Note: Descriptions are shown in the official language in which they were submitted.
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SYNTHETIC POLYPEPTIDES CORRESPONDING TO THE HEPATITIS C VIRUS (HCV) AND
APPLICATIONS
The present invention generally relates to
synthetic polypeptides, that is to say which are obtained
by preparative routes such as chemical synthesis, composed
of consecutive amino acids which are together identical to
any fragment, sequence or. region of the structural protein
of the nucleocapsid called CORE protein of the human
hepatitis C virus (HCV). These polypeptides can be used as
synthetic antigens in various applications arising from
their immunogenicity and which are specified below; at the
forefront of these applications is the detection HCV in
various body fluids such as for example a blood sample.
It has been established that the nucleocapsid
protein or CORE protein of HCV, which is composed as
established by Figure 1 of 191 amino acids (SEQ ID N0:3),
is that which exhibits the greatest homology, on the one
hand, between the sequences of the same group of viral
isolates, and, on the other hand, between the different
groups of viruses. Moreover, this CORE protein is encoded
by a structural part of the HCV genome and therefore
constitutes a structural protein. The high conservation of
the structure of this protein makes it a particularly
suitable candidate for the immunological detection of HCV.
Thus, the work of Hosein B, Fang CT, Popovsky MA,
Ye J, Zhang M, Wang CY, published in Improved sero-
diagnosis of hepatitis C virus infection with synthetic
peptide antigen from capsid protein, Proc Natl Acad Sci
USA 1991; 88: 3647-51, made it possible to determine an
immunodominant region in the CORE protein corresponding to
a polypeptide consisting of the sequence of the N-terminal
amino acids 1 to 120 of the said CORE protein.
In conformity with a publication already men
tinned, namely Hosein B, Fang CT et al., Improved sero
diagnosis of hepatitis C virus infection with synthetic
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peptide antigen from capsid protein, Proc Natl Acad Sci
USA 1991; 88: 3647-51, various synthetic peptides corres-
ponding to certain sequences of the CORE protein can be
used as antigen in detection tests in a solid phase, for
example on immunoabsorbent supports.
With the same objective of immunological detection
of HCV, the document EP-0,442,394 describes several
polypeptides comprising a polypeptide sequence belonging
to the abovementioned immunodominant region of the CORE
protein.
Among the said polypeptides, the one called VIIIE,
consisting of the sequence of N-terminal amino acids 2 to
62 of the CORE protein, was tested in an ELISA test with
respect to its immunoreactivity towards the anti-HCV
antibodies contained in sera from individuals infected
with HCV. This polypeptide demonstrated a good
immunoreactivity towards the HCV-infected sera tested.
The substitution of such known polypeptides of the
prior art, obtained by chemical synthesis, for the fusion
protein corresponding to the CORE protein itself in tests
of.detection is advantageous since it makes it possible to
reduce the risks of immunoreaction with antibodies which
may be present in a sample and which are different from
those directed against HCV.
However, it appeared essential to the Applicant to
be able to determine a minimal and sufficient sequence for
a polypeptide which, from the point of view of its
antigenic properties, is equivalent to the protein in its
entirety.
Indeed, the longer the peptide, the higher the
risks capable of interfering with the antigenicity of the
said peptide because of the higher frequency of the
following events:
- interference between the peptide and antibodies
different from those directed against HCV by cross-
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reactions or between the peptide and other biological
molecules present in the medium,
- conformational modifications relative to the
structure of the native protein which may result in a
disappearance of secondary and/or tertiary conformations
corresponding to epitopic sites, or appearance of secon
dary and/or tertiary conformations different from those
which the whole protein. adopts, which are capable of
interacting with antibodies other than the anti-HCV
antibodies.
According to EP-0,442,394, the inventors have
tried to shorten the length of the polypeptide VIIIE by
respectively amputating 9, 19, 29 and 39 amino acids from
its N-terminal part in order to prepare the polypeptides
consisting of the N-terminal amino acid sequences of the
CORE protein with a length of 10 to 62, 20 to 62, 30 to 62
and 40 to 62 respectively.
The immunoreactivity of each of these peptides was
evaluated in ELISA tests and it is observed that the
higher the number of amino acids amputated, the lower the
immunoreactivity.
In contrast to these results, the present inven-
tion provides a polypeptide, or its fragments, which
although consisting of an amino acid sequence much shorter
than that of the VIIIE polypeptide structure manifests an
immunoreactivity with all the sera of individuals or
samples infected with HCV and which carry antibodies
directed against the nucleocapsid protein.
The origin of the present invention is the
following completely unexpected discoveries, which result
from the experimental procedure outlined below:
1) an immunodominant region represented by at most the
first 45 amino acids (SEQ ID NO:1) exists in the CORE
protein of HCV;
2) this immunodominant region is sufficient by itself in
order to obtain the same sensitivity as the total
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CORE protein regarding the detection of anti-HCV
antibodies;
3) this immunodominant region must be continuous if it
is desired to react with all the sera of individuals
or blood samples infected with HCV and which possess
antibodies directed against the CORE protein;
4) this immunodorninant region clearly contains con-
formational type epitopes and linear type epitopes.
Consequently, the polypeptide used in conformity
with the invention comprises an isolated peptide sequence
which is composed of about the 45 N-terminal amino acids
of the HCV virus CORE protein (cf SEQ ID NO1).
Preferably, the polypeptide of the invention
consists of only or of an isolated peptide sequence
composed of the 45 N-terminal amino acids of the said
protein or alternatively of any homologous polypeptide
comprising about 45 amino acids and exhibiting an anti-
genic reactivity towards HCV.
Still preferably, the polypeptide of the invention
consists of a peptide sequence which is composed of the
N-terminal amino acids 2 to 45 of the CORE protein (cf SEQ
ID N02 ) .
EP-A-0 569 309 relates to an isolated peptide
sequence which is composed of 45 N-terminal amino
acids (SEQ ID NO:1) of the CORE (or capsid) protein
of the human hepatitis C virus (HCV), as represented
in Figure 1, 1 to 10 amino acids being optionally
amputated from this sequence in its N-terminal part
and/or its C-terminal part.
"Isolated peptide sequence" is understood to mean
any polypeptide not fused with another protein or another
peptide regardless of its route of production, for example
by chemical synthesis, by lysis of the CORE protein, or by
genetic recombination techniques. This polypeptide can
therefore be a synthetic peptide or a protein.
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The preferred amputations of the polypeptide of
the invention are the amputations of respectively 6 amino
acids and 11 amino acids from the N-terminal end of the
CORE protein.
5 The present invention concerns a polypeptide that
specifically binds to antibodies that specifically bind to
human hepatitis C virus consisting of an antigenic
sequence having specific immunoreactivity to the
antibodies of SEQ ID NO:1, with the proviso that said
polypeptide is different from SEQ ID NO :l, SEQ ID NO :2
and sequences resulting from the amputation of SEQ ID
NO :1 with 1 to 10 amino acids from the N-terminal part
and/or the C-terminal part of SEQ ID NO :1.
