Note: Descriptions are shown in the official language in which they were submitted.
CA 022~1904 1998-10-16
W097/39029 PCT~S97/06732
AN ANTIGENIC EPITOPE OF THE A DETERMINANT OF HEPATITIS
B SURFACE ANTIGEN AND USES THEREOF
BACKGROUND OF THE INVENTION
Technical ~ield
The present invention relates to an isolated peptide
corresponding to amino acid residues 117 to 128 of
hepatitis B surface antigen (HBsAg) and uses thereof. The
peptide is an antigenic epitope contributing to the a-
determinant and may be used, for example, as a diagnostic
reagent for the detection of hepatitis B virus (HBV) and in
the production of vaccines against HBV. Furthermore, the
invention more specifically relates to peptides containing
amino acid residues 121-124 of HBsAg, and thus having a
C(K/R)TC motif, as well as to uses of such peptides.
Backa~ound Information
Hepatitis B Virus (HBV) is a serious and widespread
human pathogen. Acute hepatitis causes significant
morbidity and mortality, and chronic infection with the
virus is associated with chronic hepatitis, cirrhosis and
hepatocellular carcinoma. Currently, approximately 1
million chronically infected people live in the United
States, and there are an estimated 300 million carriers
worldwide. HBV is a blood born pathogen which is spread by
contaminated serum and maternal-neonatal transmission.
Health care workers and others exposed to blood or blood
products are at an increased risk of acquiring HBV
infection. Transmission from acutely infected individuals
and from persistently infected carriers is well known. In
endemic regions, where HBV infection may occur in 75 to 95%
of the population and where the carrier rate exceeds 5%.
The maternal-neonatal transmission and horizontal
infections early in life are most critical because such
early acquisition of infection, which is usually
subclinical and unrecognized, is the ma~or risk factor in
. . .
CA 022~1904 1998-10-16
W097/39029 PCT~S97/06732
chronic HBV infection. Worldwide research on the prevention
of HBV infection has led to the detection of HBV carriers
and vaccine development.
The specific diagnostic marker for acute and chronic
HBV infection is the detection of the presence of hepatitis
B surface antigen (HBsAg). HBsAg is the major envelope
protein found in HBV. During the infectious cycle, a large
amount of HBsAg is produced in serum. Only a very small
portion of the total HBsAg exists as complete virions or
Dane particles [Dane, et al., Lancet 1:695-698 ~1970)]. The
majority of HBsAg assembles as small subviral particles,
without viral cores, that are in high excess over HBV
virions. The presence of HBsAg in serum is detected by
conventional sandwich immunoassays [Overby, et al., Vox
San~uinis 24: 102-113 (1973); Wands, et al., Proc. Natl.
Acad. Sci. USA 78: 1214-1218 ~1981); Usuda, et al., J.
Immunol. Methods 87: 1300. (1986)i Zuckerman eds., Viral
he~atitis and liver disease, Alan R. Liss Inc, New York,
~1988)]. These immunoassays are extensively used worldwide
for diagnosis of HBV infection and for screening blood
donors and pregnant women.
The HBsAg protein in the viral and subviral particles
displays the major B-cell antigenic determinants which can
induce a protective immune response. This has led to the
use of native or recombinant HBsAg particles as vaccines
for prevention of HBV infection [Szmuness, et al., N. Enal.
J. ~ç~. 303:833 (lg80); Zuckerman eds., Viral he~atitis and
liver disease, Alan R. Liss Inc, New York, (1988)].
Serologically, HBsAg contains a common epitope, referred to
as the a-determinant, and two sets of subtype determinants
d or y and w or r that are mutually exclusive [Le Bouvier,
J. Infect, Dis. 123: 671 (1971); Bancroft, et al., J.
Immunol. 109: 842-848 (1972)]. The combination of the
common and subtype determinants results in four major
subtypes: adw, ayw, adr and ayr. In general, the anti-
HBsAg immune response in humans mainly targets the a-
CA 022~1904 1998-10-16
W097/3~29 PCT~S97/06732
determinant associated with all subtypes of HBV [Iwarson,
et al., J. Med. Virol. 16: 89-95 (1985); Zuckerman eds.,
Viral he~atitis and liver disease, Alan R. Liss Inc, New
York, (1988)]. Immunization with one HBsAg subtype confers
protection against HBV infection with all subtypes
[Szmuness, et al., N. Encl. J. Med. 303:833 (1980)],
indicating that the a-determinant is of the greatest
clinical interest.
On a molecular level, HBsAg is a 226 amino acid
membrane protein. Primary sequence analysis suggests that
HBsAg contains four transmembrane domains and two
hydrophilic loops with one loop in the extracellular space
and one loop buried inside the HBV particle [Stirk, et al.,
Interviroloqv 33:148-158 (1992)]. The a-determinant is
located in the extracellular loop and spans amino acid
residues 101-159. This hydrophilic region (aa 101-159) is
extremely rich in cysteine, containing eight cysteine
residues. The formation of disulfide bonds among these
cysteines is crucial in defining the structure of the a-
determinant. The significance of the disulfide bonds was
clearly demonstrated in early studies that showed HBsAg
immunogenicity was greatly reduced by disulfide bond
reduction followed by alkylation of the reduced sulfhydryl
groups [Vyas, et al., Science 178:1300 (1972); Dreesman, et
al., J. Gen. Virol. 19:129 (1973)].
Studies also suggest that the a-determinant contains
several non-overlapping epitopes, indicating that it is not
a single determinant, but it is most likely composed of
several epitopes located on different regions of HBsAg
[Gerin, et al., Proc. Natl. Acad. Sci. USA 80:2365-2369
(1983); Peterson, et al., J. Immunol. 132:920-927 (1984)].
More significantly, the a-determinant can be mimicked by
synthetic peptides derived from the a-determinant region
(aa 101-159) [Lerner, et al., Proc. Natl. Acad. Sci. USA
78:3403-3407 (1981); Bhatnagar, et al., Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al., Nature
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295:158-160 (1982); Prince, et al., Proc. Natl. Acad. Sci.
USA 79:579-582 (1982); Gerin, et al., Proc. Natl. Acad.
Sci. USA, 80:2365-2369 (1983); Ohnuma, et al., J. Immunol.
