amino
acids). A panel of 40 samples which contain 25 United States HCV antibody
reactive samples, 9 Japan HCV reactive samples and 6 HCV non-reactive
negative control samples were selected for epitope mapping and cluster
analysis. The immunoassays were performed using standard ELISA
procedures.
The criteria for identifying the major epitopes were based on
the antibody reaction frequency and the antibody reaction intensity (titer) to
these epitopes. The results are presented in Figures 3 and 4.
Example 5
Peptide Derived Enzyme-linked Immunosorbent Assays
In order to deterntine the optimal immunoassay utilizing the
polypeptides described in Example 2, two separate types of polypeptide
derived enzyme-linked immunosorbent assay (ELISA) were run and the results
were compared. The first type of ELISA was the Nunc IvlaxiSorb"' on wllich
the peptides are simply adsorbed and the second type utilized Nunc Covalinl:
NHTM on which the polypeptides are covalently linked. Formation of amide
bonds between carboxylic acids and atnines is initiated by the addition of
carbodiimide. To reduce hydrolysis, the active ester can be made by addinE!
N-hydroxy-succinin3ide (NHS) to the above conjugation procedures.
The microtiter plates for the first type of ELISA was performed
as follows. The polypeptides were placed in the wells of the Nunc
/
-25- 216 22 50
MaxiSorbT"' microtiter plates at a concentration of I g/well in 100 l of
phosphate buffered saline (PBS). The polypeptides were allowed to absorb
overnight at room temperature. The microtiter plates were then washed four
times with PBS without detergent. The wells were then post-coated with 220
l SuperblockO (Pierce) for one hour and then aspirated without further
washing and vacuum dried.
The assay was performed as follows. The sera sample size of 5
l was added to the wells with 100 l of 5% nonfat milk and incubated for
one hour at 37 C. The wells were then washed five times in PBS with 0.05 k
Tween * A conjugate of affinity purified goat anti-human IgG labeled with
horse radish peroxidase (Jackson Laboratories) was then used to determine the
degree of binding of human antibodies to the polypeptides. The conjugate was
previously diluted to 5% IgG in 150 mM NaCI, PBS, 5% horse serum (heat
denatured). 100 l of the conjugate was placed in the wells and incubated for
one hour at 37 C. The wells were then washed five times with PBS/Tween*
and OPD [o-phenylene-diamine-2HCl, one tablet per developer buffer (citrate
pliosphate buffered 0.02% H202); Sigma] for thirty minutes at room
temperature and the Abs at 492 nm and 620 nm were determined. The cut off
was determined from 200 random (normal) samples where seven standard
deviations from the mean or about 0.45.
The microtiter plates for the second type of ELISA was
performed as follows. The polypeptides were placed in the wells of the Nunc
MaxiSorbTM microtiter plates at a concentration of 10 mg/well in 50 l of
water. 25 l of 0.1 M Nl'IS (sulfo-N-hydrosuccinimide, Pierce) and 25 l of
0.1 M EDC [l-Ethyl-3(3-dimethylaminopropylcarbodiirnide) Sigma] were
added to the polypeptides and mixed at room temperature for 30 minutes on a
rocking platform. The entire contents were then added to 52 ml of ice cold
0.1 M Na Carbonate pH 8.6. 100 l of the mixture was used to coat the
wells of the microtiter plates and then incubated at 4 C for 30 minutes. the
plates were then washed four times with PBS/0.1 % Triton X- I0~ - The plates
were then treated with Superblock and the assay was performed as described
above.
The results obtained are presented in the followin: Tables 5-14.
*Trademark
~ ~''
. . ~ Table 5. Serotyping Epitope Analysis from Different HCV Types C~
~o
Sequence Region: NS4 (1689-1718) ~'
Sample ID Peptide Peptide Type dependent sequences
EIA (HCV Type)
(I) Paid Donor Samples .
SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-10(la) SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-11(lb) SGRPAVIPDREVLYQFDEMEECASHLPYI
cp402-12(2a) NQRAVVAPDKEVLYEAFDEMEECASRAALI
cp402-13(2b) NDRVVVAPDKEILYEAFDEMEECASKAALI ~
* e * *
Paid Donor Samples
LL57406 4.23 cp402-10(1a) N
6.48 cp402-11(lb)
6.84 cp402-12(2a) ~
3.41 cp402-13(2b)
N
6.57 sodClOO ELISA(la) Ch
React with common epitopes la, lb, 2a & 2b
PDREVLY
K I
(II) Sample react with type-specific epitopes
84-018669 3.76 cp402-10(la)
7.88 cp402-11(lb)
6.20 cp402-12(2a)
8.94 cp402-13(2b)
6.45 sodClOO ELISA(la)
React with common epitope for la, lb, 2a & 2b
PDREVLY A
K I
w
Table 6. Serotyping Epitope Analysis from Different HCV Types
Sequence Region: NS4 (1689-1718)
Sample Description Peptide EIA Peptide from Type dependent sequences
Signal/Cutoff different HCV
types
(I) Paid Donor Samples
SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-10(la) SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-11(1b) SGRPAVIPDREVLYQFDEMEECASHLPYI
cp402-12(2a) NQRAVVAPDKEVLYEAFDEMEECASRAALI
cp402-13(2b) NDRVVVAPDKEILYEAFDEMEECASKAALI
Paid Donor Samples
IV
CF25910 (la) 5.54 cp402-10(la)
9.71 cp402-11(lb)
8.28 cp402-12(2a) e.Ti
6.63 cp402-13(2b)
6.87 sodClOO ELISA(la)
React with common epitopes la, ib, 2a & 2b PDREVLY
K I
(II) Sample react with type-specific epitopes
FF25931 5.74 cp402-10(1a)
7.43 cp402-11(lb) 8.89 cp402-12(2a)
14.5 cp402-13(2b)
~
7.25 sodC100 ELISA(la)
React with common epitope for la, lb, 2a & 2b PDREVLY
K I '=
' t
Table 7. Serotyping Epitope Analysis from Different HCV Types
V
Sequence Region: NS4 (1689-1718)
Sample Description Peptide EIA Peptides from Type dependent sequences
Signal/Cutoff different HCV
types
(I) Paid Donor Samples
SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-10(1a) SGKPAIIPDREVLYREFDEMEECSQHLPYI
cp402-11(lb) SGRPAVIPDREVLYQEFDEMEECASHLPYI
=
cp402-12(2a) NQRAVVAPDKEVLYEAFDEMEECASRAALI
cp402-13(2b) NDRVVVAPDKEILYEAFDEMEECASKAALI
* * = a ~
W
MT-32 0.68 cp402-10(la)
(2a & 2b) 0.67 cp402-11(lb)
7.81 cp402-12(2a) Reactive with type specific epitope -> CASRAAL
6.88 cp402-13(2b) CASKAAL
~
0.23 sodClOO ELISA(la)
.iiT-79 0.27 cp402-10(la)
(2a, 2b) 0.34 cp402-11(lb)
5.53 cp402-12(2a) Reactive with type specific epitope -> CASRAAL
CASKAAL ,b
4.72 cp402-13(2b)
n
0.41 sodClOO ELISA(la)
th
tI~
--
Table S. Serotyping Epitope Analysis from Different.HCV Types
4
Sequence Region: NS5 (2673-2707) w
Sample Description Peptides from Type dependent sequences
different HCV
types
(I) Chronic NANBH from Paid Donors
Peptide EIA S/CO
(1) 84-018433 3.39 cp402-4(la) IKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTT
(2b)
4.75 cp402-5(lb) IRSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTT
4.62 cp402-6(2a) IHSLTERLYVGGPMFNSKGQTCGYRRCRASGVLTT
3.56 cp402-9(3b) ISSLTERLYVGGPMFNSKGQSCGYRRCRASGVLTT
3.38 c-p402-7(2b) IHSLTERLYVGGPMTNSKGQSCGYRRCRASGVFTT
10.58 c 402-8 3a ISSLTERLYCGGPMFNSKGAQCGYRRCRASGVLPT
4.40 r-NS5 ELISA
critical sequence change altered in the epitopes in this sample C AQ P
(2) FF25951 2.28 cp402-4(la) * ~.