The polypeptide sequences according to the
invention may be such as in the native state or modified
chemically. Chemical modification is understood to mean
any chemical alteration of at least one functional group
of the peptide sequence which essentially preserves or
even develops the biological properties of the said
sequence. The replacement of an amino acid of the L series
by an amino acid of the D series, a modification of the
side chains of the amino acids such as an acetylation of
the amine functional groups, a carboxymethylation of the
thiol functional groups. or an esterification of the
carboxylic functional groups, or a modification of the
peptide bonds such as carba, retro-inverso, reduced and
methylene-oxy bonds, are especially part of the chemical
modifications considered above.
A preferred polypeptide (b) comprises or
consists of an antigenic sequence which consists of
SEQ ID NO:1 modified with at least one modification
selected from the group consisting of .
(1) substituting an amino acid of SEQ ID NO:1 for
at least one homologous amino acid, as defined below, and
(2) a chemical modification.
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A preferred chemical modification is at least one
member selected from the group consisting of .
(i) replacement of an amino acid of the L series
by an amino acid of the D series,
(ii) modification of side chains of amino acids,
(iii) modification of peptide bonds.
Said modification of side chains may be an
acetylation of amine functional groups, a
carboxymethylation of thiol functional groups or an
esterification of carboxylic functional groups.
Said modification of peptide bonds may be forming
carba, retro-inverso, reduced and methylene-oxy bonds.
According to the present invention, an amino acid
is said to be homologous to another amino acid when their
chemical characteristics, such as polarity, hydrophobicity
and/or basicity and/or acidity and/or neutrality, are
essentially the same. In particular, a classification
based on the polarity of the side chains plits up amino
acids into four groups .
- non-polar or hydrophobic amino acids
comprising .
alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophane,
methionine,
which are considered as homologous amino acids, within the
meaning of the above definition,
- amino acids having a non-charged side chain
comprising .
serine, threonine, tyrosine,
asparagine, glutamine,
cysteine,
glycine,
which are considered as homologous amino acids, within the
meaning of the above definition,
- amino acids having a negative-charged side chain
(acidic amino acids) comprising aspartic acid and glutamic
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acid, which are considered as homologous amino acids,
within the meaning of the above definition,
- amino acids having a positive-charged side chain
(basic amino acids) comprising lysine, arginine,
histidine, which are considered as homologous amino acids,
within the meaning of the above definition ; depending on
the pH, this group may further comprise asparagine and
glutamine,
- rare amino acids comprising hydroxyproline,
hydroxylysine, desmosine, isodesmosine, which are
considered as homologous amino acids, within the meaning
of the above definition.
According- to another classification, asparagine
and aspartic acid can be considered as homologous amino
acids, and glutamine and glutamic acid, as well.
Another preferred polypeptide is shorter than
SEQ ID NO:1, and shares at least one, preferably at least
two identical amino acid with SEQ ID N0:5.
Preferebly, said polypeptide shares at least two
identical amino acids with SEQ ID N0:6 and/or at least one
identical amino acid with SEQ ID N0:7.
Said polypeptide may further have at least one
amino acid which is homologous (as defined above) to at
least one amino acid of SEQ ID NO: 1.
Said polypeptide may further have at least one
amino acid which differs from at least one amino acid of
SEQ ID N0:1 by a chemical modification (as defined above)
of the side chain of said amino acid.
A preferred polypeptide is selected from the group
consisting of SEQ ID N0:8 through SEQ ID N0:18.
The inventors have settled a screening test to
determine whether a given polypeptide is an antigenic
equivalent of polypeptide (a) or not. This test can be
carried out by the one skilled in the art with routine
experimentation and is described in details at the end of
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the description (~ Antigenic polypeptide different from
but equivalent to peptide (a)).
According to this test, the given polypeptide is
screened against antibodies, at least one relevant
antibody anti-HCV and one irrelevant antibody used as a
negative control, using the usual methods to indicate the
presence or absence of an antibody reaction. Examples of
these methods include enzyme linked immunosorbent assay
(ELISA). Suitable antibodies can either be purchased as
commercially available antisera ox prepared in a suitable
host in accordance with well known procedures. The results
can be expressed as the mean OD obtained with the relevant
antibody - OD obtained with the irrelevant antibody. A
threshold value is determined to select antigenic
peptides.
In order to confirm this selection, the
immunoreactivity of the selected peptides is tested with
anti-HCV core positive human sera and with sera from
healthy individuals. The results are expressed as the mean
OD obtained with the pool of anti-core positive human
sera - OD anti-core negative human sera.
From the above-defined peptide compounds or
compositions, the invention provides a reagent for the
detection of human hepatitis C virus (HCV) comprising as
reactive substance any one of the abovementioned compounds
or compositions and optionally any additive
immunocompatible with the detection of HCV. Thus, the
detection can be carried out using a polypeptide identical
to those of the present invention or an antigenic
equivalent peptide thereof, with optionally one anti-human
immunoglobulin antibody, labelled with any conventional
marker such as a radioactive, fluorescent or enzymatic
marker or the like. Such a reagent can be used both in a
homogenous phase, for example in immunoprecipitation
assays, and in a heterogenous phase, for example in
immunoadsorption assays.
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With the abovementioned reagent, any suitable
means of detection of HCV can be obtained, whether a
detection~kit or any other equivalent system or unit. By
way of example, the abovementioned reagent is supported on
a solid support immunocompatible with the reagent as a
whole; in particular, the solid support is, without
limitation, in the form of a microtiter plate, a sheet, a
cone, a well, a bead or any other appropriate micro-
particulate substrate.
The term solid support as used here includes all
materials upon which the polypeptides according to the
invention can be immobilized. These may be synthetic
materials chemically modified or otherwise, especially
polysaccharides such as cellulose materials, for example
paper, cellulose derivatives such as nitrocellulose and
cellulose acetate; polymers such as vinyl chloride,
polyethylene, polystyrene, polyacrylate or copolymers such
as vinyl chloride and propylene polymer, vinyl chloride
and vinyl acetate polymer; styrene-based copolymers;
natural fibers such as cotton and synthetic fibers such as
nylon. Preferably, the solid support is a polystyrene
polymer, a butadiene-styrene copolymer or a butadiene-
styrene copolymer mixed with one or more polymers or
copolymers chosen from polystyrene, styrene-acrylonitrile
or styrene-methyl methylmethacrylate copolymers,
polypropylenes, polycarbonates or analogs.
Using the immunological detection reagents or
means according to the invention, anti-HCV antibodies can
be detected in any body part or fluid such as a blood
sample of an individual suspected of being infected with
HCV. For that, this body part and the above-mentioned
reagent simply have to be brought into contact under
predetermined conditions, for example of temperature,
which permit an immunological reaction where appropriate,
and to then detect the presence of an immune complex with
this reagent.
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"Body part" is understood to mean any fluid,
tissue or organ of an individual, comprising or capable of
comprising anti-HCV antibodies. These body parts may be a
blood, plasma or serum sample or various secretions and
5 the like.