145:2265-2271 (1990); Manivel, et al., J. Immunol.
5 149:2082-2088 (1992)~.
The finding that synthetic peptides can mimic the a-
determinant and elicit protective immunoresponses presents
a new approach to HBV vaccine development. Although native
or recombinant HBsAg based vaccines against HBV are wildly
available, there is still an urgent need for the
development of an appropriate vaccine for economical mass
immunization. Chemically synthesized peptides, therefore,
may have advantages in terms of cost and safety of HBV
vaccination programs. Indeed, such a strategy is being
employed for the development of potential vaccines against
HBV.
Several research groups [Bhatnagar, et al., Proc.
Natl. Acad. Sci. USA 79:4400-4404 (1982); Prince, et al.,
Proc. Natl. Acad. Sci. USA 79:579-582 (1982); Tam, ed.,
Synthetic ~e~tide: a~roaches to biolo~ical problems, Alan
R. Liss Inc, New York, (1989)] demonstrated that a
nonapeptide sequence (aa 139-147) of HBsAg represents an
essential part of the a-determinant, eliciting antibodies
crossreactive with both ad and ay subtype of HBsAg. It was
further discovered that the cyclic version of the peptide
(aa 139-147), in which there is a disulfide bond between
Cysl39 and Cysl47 as shown in Figure 6, more closely
resembles the native conformation of the a-determinant
[Tam, ed., Synthetic ~eDtide: approaches to bioloaical
problems, Alan R. Liss Inc, New York, (1989)]. The well
defined loop structure and immunogenicity of the cyclic
peptide (aa 139-147) make it an ideal candidate for a
potential synthetic vaccine. However, recent research
have established that a vaccine-induced escape mutant of
HBV contains an amino acid substitution located in the loop
region, with Glyl45 being substituted by Argl45 [Carman, et
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al., Lancet 345:1406-1407 ~1990); Carman and Thomas,
Gastroentroloov 102:711-719 (1992)]. As a result of the
Gly-Arg 145 mutation, the HBV mutant escapes from the
vaccine-induced immunity and is undetectable by monoclonal
based immunoassay [Carman, et al., Lancet 345:1406-1407
(1990); Carman and Thomas, Gastroentrolo~y 102:711-719
(1992)]. Thus, the cyclic peptide (aa 139-147) has limited
utility as a synthetic vaccine and diagnostic reagent due
to the emergence of new HBV mutants.
Dreesman, et al. [Dreesman, et al. Nature 295:158-160
(1982)] identified another cyclic synthetic peptide (aa
122-137), in which there is a disulfide bond between Cysl24
and Cysl37, as shown in Figure 6, contributing to the a-
determinant. However, the cyclic peptide showed much lower(i.e., 3 order lower) affinity than the native HBsAg
[Ionescu-Matiu, et al., J. Immunol. 130:1947-lg52 (1983)].
In addition, as shown by the sequence alignment in Figure
5, there is an extensive sequence variation in the region
covered by aa 124 to 137 among subtypes and mutants of HBV,
indicating that the whole loop structure (aa 124-137) is
less likely to be a common epitope shared by all subtypes
of HBsAg.
The same peptide sequence (aa 124-147), but presented
as an oligomerized form, was also identified as a
conformational epitope contributing to the a-determinant
[Manivel, et al., J. Immunol. 149:2082-2088 (1992);
European Patent Application WO 94/05698 (1994)]. This
oligomerized peptide elicits an immune response that
predominantly targets the a-determinant. Compared to the
corresponding monomeric peptide, the oligomerized peptide
is more immunogenic [Manivel, et al., J. lmmunol. 149:2082-
2088 (1992)]. However, the exact structural feature of the
oligomerized peptide is undefined. The undefined structure
of the oligomerized peptide and the extensive variation of
the peptide sequence limit its utility as a synthetic
vaccine and a diagnostic reagent.
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Gerin et al. ~Gerin, et al., Proc. Natl. Acad. Sci.
USA 80:2365-2369 (1983)i Milich and Chisari, U.S. Patent
No. 4,599, 230] demonstrated that a synthetic peptide (i.e.,
5 aa 110-137) can elicit a subtype specific antibody response
against HBsAg. However, because of the peptide subtype
specificity, only the HBsAg's subtype from which a peptide
is derived can be immune recognized. For instance, an
antibody elicited by a peptide sequence derived from the
ayw subtype of HBsAg, will not be able to recognize the
three other subtypes: adr, awy, awr of HBV [Gerin, et al.,
Proc. Natl. Acad. Sci. USA 80:2365-2369 (1983)]. This is
due to the fact that the peptide (aa 110-137) contains
several subtype specific amino acids (see Figure 5) which
mimic the subtype determinant more effectively than the a-
determinant. The subtype specific immune response of the
peptide sequence (aa 110-137) prevents its use as a general
vaccine against all subtypes of HBV infections.
More recently, using two shorter peptides ~i.e., aa
115-129 and aa 123-136) of HBsAg, Ohnuma, et al. found that
the peptide (aa 115-129) bears an epitope contributing to
the a-determinant while the peptide (aa 123-136) represents
mainly a subtype specific epitope [Ohnuma, et al. J.
25 Immunol. 145:2265-2271 (1990)]. Their studies showed that
30% of human serum samples from HBsAg-immunized individuals
recognized the peptide (aa 115-129), indicating that the
peptide (aa 115-129) is an immunodominant epitope.
However, similar to the longer peptide sequence (aa 110-
30 137), the shorter peptide sequence (aa 115-129) contains
the same subtype specific amino acids at position 117, 120,
122, 125, 126, 127 and 128. Therefore, on a structural
basis, it is unknown why the shorter peptide (aa 115-129)
mimics the a-determinant better than the longer peptide (aa
35 110-137).
In summary, several non-overlapping peptide sequences
derived from the region 110-160 of HBsAg have been
CA 022~1904 1998-10-16
W097/39029 PCT~S97/06732
identified as antigenic and immunogenic epitopes
contributing to the a-determinant. However, due to subtype
sequence variations of HBsAg and the emergence of HBV
mutants, there still existed a need to identify peptide
epitopes contributing to the a-determinant with consensus
- sequences shared by all subtypes of HBsAg including most
HBV mutants. Such peptide epitopes will enhance the
antigenicity and immunogenicity to the a-determinant, and
the induced immune response will target all subtypes of
HBsAg including most HBV mutants.