(la)
1.65 cp402-5(lb)
1.28 cp402-6(2a) N
2.03 cp402-9(3b)
1.47 cp402-1(2b) 10.43 cp402-8(3a)
4.81 r-NS5 ELISA(la)
n
Critical sequence change altered in the epitope
AQ
rnsponse in this sample c
. * A
r-w
Table 9. Serotyping Epitope Analysis from Different HCV Types
Sequence Region: NSS (2673-2707)
Sample Description Peptides from Type dependent sequences
different HCV
types
(I) Chronic NANBH from Paid Donors
Peptide EIA S/CO
(1) 84-018366 1.07 cp402-4(la) IKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTT
1.01 cp402-5(lb) IRSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTT
1.52 qp402-6(2a) IHSLTERLYVGGPMFNSKGQTCGYRRCRASGVLTT ~
1.16 cp402-9(3b) ISSLTERLYVGGPMFNSKGQSCGYRRCRASGVLTT
0.58 cp402-7(2b) IHSLTERLYVGGPMTNSKGQSCGYRRCRASGVFTT 1e? w
0.48 c 402-8 3a ISSLTERLYCGGPMFNSKGAQCGYRRCRASGVLPT
1.68 r-NSS ELISA ~
Critical sequence change altered in the epitopes in this sample C AQ FP
(2) 96727 3.80 cp402-4(la)
3.15 cp402-5(lb)
2.52 cp402-6(2a)
3.41 cp402-9(3b)
0.79 cp402-7(2b)
0.60 cp402-8(3a)
H
5.54 r-NS5 ELISA(la) ~
Critical sequence change altered in the epitope
response in this sample c
AQ FP
. ,
Table 10. Summary of HCV Major type Specific Epitopes in Core, NS4 and NSS
Regions
v
Core Region:
Major Conserved Epitopes type Specific Epitopes Conserved Epitopes
(15-45) (67-84) (67-84)
TNRRPQDVKFPGGGQIVGGVY HCV-la & 1b-PEGRTWAQ PGYPWP(la, ib, 2a, 2b, 3a)
LLPRRGPRLG(HCV-1) HCV-2a & 2b-STGKSWGK
HCV-3A or 4-SEGRSWAQ F(4)
NS4 Regiont
Major Conserved Epitopes type Specific Epitopes Conserved Epitopes
(1925-1935) (1689-1718) (1689-1718)
RGNHVSPTHYV(HCV-1) HCV-la CSQHLPY PDREVLY(la, lb)
HCV-lb CASHLPY K I(2a, 2b)
HCV-2a & 2b--CASRAAL
.+ K
NS5 Region:
Conserved Epitopes type Specific Epitopes
(2288-2294) (2281-2313)
WARPDYN HCV-1a & lb---PDYEPPVVHG 1V
V ~
HCV-2a PDYQPATVAG
HCV-2b PGYEPPTVLG
HCV-la FAQALPVW
HCV-lb,2a&2b-FPPQALPIW
A
p
Conserved Epitopes
(2673-2707)
RGENCGYRRCRASGVLTT-HCV-1a KGAQCGYRRCRASGVLPT-HCV-3a
K QT HCV-lb, 2a * n
K QS HCV-3b Y,GQSCGYRRCRASGVFTT-HCV-2b
u7
~o
~
,...
C ~
Table 11. Serotyping Epitope Analysis from Different Types of HCV Sequencee
(I) Chronic NANBH from Paid
Donors Sequence Region: Core (67-84)
_
Sample ID Peptide EIA S/CO Peptide (HCV tvpe) Tvpe Dependent Sequences
cp401-01(la&lb) KARR PEGRTWAQ PGYPWP
cp40l-02(2A&2b) KDRR STGKSWGK PGYPWP
cp401-04(3a) KARR SEGRSWAQ PGYPWP
cp401-05(4) KARR SEGRSWAQ PGFPWP
84-017786 7.93 cp401-O1(la&lb)
(la) 7.56 cp401-02(2a&2b) Conserved epitope PGYPWP
7.79 cp401-04(3a)
6.86 cp401-05(4) F
6.61 rC22 ELISA(2-120 aa)
84-018433 0.31 Cp401-01(la&lb) PEGRTWAQ
(2b) 7.52 cp401-02(2a&2b) ISTGKSWGK1 Type specific epitopes
1.08 cp401-04(3a) SEGRSWAQ N ~
0.99 cp401-05(4) SEGRSWAQ
,r e- N:I
R3
6.18 rC22 ELISA (2-120 aa)
96696 1.02 cp401-01(1a&lb) PEGRTWAQ
(2b) 4.75 cp401-02(2a&2b) FSTGKSWGK
1.84 cp401-04(3a) SEGRSWAQ
1.37 cp401-05(4) SEGRSWAQ
,~ * *
6.23 rC22 ELISA (2-120 aa)
~
~
w
~
. , .