The process described above can be carried out in
any detection device or apparatus comprising a vessel for
bringing the body part. analyzed into contact with a
reagent as defined above, and this with means which create
10 conditions, such as temperature, .favorable for an
immunological reaction where appropriate. And this device
comprises means, especially optical, for the detection of
the immune complex obtained with the reagent.
Another way of detecting the HCV virus using the
polypeptides according to the present invention is to
obtain monoclonal or polyclonal antibodies by any method
known per se comprising an immunological reaction between
a human or animal organism and an immunogenic agent
consisting of a polypeptide composition as defined above.
The antibodies thus obtained, for example conveniently
labelled, can be used to detect HCV or to monitor the
progression of the virus in a patient suffering from
hepatitis C.
Of course, each of the polypeptide compositions
according to the invention may constitute the active
ingredient of an active immunotherapeutic composition,
being optionally conjugated with an immunologically
suitable support. A pharmaceutically acceptable excipient
may supplement the said composition. Such a composition is
for example a vaccinal preparation.
The immunodominant character of the peptide
sequence according to the present invention was demon-
strated in conformity with the following experimental
procedure.
The strategy chosen consists in synthesizing long
polypeptide fragments of about 40 amino acids, in the
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N-terminal part of the CORE protein, which belong to the
sequence of about the first 120 amino acids.
In a first stage, three peptides were therefore
defined, beginning the synthesis at amino acid No. 2
(serine).
In conformity with Figure 1, three peptides were
synthesized, namely:
- peptide called S42G, extending from serine 2 up to
glycine 45
- peptide called P42Y, extending from proline 39 up to
tyrosine 82
- peptide called R40R, extending from arginine 75 up to
arginine 116.
It appears that these peptides have some amino
acids in common, which makes it possible to identify a
possible antigenic determinant located at the intersection
of two peptides.
The peptides were chemically synthesized by solid
phase synthesis according to the Merrifield technique
(Barany G, and Merrifield R.B, 1980, In the Peptides, 2,
1-284, Gross E and Meienhofer J, Eds Academic Press, New
York). The practical details are those described below.
~et~ti de sv~thesi s
The peptides are synthesized on a phenyl
acetamidomethyl (PAM)/polystyrene/divinylbenzene resin
(Applied Biosystems, Inc. Foster City, CA), using an
automatic "Applied Biosystems 430A" synthesizer. The amino
acids are coupled in the form of esters of hydroxy
benzotriazole (HOBT). The amino acids used are obtained
from Novabiochem (Lauflerlfingen, Switzerland) or from
BACHEM (Bubendorf, Switzerland).
Chemical synthesis of the peptides was carried out
using a double coupling procedure with N-methylpyrrolidone
(NMP) as solvent. The peptides were simultaneously cut
from their resin as well as the side protections using
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hydrofluoric acid (HF) in a suitable apparatus (type I
cutting apparatus, Peptide Institute, Osaka, Japan).
For 1 g of peptidylresin, 10 ml of HF, 1 ml of
anisole and 1 ml of dimethyl sulfide (DMS) are used, and
the mixture is stirred for 45 minutes at -2°C. The HF is
then evaporated under vacuum. After intensive washes with
ether, the peptide is eluted from the resin with 10~
acetic acid and then the peptide is freeze-dried.
The peptides are purified by preparative high
performance liquid chromatography on a_ type C18 VYDAC
column (250 x 21 mm) (The Separation Group, Hesperia, CA,
USA). The elution is performed with an acetonitrile
gradient at a flow rate of 22 ml/min. The fractions
collected are controlled by elution under isocratic
conditions on an analytical C18 VYDAC column
(250 x 4.6 mm) at a flow rate of 1 ml/min. The fractions
which have the same retention time are pooled and freeze-
dried. The predominant fraction is then analyzed by
analytical high-performance liquid chromatography with the
system described above. The peptide which is considered as
being of acceptable purity results in a single peak
representing 95~ of the chromatogram minimum.
The purified peptides are analyzed with the
objective of assessing their amino acid composition using
an automatic Applied Biosystems 420 H amino acid analyzer.
Measurement of the chemical molecular mass (mean) of the
peptides is obtained using the L.S.I.M.S. mass
spectrometer in a positive ion mode, on a dual focusing
instrument VG. ZAB.ZSEQ linked to a DEC-VAX 2000
acquisition system (VG analytical Ltd, Manchester,
England).
The reactivity of these three peptides towards the
sera of individuals infected with the hepatitis C virus,
termed (HCV) positive, was evaluated in an ELISA test
according to the procedure described below.
Det~~tion of anti-HCV antibodies bar ELISA
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The wells of a microtiter plate of "NUNC maxisorb"
trademark are saturated with 100 ~.l of a solution
containing the peptide or a mixture of peptides (10 ~,g/ml)
for 2 hours at 37°C. The plate is then emptied, then
washed with a wash buffer containing 0.05% Tween 20. The
wells are saturated with 100 ~.1 of wash buffer
supplemented with 10% goat serum (v/v), then incubated for
30 minutes at 37°C, then.washed again as above. The sera
to be analyzed are diluted to the appropriate dilution
with saturation buffer. The incubation of the sera is 1
hour at 37°C. The wells are again washed. The solution of
conjugate (goat IgG to human IgG labelled with peroxidase)
at a dilution of 1/1000 in the saturation buffer is then
added and the incubation lasts for 90 minutes at 37°C.
After washing, the solution of ortho-phenylenediamine
substrate is added. After 10 minutes, the reaction is
stopped with 50 ~,1 of HZSO4 and the optical density is read
at 492 nm. It should be noted that all the tests were
carried out in duplicate.
The reactivity of the peptides S42G, P42Y, and
R40R is measured by ELISA on HCV-positive sera (P 1 to
P 20 and B 1 to B 16) and on normal sera (SN 10, 11, 16,
17, 18, 19) .
For that, the different peptides are adsorbed on
the microplates at a concentration of 10 ~.g/ml and the
sera are used at 1/100 dilution.
The values obtained, which are collated in Table 1
below, correspond to the optical density (OD) multiplied
by 10', at 492 nm.
For each serum the experiment was carried out in
duplicate. The "" are values outside the upper scale.