All U.S. patents and publications referred to herein
are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The main object of the present invention is to provide
a common peptide epitope contributing to the a-determinant
of HBsAg that is shared by all subtypes of HBsAg including
most HBV mutants.
More specifically, the present invention encompasses
an isolated or purified linear or cyclic peptide showing
cross reactivity with anti-hepatitis B surface antigen
(HBsAg) antiserum which comprises the "a" epitope of HBsAg.
The peptide is a 12 mer of the amino acid sequence
corresponding to amino acid residues 117 to 128 of HBsAg.
This peptide may contain a disulfide bond between amino
acid residues 121 and 124. This bond yields approximately
an eight to ten fold increase in affinity as compared to
the linear peptide.
The peptide may contain the C(K/R)TC motif and have
the amino acid sequence
Xlx2x3x4c~K/R)Tcxsx6x7xs
wherein:
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x1 is selected from the group consisting of serine,
glycine, alanine, valine and an aliphatic amino acid of
from two to six carbon atoms;
X2 is selected from the group consisting of threonine,
serine, alanine and glycine;
X3 is glycine or alanine;
X4 is selected from the group consisting of proline,
serine and threonine;
Xs is selected from the group consisting of threonine,
methionine, alanine, serine and glycine;
X6 is selected from the group consisting of threonine,
serine, alanine, isoleucine and an aliphatic amino acid of
from two to six carbon atoms;
X7 is selected from the group consisting of proline,
leucine, threonine, serine, alanine and an aliphatic amino
acid of from two to six carbon atoms; and
X8 is selected from the group consisting of alanine,
glycine, and valine.
Preferably, the peptide has the amino acid sequence
STGPC(K/R)TCTT PA or AAGPC( K/R) TCATPA.
Furthermore, the present invention also includes a
vaccine against hepatitis B comprising a pharmacologically
effective dose of a cyclic peptide showing cross-reactivity
with HBsAg antiserum which comprises the "a" epitope of
HBsAg, wherein said peptide comprises amino acid residues
121-124 of HBsAg having a C(K/R~TC motif, and wherein said
peptide is prepared by synthetic means and a
pharmaceutically acceptable carrier. Preferably, the
pharmaceutically acceptable carrier is alum. The peptide
of the vaccine may further comprise a myristic acid residue
added to the amino terminus.
Additionally, the present invention encompasses an
antibody directed against the peptides of the invention as
well as any fragments thereof. More specifically, it
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includes those antibodies produced in response to peptides
comprising residues 121-124 and having a C(K/R)TC motif.
The antibody may be either monoclonal or polyclonal.
Moreover, the present invention also includes a kit
for detecting the presence of hepatitis B surface antigen
or antibody in a test sample comprising a container
containing a polypeptide comprising a cyclic peptide
showing cross-reactivity with HBsAg antiserum which
comprises the "a" epitope of HBsAg. The peptide comprises
amino acid residues 121-124 of HBsAg having a C (K/R)TC
motif. The polypeptide may be prepared by synthetic means
and may be attached to a solid phase.
Additionally, the present invention includes a method
for detecting hepatitis B virus surface antigen (HssAg) in
a test sample suspected of containing HBsAg comprising the
steps of: a. contacting the test sample with an antibody or
fragment thereof which specifically binds to a cyclic
peptide showing cross-reactivity with HBsAg antiserum which
comprises the "a" epitope of HBsAg, wherein the peptide
comprises amino acid residues 121-124 of HBsAg having a
C(K/R)TC motif, for a time and under conditions sufficient
to allow the formation of antibody/antigen complexes; and
b. detecting the complexes containing the antibody, wherein
said antibody is producing by utilizing a polypeptide
prepared by synthetic means. The antibody may be attached
to a solid phase and may be monoclonal or polyclonal.
The invention also includes a method for detecting
hepatitis B antibodies in a test sample suspected on
containing these antibodies comprising the steps of:
a. contacting the test sample with a probe polypeptide
wherein the polypeptide comprises a cyclic peptide showing
cross-reactivity with HBsAg antiserum which comprises the
- "a" epitope of HBsAg, wherein the peptide comprises amino
acid residues 121-124 of HBsAg having a C (K/R)TC motif, for
a time and under conditions sufficient to allow the
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formation of antigen/antibody complexes; and b. detecting
the complexes which contain the probe polypeptide said
antibody. The probe polypeptide may be attached to a solid
phase. This solid phase may be selected from the group
5 consisting of beads, microtiter wells, wall of test tube,
nitrocellulose strips, magnetic beads and non-magnetic
beads.
Furthermore, the present invention also includes a
10 method for producing antibodies to HBsAg comprising
administering to an individual an isolated, immunogenic
polypeptide or fragment thereof comprising a cyclic peptide
showing cross-reactivity with HBsAg antiserum which
comprises the "a" epitope of HBsAg, wherein the peptide
comprises amino acid residues 121-124 of HBsAg having a
C (K/R)TC motif, in an amount sufficient to produce an
immune response. The polypeptide may be prepared by
synthetic means.
The invention also includes a diagnostic reagent
comprising a polypeptide or fragment thereof derived from
hepatitis B surface antigen, wherein the polypeptide or
fragment thereof comprises a cyclic peptide showing cross-
reactivity with HBsAg antiserum which comprises the "a~
25 epitope of HBsAg, wherein the peptide comprises amino acid
residues 121-124 of HBsAg having a C(K/R)TC motif. The
polypeptide may be produced by synthetic means.
Further, the invention includes all of the above
30 entities and uses wherein the peptide may be, for example,
amino acids 117-128 of HBsAg or any other length of an
amino acid sequence or fragment of HBSAg provided the
peptide contains amino acid residues 121-124 and thus the
C (K/R)TC motif.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 ~A) represents binding of mAb H166 to rHBsAg
subtype ac~ and ay with equal affinity in ELISA. (B)
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11
represents inhibition of mAb H166 binding to rHBsAg (ay) by
free rHBsAg (ay and ad). Degree of inhibition was
determined using competitive ELISA as described in the
examples.