c
F(~.+
Table 12. Serotyping Epitope Analysis from Different Types of HCV Sequences
(I)
Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)
Sample ID Peptide EIA S/CO Peptide
(HCV Type) Type Dependant Sequences
I FAQALP VWARPDYNPPLVETWKKPDYEPPVVHG
FPPALP IWARPDYNPPLLESWKDPDYVPPVVHG
FPPALP AWARPDYNPPLVESWKRPDYQPATVAG
FPPALP PWARPDYNPVLIETWKRPGYEPPTVLG
(1) 96690(la) 4.59 cp402-00(la) IFAQALPVW Type specific epitope
1.57 cp402-01(lb) PP I N
1.16 cp402-02(2a) PP A tV
0.17 cp402-03(2b) PP P
5.64 r-NS5ELISA(la)
(2,) 84-01778(2a) 2.41 cp402-00(1a)
0.47 cp402-01(1b)
5.99 cp402-02(2a) Type specific epitope PDYQPATVAG
0.42 cp402-03(2b)
=d
0.42 r-NS5ELISA(la)
t/t
N
~
~
Table 13. Serotyping Epitope Analysis from Different Types of HCV Sequences
(I)
Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)
Sample ID Peptide EIA S/CO Peptide
(HCV Type) Type Dependant Sequences
(1) NAC5(lb) 7.28 cp402-00(la) FAQALPV WARPDYN PPLVETWKK PDYEPPVVHG
6.59 cp402-O1(lb) FPPALPI WARPDYN PPLLESWKD PDYVPPVVHG
0.10 cp402-02(2a) FPPALPA WARPDYN PPLLESWKR PDYQPATVAG
0.47 cp402-03(2bj FPPALPP WARPDYN PVLIETWKR PGYEPPTVLG
6.36 r-NS5ELISA PDYEPPVVHG(la) PDYVPPVVHG(lb)
PDYQPATVAG(2a) IV
PGYEPPTVLG(2b) iV
(2) Fi"25912(la) 6.44 cp402-00(la)
4.38 cp402-01(lb)
5.52 cp402-02(2a)
5.30 cp402-03(2b)
n
Non-type specific (conserved) epitopes LWARPDYN
- h+
V1
1 1 = =
tp
Table 14. Serotyping Epitope Analysis from Different Types of HCV Sequences
(I)
Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)
Sample ID Peptide EIA S/CO Peptide
(HCV Type) Type Dependant Sequences
(1) NAC5(lb) 7.28 cp402-00(la) FAQALPVWARPDYNPPLVETWKK PDYEPPVVHG
6.59 cp402-01(ib) FPPALPIWARPDYNPPLLESWKD PDYVPPVVHG
0.10 cp402-02(2a) FPPALPAWARPDYNPPLVESWKR PDYQPATVAG
0.47 cp402-03(26) FPPALPPWARPDYNPVLIETWKR PGYEPPTVLG
Type specific epitope (la & ib) PDYEPPVVHG(la)
6.36 r-NS5ELISA PDYVPPVVHG(lb)
PDYQPATVAG(2a)
PGYEPPTVLG(2b)
* * * * ~,
(2) W1(la) 7.80 cp402-00(la) Type specific epitope (la&lb) PDYEPPVVHG(la) ~
5.43 cp402-O1(lb) PDYVPPVVHG(lb)
0.56 cp402-02(2a)
0.58 cp402-03(2b) L"
6.86 r-NS5ELISA(la)
(3) MT56(la & ib) 7.68 cp402-00(la)
7.39 cp402-01(lb)
5.07 cp402-02(2a) PDYQPATVAG
0.36 cp402-03(2b) P V I
6.89 r-NS5 ELISA
N 94/27253 PCT/US94105151
-3621fi225 0
-
Example 6
Inhibition Assay
The inhibition assays were performed to determine whether the
peptides could compete with each other for binding to antibodies. Three sets
of short polypeptides from the core NS4 and NS5 regions of different types of
HCV sequences were synthesized. These polypeptides cover the sequence
regions from amino acid 1689-1695, 1696-1702 and 1711-1917. The
inhibition assays were performed by addition of 10 g of the above
polypeptides to the sample and incubation at 37 C for one hour and then
performance of the EUSA assays as described above. If inhibition was found
to be more than 50% of antibody binding the polypeptide was considered to be
inhibitory. The results obtained are presented in the following Table 15.