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TABLE 1
S42G R42Y R40R S42G P42Y R40R
P 1 ***** 101 375 B 1 ***** 869 172
***** 108 420 ***** 811 173
p 2 ***** 119 399 B 2 2302 1749 364
***** 104 391 2276 1664 345
P 3 2145 648 223 B 3 1673 623 304
1942 638 215 1686 630 341
p 4 ***** 2314 309 B 4 ***** 1688 405
***** 2105 307 ***** 1557 346
p 5 ***** 234 129 B 5 ***** 1639 360
***** 243 1?6 2308 1699 281
P 6 116 184 87 B 6 1671 810 172
130 185 100 1778 791 163
P 7 ***** 2295 496 B 7 ***** 1657 418
***** 2389 478 ***** 1489 457
p 8 ***** 983 282 B 8 1567 611 271
***** 903 328 1543 620 259
p 9 186 238 159 B 9 ***** 957 235
183 231 158 ***** 913 229
P 10 169 194 218 B 10 360 22? 108
177 204 216 386 223 98
P 11 ***** ***** 1191 B 11 1749 813 164
***** ***** 1377 1849 789 184
P 12 ***** ***** 1121 B 12 ***** 755 136
***** ***** 1231 ***** 407 117
P 13 114 64 113 B 13 1341 746 140
106 108 116 1142 609 99
P 14 ***** 362 280 B 14 455 246 130
***** 349 270 450 259 125
P 15 ***** ***** 2305 8 15 ***** 313 301
***** ***** 2335 ***** 312 303
P 16 ***** 1742 938 B 16 ***** 222 117
***** 1667 964 ***** 153 125
p 17 ***** 799 217 SN 10 205 237 163
***** 736 212 192 205 154
p lg ***** 2253 1427 SN 11 107 156 150
***** 2339 1327 100 141 138
P 19 105 106 84 SN 16 551 657 426
112 105 89 537 667 439
P 20 ***** 1701 714 SN 17 129 156 104
***** 1679 740 122 144 74
SN 18 218 332 119
173 279 87
139 167 480
SN 19 120 161 496
SUBSTITUTE SHEET (RULE 26)
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Table 1 shows that the peptides react differently
with the sera.
It appears clearly that the most reactive peptide
is peptide S42G which detects 31 sera out of 36.
5 None of these peptides detects normal sera, which
confirms their specificity.
Finally, no serum which is negative with peptide
S42G is positive with peptides P42Y or R40R, which shows
that on its own, peptide S42G detects the sera without the
10 help of the other two peptides.
The study was then continued in order to know more
precisely the antigenic determinants) located on peptide
S42G.
For this purpose, two peptides were prepared under
15 the same conditions as above.
These two peptides are, in conformity with Figure
1:
1) a peptide of 20 amino acids, called S18D, covering
sequence 2 to 21 of the CORE protein
2) a peptide of 24 amino acids, called V22G, covering
sequence 22 to 45 of the CORE protein.
The reactivity of these two peptides (separated
and combined) was evaluated by comparing it with that of
peptide S42G in an ELISA test as described above.
The reactivity of peptides S42G, S18D, V22G,
S18D + V22G is measured by ELISA on HCV-positive sera. The
different peptides are absorbed onto microplates at a
concentration of 10 ~g/ml and the sera are used at the
dilution stated.
The values obtained, which are collated in Table 2
below, correspond to the optical density at 492 nm. All
the experiments were carried out in duplicate.
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TABLE 2
SERU S42G S18D-V22G S18D V22G
M
~
1 __ _ 2.500 0.438 0.012 0.426
_
~P1 ~~
1 100
2 P2 1 100 2.500 0.310 0.020 0.290
3 P3 1 10 0 2.500 0.665 0.162 0.503
4 P4 1 100 2.500 2.500 0.982 1.739
5 P5 1 100 2.500 2.500 2.500 2.50 0
6 1 1000 2.500 2.500 2.317 2.29 3
7 1 10000 2.093 0.977 0.399 0.57 8
8 P6 1 100 0.000 0.007 0.007 0.00 0
9 P7 1 100 2.500 2.500 2.500 2.500
10 1 1000 2.500 2.500 0.854 1.590
11 1 10000 1.916 0.402 0.165 0.23 7
12 P8 1 100 2.500 2.500 2.500 2.24 1
13 1 1000 2.500 2.500 2.500 2.47 6
14 1 10000 1.565 0.730 0.681 0.049
15 P9 1 100 0.090 0.027 0.019 0.008
16 P10 1 0.172 0.054 0.028 0.026
100
17 P11 1 2.500 2.500 0.383 2.500
100 '