Figure 2 represents inhibition of mAb H166 binding to
rHBsAg (ay) by cyclic peptides derived from HBsAg sequence.
Degree of inhibition was determined using competitive ELISA
as described in the examples.
Figure 3 represents inhibition of mAb H166 binding to
rHBsAg (ay) by the linear and the cyclic peptide I
(STGPCKTCTTPA). Degree of inhibition was determined using
competitive ELISA as described in the Examples.
Figure 4 represents a plot of ~GmUt-wt at the various
alanine substitution sites in peptide I derived from HBsAg.
Figure 5 represents a sequence alignment of the region from
residues 101 to 160 of subtypes of HBsAg and HBsAg mutants.
The alignment was performed as described in the Examples.
All sequences are denoted by their Genbank accession codes
(source). The original sequences and their authors can
easily be retrieved from the NCBI (National Center for
Biotechnology Information, Bethesda, MD) or from GCG
(Genetic Computer Group, Madison, WI) using the Genbank
accession codes as keywords. The listed subtypes (SUBT) and
genotypes (GTYPE) are from the original references. HBsAg
mutants are denoted as 'mut' and several undefined subtypes
of HBsAg sequences are labeled as 'nd'. The C(K/R)TC motif
is shadowed.
Figure 6 illustrates the proposed structures of the HBsAg a
determinant. (A) Amino acids 124-147 of H3sAg form two
putative loops via the disulfide bridges between cysteines
at 124-137 and 139-147 [Bhatnagar, et al., Proc. Natl.
Acad. Sci. USA 79: 4400-4404, (1982); Dreesman, et al.,
Nature 295:158-160, ~1982); Brown, et al., J. Immunol
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12
Methods, 72: 41-48, (1984)]. (B) The C(K/R)TC motif forms
a loop structure via the disulfide bridging between Cysl21
and Cysl24.
DETAILED DESCRIPTION OF THE INVENTION
The subject invention relates to an isolated peptide
corresponding to amino acid residues 117 to 128 of
hepatitis B surface antigen (HBsAg) and uses thereof. The
peptide is an antigenic epitope contributing to the a
determinant and may be used as a diagnostic reagent for the
detection of hepatitis B virus (HBV) and in the production
of vaccines against HBV. Further, the invention relates to
the C(K/R)TC motif (aa 121-124) within the epitope which is
the main binding site for recognition by anti-a monoclonal
antibody. This motif was discovered to be a common epitope
shared by all subtypes of HBsAg including most HBV mutants.
The present invention also encompasses peptides and kits
containing this motif.
Initially, the peptides of the invention were
identified by using an anti-a monoclonal antibody (H166).
The peptide sequence corresponding to amino acid residues
117 to 128 of HBsAg specifically binds to this anti-a
monoclonal antibody. In particular, the peptide
specifically inhibits the native protein HBsAg binding to
the anti-a monoclonal antibody with a IC50 (50% inhibition
concentration) in the range of 10-6 to 10-7 M, and the
maximum inhibition by the peptide is about 80% (Figure 2
and Figure 3). Therefore, the peptide (aa 117-128)
contains an antigenic epitope contributing to the a-
determinant.
In addition to the peptides noted above, a previously
unidentified disulfide bond between residues Cysl21 and
Cysl24 of HBsAg, as shown by Figure 6B, is disclosed by the
present invention. As stated in the background, disulfide
bond formation among the cysteine residues in the a-
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W097/39029 PCT~S97/06732
13
determinant is crucial in defining the structure of the a-
determinant. Formation of a correct disulfide bond will
enhance the ability of the peptide to mimick the a-
determinant, resulting in increased binding affinity of the
peptide for anti-a monoclonal antibody. Indeed, compared
to the native protein HBsAg, the cyclic form of the peptide
(aa 117-128) is only 20-fold less effective, whereas the
linear form of the peptide is 160-fold less effective in
the inhibition of the anti-a monoclonal antibody binding to
HBsAg. Therefore, the loop structure constrained by the
disulfide bond between Cysl21 and Cysl24 represents more
closely the native conformation of the a-determinant.
It is well known in the art that appropriate
restriction of the conformational freedom of synthetic
peptides lead to their enhanced performances.
Conformational restriction is usually achieved by
crosslinking amino acids within the synthetic peptide. The
simplest way to achieve this is to cyclize the peptide via
disulfide bridges. There are numbers of examples in which
the procedure has improved the antigenic and/or immunogenic
properties of synthetic peptides (Kennedy, et al., J.
Virol. 46:653-655 (1983), Ferguson, et al., Virolo~v
143:505-515 (1985)). Similarly, formation of the disulfide
bond between Cysl21 and Cysl24 imparts an eight fold
increase in affinity to the linear peptide (aa 117-128) for
immune recognition by the anti-a monoclonal antibody
(F igure 3).
Similar to the previously identified peptide sequences
(aa 110-137 and aa 115-129), both linear and cyclic peptide
sequences (aa 117-128) of the present invention contain the
same subtype specific amino acids at position 117, 120,
122, 125, 126, 127 and 128. The effect of these subtype
specific amino acids on the peptides mimicking the a-
determinant is unknown. In order to identify the most
critical residues in the peptide (aa 117-128) for mimicking
the a-determinant, a set of alanine-substituted analogs of
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14
the peptide (aa 117-128), which represent single amino acid
substitutions by alanine, were used. Using the alanine-
substituted analogs of the peptide (aa 117-128), three
residues Cysl21, Thrl23 and Cysl24 were found to be the
most critical residues for the anti-a monoclonal antibody
recognition (see Example VII). Substitution of alanine for
any one of the three residues completely eliminated the
peptide (aa 117-128) binding to the anti-a monoclonal
antibody (see Table I and Figure 4). Minor effects (i.e.,
a less than 10 fold decrease or increase in binding
affinity) were observed for the remaining residues in the
sequence (aa 117-128) (see Table I and Figure 4). These
results indicate that the CXTC motif, where position 122
can be any amino acid as represented by the x residue (see
Summary section), is the most important structural feature
for antibody recognition and for mimicking the a-
determinant. In other words, it is the CXTC motif that
contributes to the a-determinant, not the remaining
residues in the peptide sequence.