30
Table 15.
t=Z
1. SQHLPY CHIEN-101 NS4 Region 1712-1717 la
2. ASRAAL CHIEN-102 1712-1717 2a
3. ASKAAL CHIEN-103 1712-1717 2b
4, PDREVLY CHIEN-104 1696-1972 la, lb, 2a
5. PDYRPPVVHG CHIEN-105 NS5 Region 2304-2313 la
6. PDYQPATVAG CHIEN-106 2304-2313 2a
7. FAQALPVW CHIEN-107 2281-2288 la
8. FPPQALPPW CHIEN-108 2281-2288 2b
9. STGKSWGK CHIEN-109 Core 71-78 2a & 2b
10. SEGRSWAQ CHIEN-110 Core 71-78 4
W
! V
~
Aj
~
H
~
v~
-r
~
...
. ,
.~.
Table 16. HCV Major Type Specific Epitopea in Core, NS4 & NSS Regiona
Major Conserved Type Specific Conserved
E ito es Epitopes E ito es
Core Region: (15-45) (67-84) (67-84)
TNRRPQDVKFPGGGQIVGGVY HCV-la & lb PEGRTWAQ PGYPWP (la,lb,2a,2b,3a)
LLPRRGPRLG (HCV-1) HCV-2a & 2b STGKSWGK
HCV-3a or 4 SEGRSWAQ F (4)
NS4 Region: (1925-1935) (1689-1718) (1689-1718)
[RcNHvsPTitvl(Hcv_1) ~HCV-la CSQHLPY PDREVLY (la,lb)
HCV-lb CASHLPY K I (2a,2b)
HCV-2a & 2b CASRAALI
K
NS5 Region: (2288-2294) (2281-2313)
WARPDYN HCV-la & lb IP1fvHG1 ~
U
HCV-2a PDYQPATVAG
HCV-2b. PGYEPPTVLG ~
HCV-la FAQALPVW
HCV-1b,2a&2b FPPQALPZW
H
(2673-2707)
[CENCGYRRCRASGVLTf] HCV-la KGAQCGYRRCRASGVL P T--HCV-3a *
K QT HCV-lb,2a
K QS HCV-3b KGQSCGYRRCRASGVFTT-HCV-2b
i = . .
~94127153 PCT/US94/05151
2 6 2 2 5 0
-39-
Example 7
HCV Clinical Sample Ty,pinQ
The type or type-cluster specific epitopes given in Table 16
above were used in the ELISA assay of Example 5 to- test 13 clinical samples
" from non-A, non-B hepatitis patients (10 paid donors, 3 transfusion-related
chronic non-A, non-B patients). Table 17 shows the results of these assays.
Each clinical sample was assayed for reactivity with twelve different peptides
corresponding to the given regions (aa 67-84, 1689-1718 or 2281-2313)
representing the various type-specific or type-cluster epitopes. Each sample
gives a non-reactive (NR), weak reactive (WR) or reactive (R) response with
each typing peptide. These results predict an HCV genotype for each sample
that correlates with the HCV genotype determined by PCR, given in the right-
hand column.
30
.. =
~..
HCV LI L SAMPLE i GENWES
SAMPLE CORE(67-84 aa NS4 1689-1718 NS5 2281-231 GENOTYPES SEROTYP.XLS
DESCRIPTION 1 adcl b 2adc2b 3a 4 1 a lb 2a 2b 1 a lb 2a 2b
NANBN PATIENTS
FROM PAID DONORS
FF25951 NR NR NR NR R NR NR NR R NR NR NR 1a
96727 NR NR NR NR NR NR NR NR R NR NR NR 1a
84-017786 R R R NR NR NR NR NR R NR NR NR la
cn 96696 NR NR NR NR NR WR NR NR NR NR NR NR lb
CA FF25910 NR NR NR NR R R R R R WR WR NR la
=-+
83-018433 NR R NR NR WR WR R R NR NR NR R 2b
cn
m 84-018699 NR NR NR NR R R NR NR NR NR NR NR la
rn
--~
25931 NR NR NR NR R R R R NR NR NR NR la
m FF25912 NR NR NR NR R WR NR NR R NR NR WR la
84-017778 NR NR NR NR NR NR NR NR WR NR R NR 2a
TR/WSFUSION RELATED
CHRONIC NANBN
NAC5 NR NR NR NR NR NR NR NR NR NR NR NR ib n
H
N N N N b
N R N N R N N N NR N NR 30 CUTOFF OD =1.0 0D R=OD SIGNAL> 1.5 0D
WR=1.00D<SIGNAL<1.5 0D TAeLE 17