18 P12 1 2.500 2.500 2.500 2.500
100
19 1 1000 2.500 2.500 2.500 2.500
20 1 10000 2.500 0.560 0.454 0.106
21 P13 1 0.000 0.025 0.012 0.013
100
22 P14 1 2.500 2.500 0.907 1.778
100
23 P15 1 2.500 2.500 2.500 1.810
100
24 P16 1 2.500 2.500 0.225 2.500
100
25 P17 1 2.500 2.129 0.297 1.832
100
26 P18 1 2.500 2.500 2.500 2.500
100
27 1 1000 2.50 0 0.895 0.297 0.598
28 1 1 0000 1.00 6 0.167 0.095 0.072
29 P19 1 100 0.000 0.021 0.011 0.000
30 P20 1 100 2.500 2.500 1.433 2.249
31 P21 1 100 2.500 2.383 0.111 2.272
32 P22 1 100 2.500 2.500 2.500 2.500
33 1 1000 2.500 1.844 1.142 0.702
34 1 10000 0.894 0.234 0.146 0.088
35 P23 1 0.000 0.030 0.015 0.015
100
36 P24 1 2.500 0.594 0.015 0.579
100
37 P25 1 2.500 2.500 2.500 2.252
100
38 1 1000 2.500 2.500 2.199 0.695
39 1 10000 1.550 0.418 0.329 0.089
40 P26 1 2.500 2.500 2.500 2.500
100
41 1 1000 2.500 2.500 2.500 1.541
42 P26 1 2.500 1.156 0.95 7 0.199
10000
4 3 P27 1 2.500 2.500 1.42 5 1.600
100
4 4 P28 1 2.500 2.500 0.11 5 2.500
100
45 P29 1 0.33 1 0.905 0.000 0.005
100
46 P30 1 2.50 0 2.500 0.483 2.500
100
47 P31 1 2.50 0 2.500 2.500 2.500
100
48 1 1000 2.500 2.500 1.975 2.500
49 1 10000 2.071 1.030 0.183 0.8 47
50 P32 1 2.500 2.500 1.046 1.6 39
100
51 P33 1 2.500 2.500 1.307 1.9 8?
100
52 P34 1 2.500 2.500 2.500 1.618
100
53 P35 1 2.500 2.500 2.500 1.504
100
54 P36 1 2.500 1.341 0.115 1.228
100
55 P37 1 2.500 2.500 2.500 2.500
100
56 1 10 00 2.500 1.388 0.523 0.8 65
57 I 10000 1.088 0.230 0.102 0.1 28
58 P38 1 2.500 2.500 2.500 2.5 00
100
59 1 1000 2.500 2.3 35 0.753 1.5 82
60 1 10000 1.477 0.1 56 0.099 0.0 57
61 P39 1 2.500 2.500 2.500 1.8 35
100
62 P40 1 2.500 2.500 2.500 2.5 00
100
63 P41 1 2.500 2.500 1.579 2.218
100
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64 P42 1 100 0.034 0.000 0.000 0.000
65 CTS 1 100 2.500 2.500 2.500 2.500
66 1 1000 2.500 2.500 2.500 1.155
67 1 10000 2.500 0.338 0.295 0.043
68 1 100 2.500 2.500 2.500 2.500
6g 1 1000 2.500 2.500 2.500 1.605
70 1 10000 1.336 0.602 0.347 0.255
71 Bl 1 100 2.500 2.500 1.859 1.225
72 B2 1 100 2.500 2.500 1.781 0.756
73 83 1 100 2.500 1.573 1.244 0.329
74 B4 1 100 2..500 2.500 2.500 2.500
75 1 1000 2.500 2.464 1.250 1.214
76 1 1000 1.021 0.315 0.171 0.144
77 B5 1 100 2.500 2.500 2.032 0.863
78 B6 1 100 2.500 2.500 2.500 0.749
79 B7 1 100 2.500 2.500 2.500 2.102
80 BS 1 100 2.500 1.720 1.362 0.358
gl B9 1 100 2.500 2.500 0.808 2.082
82 B10 1 100 0.721 0.324 0.099 0.225
83 B11 1 100 2.084 2.500 2.324 0.616
84 B12 1 100 2.500 2.392 1.375 1.017
85 B13 1 100 1.809 0.674 0.370 0.304
86 B14 1 100 0.698 0.258 0.072 0.186
87 B15 1 100 2.500 1.044 0.090 0.954
88 B16 1 100 2.500 2.500 2.500 2.500
Each serum was, in a first instance, tested at the
5 dilution 1/100. In the event where the response proved
saturating (value 2500) for all the peptides (example:
serum P 5) a 1/1000 dilution, and if necessary a 1/10,000
dilution, was carried out.
It appears that for all the HCV-positive sera, the
10 reactivity of peptide S42G is substantially greater than
the reactivity of peptides S18D and V22G, and than that of
S18D + V22G.
The sera P6, P9, P10, P13, P19, P23, P24 are sera
which do not possess antibodies against the CORE protein
15 of HCV.
Although these results as a whole are unambiguous,
the attachment of the different peptides to the wells of
the microtiter plates can modify the epitopes or deter-
minants of the peptide tested. The plates used (NUNC
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Maxisorb) are polystyrene plates irradiated with gamma
rays, which bind the peptides in a noncovalent manner via
electrostatic type bonds but also hydrophobic bonds. It is
possible that peptides, depending on their sequence, are
selectively adsorbed, thus favoring a well defined part
and thus preventing immunogenic reactivity towards another
part which may have become less acceptable.
To evaluate this hypothesis, inhibition tests,
whose usefulness lies in the fact that they allow the
formation of the antigen-antibody complex in_ liquid
medium, were carried out, thus dispensing with possible
artifacts linked to the adsorption of peptides onto a
solid support.
The methodology is that described below.
Inhibition test
The inhibition experiments were carried out by
reaction, in liquid phase, of the HCV sera with the
peptides followed by reaction of the remaining antibodies
with the peptide adsorbed onto the microplates. The
inhibitory peptides are incubated at a concentration of
0.1 mg/ml with sera of appropriate dilution. The rest of
the manipulation is identical to that described for the
ELISA test.
Peptide S18D, or V22G, or a mixture of both, is
preincubated overnight in the presence of the serum to be
tested. The antibodies can bind onto the corresponding
sites. The mixture (peptide + serum) is then incubated
with the peptide S42G adsorbed onto the microtiter plates.
If all the antibodies reacted during the incubation with
the peptides S18D, V22G, or with the mixture, no
reactivity will be observed, which will result in a 100
inhibition. In contrast, if antibodies specific for
peptide S42G remain, they will then be able to react.
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A control is carried out by preincubating each
serum with peptide S42G, which makes it possible to
calculate the percentage inhibition.
Table 3 collates the results of the inhibition of
the binding of anti-HCV antibodies (dilution 1/10,000)
onto peptide S42G, by preincubation of HCV sera with
peptide S42G, peptide S18D, peptide S22G and the mixture
of peptides S18D + S22G..
TAELE 3
Serum Inhibition Inhibition Inhibition Inhibition
S42G S18D V22G pool S18D
+
V22G
1 P5 100% 3.5% 83.0% 77.0%
2 P7 100% 8.4% 81.0% 86.0%
3 P8 100% 1.4% 77.4% 53.0%
4 P18 100% 10.6% 48.0% 52.0%
5 P22 100% 5.6% 65.5% 57.0%
6 P25 100% 14.1% 71.8% 70.0%
? P31 100% 2.0% 39.9% 27.0%
8 P37 100% 43.6% 88.7% 73.0%
9 P38 100% 8.9% 70.3% 65.0%
B4 100% 16.8% 83.6% 72.0%
10 As shown in Table 3, no peptide completely
inhibits the reactivity of the sera towards peptide S42G.
In other words, this experiment proves that
antibodies specific for peptide S42G exist which do not
react with either peptide S18D or with peptide V22G, the
sum of both representing the total sequence of peptide
S42G.
A final hypothesis to be evaluated consists in
verifying that the antibodies specific for peptide S42G
were not directed against the central part of peptide
S42G, that is to say at the junction of peptides S18D and
V22G.
A peptide was therefore prepared (cf Figure 1)
whose sequence comprises the C-terminal part (6 amino
acids) of peptide S18D and the N-terminal part (6 amino
acids) of peptide V22G.
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19p
Although this peptide exhibits reactivity with
HCV-positive sera, the level obtained is in no case
comparable to that obtained with peptide S42G.
The set of results presented above makes it
possible to draw the following conclusions.
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In the 120 N-terminal amino acids of the CORE
protein, and more particularly in the first 62 amino
acids, the first 45 amino acids are the most reactive
towards HCV-positive sera.
5 The first 21 amino acids (peptide S18D) react,
which shows the presence of one or more antigenic deter-
minants on this peptide.
The amino acids 22 to 45 (peptide V22G) also carry
one or more epitopes.
10 The junction of these two sequences is also
reactive.
Consequently, the sequence 1-45 of the CORE
protein is pluriepitopic.
Furthermore, one or more antigenic determinants
15 exist which are reactive only insofar as the entire
sequence 2-45 is available and not in a discontinuous
manner (peptides S18D + V22G). These epitopes, which are
specific to peptide S42G, are without any doubt conforma
tional type epitopes which can exist only insofar as this
20 sequence of 44 amino acids (peptide S42G) has a suitable
structure, a structure which is not obtained with smaller-
sized peptides.
If the amino acid sequence of peptide S42G should
not be discontinuous in order to preserve all the epi
topes, it can be asked if the N- and/or C-terminal parts
of peptide S42G are involved in the epitopic conformations
of S42G or carry the epitopes themselves.
In order to try to respond, five peptide fragments
derived from S42G by N- and/or C-terminal amputations were
deffined.