Furthermore, sequence analysis indicated that the CXTC
motif is highly conserved among 100 subtypes and mutants of
HBsAg isolates. As shown by the sequence alignment (see
Figure 5), although there are many sequence variations in
the region of residues 101 to 160, the CXTC motif is fully
conserved in all of the sequences shown in Figure 5. In
addition, the residue at position 122 is relatively
conserved; it is either Lys or Arg in all of the sequences
shown in Figure 5. Also, the alanine-substituted alalogs
showed that keeping Lys or Arg at position 122 imparted
approximately a ten-fold increase in affinity (see Table
I). Therefore, using both alanine-substituted alalogs and
computational sequence analysis, the CXTC, or more
accutately the C(K/R)TC motif was identified as the key
element or the essential core epitope within the peptides
~aa 117-128 or aa 115-129) for mimiking the a-determinant.
Sequence analysis also indicated that the C(K/R)TC motif is
a common epitope shared by all subtypes of HBsAg including
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W097/39029 PCT~S97/06732
most HBV mutants. Thus, the conservation of the C(K/R)TC
motif explained on a structural basis why immunization with
one subtype of HBsAg can confer protection against HBV
infection with all subtypes. Since all subtypes contain
the C(K/R)TC motif, immunization with one subtype will
induced neutralizing antibodies targeting the C(K/R)TC
motif. Thus, the antibodies will target all HBsAg subtypes
as long as they contain the C(K/R)TC motif.
The highly conserved C(K/R)TC motif and its antigenic
nature permit the use of the peptide (aa 117-128) or
synthetic peptides containing the C(K/R)TC motif as
synthetic vaccines. Since the loop structure of the
C(K/R)TC motif is well constrained by the disulfide bond
between Cysl21 and Cys 124, synthetic peptides containing
this motif will mimic the native structure of the a-
determinant on HBsAg and elicit neutralizing antibodies
that target the C(K/R)TC motif on all subtypes and most
mutants of HBsAg. Furthermore, the finding that the high
binding affinity can be maintained by the C(K/R)TC motif
establishes the feasibility of using the C(K/R)TC motif in
tracer epitopes for the detection of HBsAg in a homogeneous
immunoassay format. These potential utilities are
described below.
Vaccine Pre~aration
The peptides of the invention can serve as synthetic
vaccines by conjugating the peptides to immunogenic
carriers. Suitable carriers include proteins,
polysaccharides such as latex functionalized sepharose,
agarose, cellulose, cellulose beads, and polymeric amino
acids such as polyglutamic acid and polylysine. Examples
of protein substrates or carriers include serum albumins,
keyhole limpet hemocyanin, immunoglobulin molecules,
thyroglobulin, ovalbumin, tetanus toxoid, and yet other
proteins known to those skilled in the art.
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16
Conjugation methods are known in the art and include
but are not limited to using N-succinimidyl-3-(2-
pyridylthio)propionate (SPDP) and succinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Either
the amino or the carboxyl terminal of the peptides
disclosed here can be modified by adding a cysteine
residue. These reagents create a disulfide linkage between
themselves and peptide cysteine residues on one protein and
an amide linkage through the epsilon-amino on a lysine, or
other free amino group in the other. A variety of such
disulfide/amide-forming agents are known. Other
bifunctional coupling agents form a thioester rather than a
disulfide linkage. Many of these thio-ether-forming agents
are commercially available and are known to those of
ordinary skill in the art. The carboxyl group of the
peptides also can be activated by combining them with
succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium
salt, and the conjugation of the peptides to carriers can
be achieved by the formation of an amide bond.
Typically, such synthetic vaccines are prepared as
injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in or suspension in
liquid prior to injection also may be prepared. The
preparation may be emulsified or the protein may be
encapsulated in liposomes. The active immunogenic
ingredients often are mixed with pharmacologically
acceptable excipients which are compatible with the active
ingredient. Suitable excipients include but are not
limited to water, saline, dextrose, glycerol, ethanol and
the like; combinations of these excipients in various
amounts also may be used. The vaccine also may contain
small amounts of auxiliary substances such as wetting or
emulsifying reagents, pH buffering agents, and/or adjuvants
which enhance the effectiveness of the vaccine. For
example, such adjuvants can include aluminum hydroxide, N-
acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-
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17
acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also
referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-
isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-
hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred
to as MTP-PE), and RIBI (MPL + TDM+ CWS) in a 2%
squalene/Tween-80~ emulsion.
The vaccines usually are administered by intravenous
or intramuscular injection. Additional formulations which
are suitable for other modes of administration include
suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include
but are not limited to polyalkylene glycols or
triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of
about 0.5% to about 10%, preferably, about 1% to about 2%.
oral formulation include such normally employed excipients
as, for example pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate and the like. These compositions may
take the form of solutions, suspensions, tablets, pills,
capsules, sustained release formulations or powders and
contain about 10% to about 95% of active ingredient,
preferably about 25% to about 70%.
Vaccines are administered in a way compatible with the
dosage formulation, and in such amounts as will be
prophylactically and/or therapeutically effective. The
quantity to be administered generally is in the range of
about 5 micrograms to about 250 micrograms of antigen per
dose, and depends upon the subject to be dosed, the
capacity of the subject~s immune system to synthesize
antibodies, and the degree of protection sought. Precise
amounts of active ingredient re~uired to be administered
also may depend upon the ~udgment of the practitioner and
may be unique to each subject. The vaccine may be given in
a single or multiple dose schedule. A multiple dose is one
in which a primary course of vaccination may be with one to
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ten separate doses, followed by other doses given at
subsequent time intervals required to maintain and/or to
reinforce the immune response, for example, at one to four
months for a second dose, and if required by the
individual, a subsequent dose or doses after several
months. The dosage regimen also will be determined, at
least in part, by the need of the individual, and be
dependent upon the practitioner~s judgment.
Pre~aration of antibodies a~ainst the C(K/R)TC motif
The peptides prepared as described herein are used to
produce antibodies against the C(K/R)TC motif, either
polyclonal or monoclonal. The peptide could be the
sequence aa 117-128 of HBsAg, it also could be any peptide
as long as it contains C(K/R)TC motif (aa 121-124). When
preparing polyclonal antibodies, a selected mammal (for
example, a mouse, rabbit, goat, horse or the like) is
immunized with the peptide-carriers disclosed herein.