In conformity with Figure 2, the following five
fragments were synthesized according to the Merrifield
technique in conformity with the procedure described
above:
- peptide called P37G corresponding to the amino acid
sequence 7 to 45 of the CORE protein and to an
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21
amputation of 5 amino acids from the N-terminal part
of S42G,
- peptide called K32G corresponding to the amino acid
sequence 12 to 45 of the CORE protein and to an
amputation of 10 amino acids from the N-terminal part
of S42G,
- peptide called S32Y corresponding to the amino acid
sequence 2 to 35 of the CORE protein and to an
amputation of 10 amino acids from the C-terminal part
of S42G,
- peptide called P27Y corresponding to the amino acid
sequence 7 to 35 of the CORE protein and to an
amputation of 5 amino acids from the N-terminal part
and of 10 amino acids from the C-terminal part of
S42G,
- peptide called K22Y corresponding to the amino acid
sequence 12 to 35 of the CORE protein and to an
amputation of 10 amino acids from the N-terminal part
and of 10 amino acids from the C-terminal part of
S42G.
The reactivity of the five peptides towards sera
of individuals infected with HCV was evaluated in ELISA
tests in conformity with the procedure described above for
measuring the activity of the peptides S42G, P42Y and
R40R.
Table 4 below collates the results obtained for
the peptides S42G, S32Y, P27Y, K22Y in order to examine
the influence of an amputation of the N-terminal part and
the C-terminal part of the peptide S42G.
These results are expressed in optical density
values read at 492 nm multiplied by a factor of 10'.
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T~18LE 4
-~.,
Serum Dilution RIBA C22 S42G S32Y P27Y K22Y
P 1 1 100 D 703 620 435
P 2 1/100 D 1177 891 666
P 3 1/100 D 1576 1470 1031
P 23 1/100 146 157 138 125
P 10 1/100 56 78 60 66
P 17 1/100 D D 2300 1930
P 24 1/100 D 1214 1063 735
P 29 1/100 594 400 347 310
P 30 1/100 D D 2274 1805
P 32 1/100 D D D D
B 3 1/100 D D D 2033
P 4 1/100 D D D D
1/1000 D 2165 2111 1959
1/10,000 588 369 324 300
P 5 1/100 D D D D
1/1000 D D D D
1/I0,000 1880 1242 856 394
P 7 1/100 D D D D
1/1000 D D D D
1/10,000 2024 1932 1667 1487
P 8 1/100 D D D D
1/1000 D D D 2257
1/10,000 1423 1068 654 335
P 14 1/100 D D D D
1/1000 D D D D
1/10,000 1296 601 566 398
P 16 1/100 D D D D
1/1000 D 983 913 610
1/10,000 750 124 104 75
A 8 1/100 4 D D D D
1/1000 D 1372 1198 431
1/10,000 620 173 118 56
A 9 1/100 4 D D D 2152
1/1000 D 1941 1388 282
1/10,000 394 297 182 43
A 10 1/100 4 D D 2308 2114
1/1000 D 373 334 207
1/10,000 819 33 31 26
A 11 1/100 4 D 2240 1937 1878
1/1000 1865 228 226 212
1/10,000 223 36 33 31
A 12 1/100 4 D D D D
1/1000 D D D 646
1/10,000 848 588 365 62
The sera P23 and P10 are sera which do not possess
antibodies against the CORE protein of BCV.
From this Table, it can be deduced that when the
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23
amino acids of its C-terminal part are amputated, S42G
loses its reactivity and that furthermore if 5 and 10
amino acids are respectively amputated from its N-terminal
part, this results in a reduction in immunoreactivity
5 which increases as a function of decreasing peptide
length. (cf Fig. 2, and in particular the sera P2, P5 and
P8 of Table 4).
Table 5 collates the results of the tests of
immunoreactivity of the peptides S42G, P37G, K32G, during
10 ELISA tests, to examine the influence_of amputation of the
N-terminal part of peptide S42G.
The values given correspond to the optical density
read at 492 nm multiplied by the factor 103.
TABLE 5
Dilution S42G P37G~~ K32G
Serum
A 8 1 1000 D D D
1/10,000 921 525 666
A 9 1/1000 D 2059 1675
1/10,000 672 275 229
A10 1/10,000 1485 1215
All 1/1000 D D 2108
1/10,000 397 321 343
A12 I/1000 D D D
1/10,000 1418 823 598
A13 1/1000 D D D
1/10,000 1519 1061 1247
A14 1/1000 1407 539 998
1/10,000 149 65 103
A15 1/1000 D D D
1/10,000 1357 905 715
A16 1/1000 D D D
1/10,000 D 2003 D
A19 1/1000 D D D
1/10,000 620 446 594
A20 1/1000 D D D
1/10,000 D 2338 D
A21 1/1000 1319 652 993
1/10,000 177 90 123
A22 1/1000 1216 702 876
1/10,000 164 102 129
A23 1/1000 D D D
1/10,000 860 557 774
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These results strengthen us regarding the
hypothesis according to which peptide S42G must be present
in its entire sequence from 2 to 45 in order to exhibit
maximum immunoreactivity.
In all cases, S42G is higher than P37G, which
indicates that the 5 N-terminal amino acids play a role in
the antigenicity.
However, in certain cases, little or no difference
is observed in reactivity between peptides P37G and K32G,
which would tend to prove that amino acids 7 to 11 are not
of major importance for the antigenicity of peptide S42G.
Moreover, a comparison of Tables 4 and 5 makes it
possible to demonstrate the importance of the 10
C-terminal amino acids of peptide S42G in the immuno-
reactivity itself.
Finally, the entire CORE protein (191 amino acids)
was replaced by peptide S42G (44 amino acids), to detect
the anti-HCV antibodies.
For this, the choice was made to compare the
sensitivity of peptide S42G to that of the 2nd generation
ORTHO HCV ELISA test; it is a test marketed by the company
ORTHO which comprises a fusion protein incorporating the
CORE protein of HCV, called C22-3; cf Vanderpoel, C.L.,
HTM Cuypers, H.W Reesink et al., 1991, Confirmation of
hepatitis C virus infection by new four antigen
recombinant immunoblot assay, Lancet 337; 317-319.
The comparison was carried out on 173 samples
which were positive with the 2nd generation ORTHO HCV
ELISA test.
Of 173 samples, the peptide S42G detected 151 of
them, which gives a sensitivity of 87.28%. The 22 discor-
dant sera were then analyzed using another 2nd generation
test, namely CHIRON RIBA HCV. It is an immunoblotting
intended for the detection of antibodies directed against
the hepatitis C virus antigens in human serum or plasma.
This test comprises five recombinant antigens (proteins).
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One of them is the recombinant CORE protein C22-3 obtained
in the form of a fusion protein with human superoxide
dismutase and expressed by a yeast.
It is found at the end of this confirmatory test
5 that none of the 22 sera exhibits reactivity towards the
band C22-3 (CORE).
Consequently, the sensitivity of peptide S42G is
100% relative to the CORE protein (C22-3) of the 2nd
generation CHIRON RIBA HCV test.
10 In conclusion, the CORE protein can be replaced by
the peptide S42G for the serological detection of HCV.