Serum from the immunized animal is collected after an
appropriate incubation period and treated according to
known procedures. If serum containing polyclonal
antibodies to the C(K/R)TC motif contains antibodies to
other antigens, the polyclonal antibodies can be purified
by, for example, immunoaffinity chromatography. Techniques
for producing and processing polyclonal antibodies are
known in the art and are described in, among others, Mayer
and Walker, eds., Immunochemical Methods In Cell and
Molecular BioloaY, Academic Press, London (1987).
Antibodies specifically against the C(K/R)TC motif also may
be obtained from a mammal previously immunized with HBsAg.
An example of a method for purifying antibodies specific to
the C(K/R)TC motif from serum of an individual immunized
with HBsAg using affinity chromatography is provided
herein.
Monoclonal antibodies directed against the C(K/R)TC
motif also can be produced hy one skilled in the art. The
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19
general methodology for producing such antibodies is well-
known and has been described in, for example, Kohler and
Milstein, Nature 256:494 (lg75) and reviewed in J.G.R.
Hurrel, ed., Monoclonal Hvbridoma Antibodies: Techniaues
and A~lications, CRC Press Inc., soca Raton, FL (1982), as
well as that taught by L. T. Mimms et al., Viroloov
176:604-619 (1990). Immortal antibody-producing cell lines
can be created by cell fusion, and also by other techniques
such as direct transformation of B lymphocytes with
oncogenic DNA, or transfection with Epstein-Barr virus.
See also, M. Schreier et al., Hybridoma Techniques, Scopes
(1980) Protein Purification, Principles and Practice, 2nd
Edition, Springer-Verlag, New York (1984); Hammerling et
al., Monoclonal Antibodies and T-Cell Hybridomas (1981);
Kennet et al., Monoclonal Antibodies (1980). Examples of
uses and techniques of monoclonal antibodies are well known
to those of skill in the art.
Monoclonal and polyclonal antibodies thus developed,
directed against the C(K/R)TC motif, are useful in
diagnostic and prognostic applications as well as in
passive immunotherapy. Monoclonal antibodies especially
can be used to produce anti-idiotype antibodies. These
anti-idiotype antibodies are immunoglobulins which carry an
~linternal image~ of the antigen of the infectious agent
against which protection is desired. See, for example, A.
Nisonoff et al., Clin. Immunol. Immuno~ath. 21:397-406
(1981), and Dreesman et al., J. Infect. Dis. 151:761
(1985). Techniques for raising such idiotype antibodies
are known in the art and exemplified, for example, in Grych
et al., Nature 316:74 (1985); MacNamara et al., Science
226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225
(1985). These anti-idiotypic antibodies also may be useful
for treatment of HBV infection.
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Immunoassa~ and Diaanostic Kits
Both the peptides of the present invention and the
antibodies raised against the C(K/T)TC motif are useful in
immunoassays to detect the presence of HBsAg or HBV in
biological test samples. The design of these immunoassays
is subject to variation, and a variety of these are known
in the arti a variety of these have been described herein.
Examples of assays which utilize labels as the signal
generating compound and those labels are described herein.
Signals also may be amplified by using biotin and avidin,
enzyme labels or biotin anti-biotin systems.
One of the competitive assay formats using solid phase
can be designed which utilize the signal labeled ( with
radioactive isotope, such as 1125, or with an enzyme, or
with biotin) synthetic peptides detailed herein and a
monoclonal or polyclonal antibodies directed against the
C(K/T)TC motif. In the assay format to detect the presence
of HBsAg in a human test sample, a known amount of signal
labeled peptides is first added to the human test sample,
then the human test sample containing certain amount signal
labeled peptides is contacted and incubated with a solid
phase coated with a monoclonal or polyclonal antibodies
directed against the C(K/T)TC motif. If HBsAg and HBV
particles are present in the test sample, they will compete
with the labeled peptides binding to the monoclonal or
polyclonal antibodies on the solid surface. After removal
of unbound materials and peptides by washing the solid
phase, the amount of peptides bound by the antibodies can
be determined by determining the level of radioactivity or
by adding an enzyme substrate or by adding the anti-biotin
conjugate following the addition of substrate. ~he reduced
signal is proportional to the amount of HBsAg and HBV
particles in the human test sample.
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In fluorescence polari~ation immunoassay (FPIA), which
is a homogeneous competitive assay,; i.e. it does not
reguire wash or separation steps, synthetic peptides,
particularly the C(K/R)TC motif, of the present invention
may be used as tracer epitopes for the detection of HBsAg.
Using synthetic peptides as tracer epitopes in FPIAs of
high molecular weight proteins is known in the art and is
described in, among others, Geysen, et al., J. Immunol.
Methods 102:259-274 (1987); Houghten, et al., Nature
354:84-86 (1991); Lam, et al., Nature 354:82-83 (1991);
U.S. Patent No. 4,833, 092. In the FPIA, peptides of the
present invention covalently linked to labeling group (such
as fluorescein) will be used as a tracer; and an antibody
(monoclonal or polyclonal) against the C(K/R)TC motif will
be used to form a complex with the peptide epitope tracer.
The system containing the complex of the epitope tracer and
the antibody then can be used to detect and quantitate the
presence of HBsAg in human test sample by monitoring the
change of fluorescence polarization.
The present invention can be illustrated by the use of
the following non-limiting examples: '
EXAMPLES
EXAMPLE I
PEPTIDE SYNTHESIS
All Fmoc protected amino acid and reagents used for
peptide synthesis were purchased from Applied Biosystem
(Foster City, CA) or Rainin Instrument Co. Inc.
(Emeryville, CA) Monoclonal antibody (mAb) H166 [Peterson,
et al., J. Immunol. 132: 920927 (1984)] was obtained from
the Abbott Monoclonal Antibody Development Group, Abbott
Park, Illinois. Recombinant HBsAg [Mimms, et al., J Virol.
Methods 25:211-232, (1989)], subtype ad and ay, was
obtained from Abbott Rare Reagent Development Group (Abbott
Park, IL). Alkaline phosphates-conjugated goat anti-mouse
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22
IgG and IgM were purchased from Pierce Chemical Co.,
(Rockford,IL).