At this stage of the description of the invention,
it is appropriate to demonstrate the advantageous use of
synthetic peptides relative to that of recombinant protein
15 fragments. For that, the results and experimental
observations according to the invention were compared with
those of the publication, namely: Nasoff MS, Zebedee SL,
Inchauspe G, Prince AM, Identification of an immuno-
dominant epitope within the capsid protein of hepatitis C
20 virus, Proc Natl Acad Sci USA 1991; 88: 4641-5. This
publication relates to the production of recombinant
protein fragments of the CORE protein expressed in E.
coli, in conformity with Figure 3, and reports results
which are both similar to and different from those
25 reported above.
Indeed, the authors have expressed a recombinant
protein comprising the first 74 amino acids of the CORE
protein. The cloning strategy used results in the produc-
tion of fusion proteins. In other words, the 74 N-terminal
amino acids of the HCV CORE protein called CAP-A are
preceded by 308 amino acids of which the first 221
correspond to glutathione S-transferase. The reactivity of
this protein of 382 amino acids, of which only 20%
represent the CORE protein, towards HCV-positive sera is
good only in appearance given the very small number of
sera tested (5 human sera).
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In contrast, a protein comprising the sequence
69-120 of the CORE protein, called CAP-B, exhibits no
reactivity towards these same sera. This last result is in
relative contradiction with those of the present invention
since the peptide R40R which comprises the sequence 75-116
of this same protein reacts nevertheless with some sera
(about 10%, cf Table 1).
These same authors pursued their work, producing
other recombinant fusion proteins with, as sequence of the
CORE protein, the amino acids 1-20, 21-40, 41-60
respectively, called CAP-1, CAP-2 and CAP-3 respectively.
Their results, still obtained on a very small
number of human sera (9 sera), show that the sequence
21-40 reacts better than the sequence 1-20. The sequence
41-60 exhibits for its part no reactivity. This last
result is also in relative contradiction with those
presented according to the invention since the peptide
P42Y (amino acids 39-82) exhibits (cf Table 1) a high
reactivity although less than the peptide S42G.
Furthermore, another publication, namely: Okamoto
H, Munekata E, Tsuda F, Takahashi K, Yotsumoto C, et al.,
1990, Jpn, J. Exp Med 60, 223-233, has shown that a
peptide of 36 amino acids comprising the sequence 39-74 of
the CORE protein reacts with at least 70% of HCV-positive
sera.
Regarding the reactivity of the sequence 21-40,
these authors assert that in several cases, the reactivity
of this recombinant CORE protein fragment is greater than
that comprising the sequence 1-74.
The results obtained according to the invention
are not in agreement with the results of these authors
since it has been demonstrated that in all the cases of
HCV sera studied (cf Table 2), the reactivity of the
peptide S42G (sequence 2-45) is substantially greater than
that of the peptides S18D (sequence 2-21) and V22G
(sequence 22-45).
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The explanation proposed to explain these diver-
gent results relates to the production of different
fragments of the CORE protein in both cases.
According to the invention, the peptides obtained
by chemical synthesis comprise only the sequence mentioned
for each of them, and as explained above, this is one of
the advantages linked to these synthetic peptides.
In the case of the recombinant proteins obtained
by NASOFF et al., they are fusion proteins in which 308
amino acids, which are completely foreign to the CORE
protein, are present in the N-terminal position.
The recombinant proteins which comprise the
sequences 1-20 and 21-40 of the CORE protein are therefore
composed of a foreign sequence by more than 90~.
Although the authors stipulate that for the
detection tests, the glutathione S-transferase part of the
fusion protein does not disrupt the reaction since none of
the sera tested reacts with isolated glutathione
S-transferase (therefore no false positives), it appears
difficult to admit that these 90$ of fusion protein do not
in any way interfere in the reactivity with the anti-HCV
antibodies.
Indeed, the fact that the N-terminal part of the
sequences 1-20 or 21-40 of the CORE protein is linked to
the C-terminal part of the fusion protein contributes
towards restricting the accessibility of this N-terminal
region. In contrast, the C-terminal part is for its part
detected. It is highly probable that it is these struc-
tural stresses, imposed by the production of a recombinant
fusion protein in which the immunogenic part (the CORE
part) represents the minor part of the fusion protein
(less than 10% in this case), which lead to results
contrary to those presented.
The amino acids are represented according to
Figures 1 and 2, according to the convention of the Table
below:
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TABLE 6
3 LETTER CODE MOLECULAR WEIGHT
AMINO ACID
ALANINE Ala S9
CYSTEINE Cys 121
ASPARTIC AC. Asp 133
GLUTAMIC AC. Glu 147
PHENYLALANINE Phe 165
GLYCINE Gly 75
HISTIDINE His 155
ISOLEUCINE Ile 131
LYSINE Lys 146
LEUCINE Leu 131
I~iETHI ONINE Met 14 9
ASPARAGINE Asn 132
PROLINE Pro 115
GLUTAMINE Gln 146
ARGININE Arg 174
SERINE Ser 105
THREONINE Thr 119
VALINE Val 117
TRYPTOPHAN Trp 204
TYROSINE Tyr 181
According to the invention, the complete experi-
mental procedure described above clearly demonstrates, by
the types of antigen-antibody reaction carried out either
in solid phase (direct ELISA, cf Table 2), or by inhibi-
tion (cf Table 3), that the sequence 2-45 of the CORE
protein of the HCV virus obtained by solid phase chemical
synthesis not only proves substantially greater than
smaller sequences (S18D or V22G), but also that it
exhibits a sensitivity equivalent to the CORE protein
itself (protein C22-3 of the 2nd generation CHIRON RIBA
HCV test), and that consequently the synthetic peptide
S42G can be used in serological diagnostic tests in place
of the CORE protein.
It is evident from all these results that peptide
S42G appears to be the minimum but sufficient structure
which, from the point of view of its antigenic properties,
is equivalent to the CORE protein in its entirety and can
therefore replace it in a reagent for the detection of
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HCV.
AnticLenic polypeptide different from but
equivalent to peptide (a),
Structural analysis and molecular modeling of the
peptide S42G allowed the characterization of a
tridimensional motif that is composed of 2 a-helix
separated by a loop. This structure could define a
conformation-dependent antigenic domain. As an attempt to
obtain mimotopes which could mimic this domain, we have
screened a dodecapeptide library displayed on phage
(Ph.D.-12TM Phage Display Peptide Library Kit, New England
BioLabs Inc) with the mouse monoclonal antibody (Mab)
19D9D6. This Mab was directed against a conformational
epitope present within this region and also recognized by
human sera.
Four rounds of biopannings with decreasing amounts
of Mab 19D9D6 were performed according to the instructions
of the manufacturer manual. Then 72 clones were randomly
selected and their inserts were sequenced. Twenty three
different deduced aminoacid sequences were obtained.
However, among these different sequences, 5 were
represented 35, 10, 4, 3 and 2 times respectively. The
different peptides displayed on phage were tested for
target binding by ELISA with either Mab 19D9D6 (anti-HCV)
or an irrelevant Mab (anti OSPA Mab) used as a negative
control. Eleven mimotopes were specifically recognized by
Mab 19D9D6 and not by the anti OSPA Mab. However, 1 out of
the 11 mimotopes, the sequence of which was
overrepresented (WPHNWWPHFKVK), appeared to be highly
immunoreactive compared to the other peptides.