All peptides were synthesized by the stepwise solid-
phase method of Merrifield ~Merrifield, J. Am. Chem. Soc.
85:2149-2154, (1963)] on an Applied siosystem 431A
Synthesizer (Foster City, CA) or a Rainin Symphony
Synthesizer (Emeryville, CA) using standard Fmoc (9-
fluorenylmethoxycarbonyl) chemistry. Peptides were cleaved
and deprotected with a mixture of 82% trifluoroacetic acid
(TFA), 5% phenol, 5% H2O, 5% thioanisole and 2.5%
ethanedithiol for 2-4 hours at 25~C and precipitated by
addition of cold ether. Crude peptides were purified by
reverse phase HPLC using a 5-50% acetonitrile gradient
containing 0.1% TFA. The homogeneity and identity of the
purified peptides were confirmed by electro-spray mass
spectrometry. All peptides were determined to be at least
95% pure. Cyclic peptides formed by intramolecular
disulfide bridging were synthesized using the air oxidation
method [Tam, et al., Proc. Natl. Acad. Sci. USA 83: 8082,
(1986)]. Briefly, purified linear peptides were dissolved
in 0.1 mM Tris-HCl buffer (pH 8.4) at a concentration of
0.5 mg/ml and stirred at 25 C for varying periods of time
exposed to air. Cyclization was monitored by HPLC. Cyclic
peptides were purified as described above and formation of
intramolecular disulfide bonds was confirmed by electro-
spray mass spectra.
EXAMPLE II
DETERMINATION OF AFFINITY
Enzyme-linked immunoassays (ELISA~ were used to
evaluate the affinity of monoclonal antibody H166 to the
rHssAg, subtype (ad and ay, 5 ~g/ml) in 0.2 M carbonate-
bicarbonate buffer (pH 9.0) and blocked with 1% ssA in Pss
buffer (pH 7.4). Twofold dilutions of H166, starting
concentration (1 ~g/ml), were subsequently added (100
~l/well) and the ad and ay. Briefly, microtiter plates
were coated with ~l/well of rHBsAg 100 plates were
incubated for 1 hour at 25 C. The wells were washed with
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PBS buffer containing 0.5% Tween 20, and 100 ~Ll/well of
alkaline phosphates-conjugated goat antimouse Ig at 5000
fold dilution was added and incubated for 1-2 hr at 25 C.
After washing, 100 ~l/well of p-nitrophenyl phosphate
substrate was added, and the absorbance was measured at 405
nm using an automated microtiter plate reader (Molecular
Devices Corp., Menlo Park, CA). ELISA results were used to
fit an analog of the Michaelis-Menten equation [Langone and
Van Vunakis eds, Methods in Enz~moloov, Academic Press, San
Diego, (1986)]:
[Ag--A~] = [Ag--Ab]ma~. x [Ab] + K
Where [Ag-Ab] is the antigen-antibody complex
concentration, [Ag-Ab] maX is the maximum complex
concentration, [Ab] is the antibody concentration, and Kd
is the dissociation constant. During the curve fitting,
the [Ag - Ab] was substituted with the value of OD405mm at
the given concentration of antibody. Both Kd and
[OD405nm]max~ which corresponds to the [Ag - Ab]~aX, were
treated as fitted parameters.
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EXAMPLE III
COMPETITIVE ELISAs
Competitive ELISAs were used to identify the potential
antigenic determinants and to compare their affinity
through the determined IC50s. Microtiter plates were
coated with rHBsAg and blocked with ~SA as described above.
Aliquots of peptides or rHBsAg competitors (50 ~l/well) at
varying dilutions were added together with 100 ~l of H166
at 0.4 ~l/ml, and incubated for 1 hour at 25'C. The wells
were washed with phosphate buffered saline (PBS) containing
0.5% Tween 20, and alkaline phosphatase-conjugated goat
anti-mouse Ig at 5000 fold dilution (100 ~l/well) was
added. The plates were incubated for 1-2 hr at 25 C,
washed, and 100 ~l/well of p-nitrophenyl phosphate
substrate was added, and the absorbance was measured at 405
nm. The percent inhibition of mAb H166 binding to rHBsAg
by peptides was calculated according to the 'following
equation:
. . OD40swith peptide - bkgd
Inhlbltlon% = 100 x 1- . .
OD40swlthout peptlde-bkgd
where the bkgd is the absorbance from the well without
coating of rHBsAg.
EXAMPLE IV
SEOUENCE ANALYSIS
Nucleotide sequences corresponding to ~BsAg coding
sequences were obtained from Genbank (release 89.0, 6/95)
and EMBL (release 42.0, 3/95). The nucleotide sequences
were translated into protein sequences and analyzed using
programs from the Wisconsin Genetic Computer Group sequence
analysis package (GCG, Version 8.0). Multiple sequence
alignment of protein sequence was performed using
progressive pairwise alignment (PILEUP, GCG), and sequences
were displayed using the program PRETTY ( GCG). Final
alignment and editing were performed manually.
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EXAMPLE V
IDENTIFICATION OF PEPTIDE EPITOPE CONTRIBUTING TO THE A-
DETERMINANT
Monoclonal antibody H166 is specific for the a-
determinant, recognizing nine different subtypes of HBsAg
as observed by Peterson et al.[Peterson, et al., J.
Immunol. 132: 920927 (1984)]. In order to quantitatively
compare the affinity of mAB H166 for different HBsAg
subtypes, the apparent dissociation constants (Kd) derived
from the fitting curves for both subtype ad and ay of
rHBsAg were determined via ELISA. As shown in Figure lA,
mAb H166 binds the ad and ay subtypes with equal affinity.
The Kd derived from the fitting curves for both subtypes ad
and ay, is approximately 0.9 ~g/ml, corresponding to lx10-
8M. Competitive ELISAs were also used to evaluate the
crossreactivity of mAb H166 with subtype ad and ay .
Figure lB shows the inhibition curves using free ad and ay
subtypes as competitors. It is clear that both subtypes
inhibit mAb H166 binding to rHBsAg with similar affinities.
The IC50s for subtype ay and ad are 0.026 ~M and 0.030 ~M
respectively. Thus, H166 recognizes a common epitope
shared by the ad and ay subtypes of HBsAg.
Since H166 is a monoclonal anti-a antibody, it can be
used to screen for an epitope that contributes to the a -
determinant of HBsAg. Identifying the epitope of H166 will
help to define the a determinant, or part of the a-
determinant, on HBsAg. To identify the H166 epitope, three
cyclic peptides derived from the extracellular hydrophilic
region of HBsAg were synthesized. The peptide sequences
are shown in Figure 2. Although the cyclic peptides II (aa
124-137) and III (aa 139-147) are known to be a major part
of the a-determinant [Bhatnagar, et al. Proc. Natl. Acad.
Sci. USA 79:4400-4404 (1982); Dreesman, et al. Nature,
295:158-160 (1982)] only cyclic peptide I (aa 117-128~
binds specifically to H166. As shown in Figure 2, the
cyclic peptide I inhibits 70% of HBsAg binding to the H166
-
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26
at a concentration of 2 mM. Therefore, it is clear that
peptide I represents the epitope of H166. It is
interesting to note that peptide II exhibits about 20%
inhibition at a concentration of 2 mM, probably because
part of its se~uence (aa 124-128) overlaps with peptide I
(aa 117-128).
EXAMPLE VI
AFFINITY OF LINEAR VS CYCLIC PEPTIDE I (aa 117-128)
To further characterize the identified epitope of mAb
H166, the IC50 of the cyclic peptide I (aa 117-128) was
compared to the IC50s of its linear sequence and the native
protein, rHBsAg (subtype ad), using competitive ELISA. As
shown in Figure 3, the IC50 of cyclic peptide I is 0.54 ~1,
only 20 fold less than the IC50 of the native protein,
rHBsAg (0. 026~1M). The high potency of the cyclic peptide
indicates that it contains the essential part of the
epitope on HBsAg recognized by the mAb. More importantly,
the IC50 of the linear peptide (4.0 ~M) is 8 fold higher
than the IC50 of the cyclic peptide, although both peptides
exhibit similar inhibitions at higher concentration (>0.1
mM). These result indicate that the cyclic peptide I
contains a micro conformation that is closer to the native
structure of the a determinant on HBsAg compared to the
linear peptide I.
An experiment was performed to verify that the
sulfhydryl groups of the linear peptide I (aa 117-128)
remained in a reduced form under the experimental
conditions. Linear peptide I was first incubated with H166
in PBS buffer for 1 hour, and the mixture was analyzed by
electro-spray mass spectrometry. No trace of the oxidized
molecular ion was observed in the mass spectra, confirming
that no oxidation of the sulfhydryl groups occurred during
the time course of the ELISA. These results indicate that
the linear peptide can be recognized by the mAb H166,
although the cyclic form is the favorable confirmation.
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27
EXAMPLE VII
RESIDUES IN PEPTIDE I (aa 117-128) CRITICAL FQR BINDING
AFFINITY
The cyclic peptide I (aa 117-128) contains a critical
residue at position 122 that differentiates the d and y
subtypes. For the d subtype, position 122 is Lys, and Arg
for the y subtype [Okamoto, et al. J. Virol. 61: 3030-3034,
(1987)]. The insensitivity of mAb H166 to the subtype-
specific residue implies this residue is not critical for
the binding affinity. To prove this hypothesis and to
identify the critical residues for mAb H166 recognition, a
set of alanine-substituted analogs of peptide I, which
represent single amino acid substitutions by alanine, were
synthesized. Inhibition of H166 binding to HBsAg by the
alanine-substituted peptides were determined using
competitive ELISA. Table 1 shows the alanine-substituted
peptide sequences and their determined IC50s.
Substitutions of Cysl21 and Cysl24 with Ala almost
completely eliminate specific binding to H166. IC50s could
not be obtained for these two peptides (121C and 124C)
because no concentration dependent inhibition curves were
observed in the concentration range 2.9x10-9 to 2.9x10-4 M.
Both peptides (121C and 124C) exhibited only 20% inhibition
at the highest peptide concentration (3 mM).
Under the assay conditions, the IC50 is approximately
inversely proportional to the binding affinity constant Ka
[Cheng, et al., Biochem. Pharmacol. 22: 3099. (1973);
Munson, et al., ~n~l. Biochem. 107:220-239, (1980)]. Thus,
the differences in binding free energy between alanine-
substituted peptide and the wild type peptide (~Gmut-wt)
were derived based on the equation:
~Gm~t-WI = - RT lnlc5ow~
where R is the gas constant and T is the temperature.
Figure ~ shows the plot of ~GmUt-wt at the alanine-
substitution site. It is very clear that Cysl21, Thrl23
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W097/39029 PCT~S97/06732
28
and Cysl24 are the most critical residues for H166
recognition, since alanine substitutions at these three
residues cause substantial loss in binding energy (>4.5
kcal/mol). Minor effects (0.5-1.5kcal/mol) were observed
for the remaining residues, including residue 122. These
results indicate that the CXTC motif is the main binding
site or the essential core epitope of H166 on HBsAg where
position 122 can be any amino acid as represented by the X
residue. These results also explain why mAb H166 binding
is insensitive to the ad and ay subtype-specific residue.
Since H166 recognition only requires the CXTC motif, it
will recognize all subtypes of HBV as long as the CXTC
motif is conserved in their HBsAg sequences.
EXAMPL E VI I I
SEOUENCE ALIGNMENT OF HBsAa
In order to evaluate the conservation of the CXTC
motif in all subtypes of HBsAg and HBsAg mutants, analysis
of 100 HBsAg sequences derived from human HBV genomes
retrieved from the Genbank and EMBL databases was
performed. The retrieved nucleotide sequences were
translated into protein sequences and aligned using PILEUP.
From the sequence analysis, 43 unique sequences in the
extracellular hydrophilic region from residues 101 to 160
HBsAg were identified and the sequence alignment is shown
in Figure 5. Although there are many sequence variations
in the entire region, it is very clear that the CXTC motif
is fully conserved in all retrieved sequences regardless of
the genotypes or subtypes. The alignment also shows that
residue 122 is relatively conserved; it is either Lys or
Arg in all sequences shown in Figure 5. From the sequence
analysis results, it can be concluded that the CXTC, or
more accurately the C(K/R)TC motif, is a common epitope
shared by all subtypes of HBsAg including HBsAg mutants.