Interestingly, homology search using the clustalw aligment
(Mac Vector software 6Ø1, Oxford Molecular group PLC)
indicated that this mimotope did not share any obvious
homology with the core region previously defined by
spotscan as critical for MAb 19D9D6 binding whereas only a
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few homologies with aminoacids contained within this
region were found with other mimotopes (figure 4). They
allowed to localize the MAb 19D9D6 epitope on the
tridimensional structure of S42G.
5
Table 7
N of Clone sequence Nb of clones OD MAb 19D9D6-
clone /72 OD antiOSPA
MAb
1 WPHNWWPHFKVK 35 1.956
SEQ ID N0:8
2 HLNILSTLWKYR 1 1.114
SEQ ID N0:9
3 HKHAHNYRLPFS 4 0.714
SEQ ID NO:10
4 PKVLIVVLEASG 1 0.672
SEQ ID NO:11
5 QVYAEFKTSFRS 1 0.592
SEQ ID N0:12
6 GHIHSMRHHRPT 10 0.559.
SEQ ID N0:13
7 TSFHSKAEYTGI 1 0.559
SEQ ID N0:14
8 KDVDTSQDKMYW 1 0.507
SEQ ID N0:15
9 QNSSMMLVPWRT 1 0.481
SEQ ID N0:16
10 AETVESGLAKSH 1 0.456
SEQ ID NOsl7
11 SLRLGIVLLWKL 1 0.444
SEQ ID N0:18
The immunoreactivity of phage clones was tested
10 according to the manufacturer manual. The results were
expressed as the mean OD obtained with the MAb 19D9D6
against 2.5 x 108 phages - the OD obtained with the anti
borrelia burgdorferi OSPA MAb against the same number of
phages.
Clones N°1-6 were then tested in parallel with a
pool of 10 anti HCV core positive sera and a pool of 10
sera from healthy individuals
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Table 8
N of close Clone sequence OD (sera Core +) - OD
(sera Core-
~
1 WPHNWWPHFKVK 0.1
SEQ ID N0:8
HLNILSTLWKYR 0.350
SEQ ID N0:9
3 HKHAHNYRLPFS 0.286
SEQ ID NO:10
pKVLIVVLEASG 0.362
SEQ ID N0:11
QVYAEFKTSFRS 0
SEQ ID N0:12
GHIHSMRHHRPT 0.310
SEQ ID N0:13
The immunoreactivity of phage clones was tested
5 according to the manufacturer manual. The results were
expressed as the mean OD obtained with the pool of anti
core positive human sera against 2.5 x 108 phages - the OD
obtained with the pool of anti core negative human sera
against the same number of phages. As shown in table 8,
compared to the response obtained with negative sera, 5
out of the 6 tested clones gave a positive signal with the
pool of positive sera. This results are in agreement with
previous results, indicating that the epitope recognized
by MAb 19D9D6 was also recognized by human sera.
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1
SEQUENCE LISTING
<110> BIO MERIEUX
5 <120> Synthetic polypeptides equivalent to polypeptides
belonging to the hepatitis C virus (HCV) and
applications
<130> Hepatite C 2
<140>
<141>
<150> US N°09/196,155
15 <151> 1998-11-20
<160> 18
<170> PatentIn Ver. 2.1
<210> 1
<211> 45
<212> PRT
<213> Homo Sapiens
<400> 1
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
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Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
35 40 45
<210> 2
<211> 44
4 0 <212> PRT
<213> Homo Sapiens
<400> 2
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly
20 25 30
50 Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
35 40
55 <210> 3
<211> 191
<212> PRT
<213> Homo Sapiens
CA 02354152 2001-05-18
WO 00/31130 PCT/IB99/01933
2
<400> 3
Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
10 35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
15 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly
65 70 75 80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
2 5 115 120 12 5
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala
180 185 190
<210> 4
<211> 60
<212> PRT
<213> Homo sapiens
<400> 4
Met Ser Thr Ile Pro Leu Pro Gln Ala Leu Thr Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Glx Asx Val Lys Phe Pro Gly Gly Gly Glx Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
Thr Arg Lys Thr Ser Glx Arg Ser Glx Pro Arg Gly
50 55 60
CA 02354152 2001-05-18
WO 00/31130 PCT/IB99/01933
3
<210> 5
<211> 29
<212> PRT
<213> Homo Sapiens
<400> 5
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
1 5 10 15
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
25
<210> 6
<211> 16
<212> PRT
2 0 <213> Homo Sapiens
<400> 6
Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
1 5 10 15
<210> 7
<211> 13
3 0 <212> PRT
<213> Homo sapiens
<400> 7
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln
1 5 10
<210> 8
<211> 12
<212> PRT
<213> Homo sapiens
<400> 8
4 5 Trp Pro His Asn Trp Trp Pro His Phe Lys Val Lys
1 5 10
<210> 9
<211> 12
<212> PRT
<213> Homo Sapiens
<400> 9
His Leu Asn Ile Leu Ser Thr Leu Trp Lys Tyr Rrg
1 5 10
CA 02354152 2001-05-18
WO 00/31130 PCT/IB99/01933
4
<210> 10
<211> 12
<212> PRT
<213> Homo sapiens
<400> 10
His Leu Asn Ile Leu Ser Thr Leu Trp Lys Tyr Arg
1 5 10
<210> 11
<211> 12
_<212> PRT
<2i3> Homo Sapiens
<400> I1
2 0 His Leu Asn Ile Leu Ser Thr Leu Trp Lys Tyr Arg
1 5 10
<210> 12
<211> 12
<212> PRT
<213> Homo Sapiens
<400> 12
Gln Val Tyr Ala Glu Phe Lys Thr Ser Phe Arg Ser
1 5 10
<210> 13
<211> 12
<212> PRT
<213> Homo Sapiens
<400> 13
Gly His Ile His Ser Met Arg His His Arg Pro Thr
1 5 10
<210> 14
<211> 12
<212> PRT
50 <213> Homo Sapiens
<400> 14
Thr Ser Phe His Ser Lys Ala Glu Tyr Thr Gly Ile
1 5 10
<210> 15
CA 02354152 2001-05-18
WO 00/31130 PCT/IB99/01933
<211> 12
<212> PRT
<213> Homo Sapiens
5 <400> 15
Lys Asp Val Asp Thr Ser Gln Asp Lys Met Tyr Trp
1 5 10
15
<210> 16
<211> 12
<212> PRT
<213> Homo Sapiens
<400> 16
Gln Asn Ser Ser Met Met Leu Val Pro Trp Arg Thr
1 5 10
<210> 17
<211> 12
<212> PRT
2 5 <213> Homo Sapiens
<400> 17
Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Ser His
1 5 10
<210> 1a
<211> 12
3 5 <212> PRT
<213> Homo Sapiens
<400> 18
Ser Leu Arg Leu Gly Ile Val Leu Leu Trp Lys Leu
4 0 1 5 10
