Note: Descriptions are shown in the official language in which they were submitted.
WO 94/19011 215641. 6 PCT/US94/02195
1
PEPTIDES FOR INDUCING CYTOTOXIC T LYMPHOCYTE
RESPONSES TO HEPATITIS B VIRUS
Government Support
The U.S. Government may have certain rights in this
invention pursuant to grants awarded by the National
Institutes of Health.
Background of the Invention
Cytotoxic T lymphocytes (CTLs) play an essential role
in fighting cells infected with viruses, intracellular
bacteria and parasites, and tumor cells. They do so by direct
cytotoxicity and by providing specific and nonspecific help to
other immunocytes such as macrophages, B cells, and other T
cells. Infected cells or tumor cells process antigen through
intracellular events involving proteases. The processed
antigen is presented on the cellular surface in the form of
peptides bound to HLA class I molecules to T cell receptors on
CTLs. MHC class I molecules can also bind exogenous peptides
and present them to CTLs without intracellular processing.
At the present time it is difficult to accurately
predict from the sequence of an antigenic protein how the
protein will be processed and which peptide portions will bind
HLA class I molecules and be presented to CTLs. Binding
motifs have been predicted for some HLA class I molecules
based on sequence analysis of peptides eluted from these
molecules (Falk et al., Nature 351:290 (1991)). Further, of
the peptides that are processed and do bind to HLA class I,
which ones will contain CTL-recognizable epitopes is not yet
predictable.
Hepatitis B Virus ("HBV") is a non-lytic virus which
has currently infected approximately 250 million people
worldwide. HBV infection in adults typically leads to an
PCT/US94/02195
WO 94/19011 21S6416
2
acute disease in the majority of cases, and to a chronic
disease state in a minority of patients. This ratio of acute
to chronic is reversed when the infection occurs close to the
time of birth. There is an increased incidence of
hepatocellular carcinoma in chronic HBV infection. A small
percentage of individuals who are infected with HBV in
adulthood develop fulminant hepatitis associated with a strong
immune response with high lethality.
While there is no effective treatment for HBV
infection, vaccines have been developed in recent years to
prevent HBV infection. The vaccines employ either HBV surface
antigen (HBsAg) purified from the plasma of chronic HBV
carriers, or HBsAg produced by recombinant DNA technology.
Synthetic HBsAg peptide-based vaccines have also been
proposed. See, for example, U.S. Patent Nos. 4,599,230 and
4,599,231. The anti-HBsAg vaccines, however, afford
protection in only about 90% of immunized individuals. Those
who are unimmunized, or immunized but unprotected, provide a
significant reservoir of potential infection.
The contribution of CTLs to immunity to HBV antigens
has been difficult to assess. Chisari et al. (Microbial
Pathogen. 6:31 (1989)) have suggested that liver cell injury
may be mediated by an HLA-Class I restricted, CD8+ cytotoxic T
cell response to HBV encoded antigens. Class I major
histocompatibility (MHC) -restricted cytotoxic T lymphocyte
responses have been identified for a variety of other viruses,
such as influenza. For example, Townsend et al., Cell 44:959
(1986) reported that epitopes of an influenza virus
nucleoprotein recognized by cytotoxic T lymphocytes could be
defined by synthetic peptides. In attempting to define the
cytotoxic T lymphocyte response to HBV, it has been shown that
peripheral blood lymphocytes from patients with acute and
chronic HBV may be able to kill autologous hepatocytes in
vitro, but the specificity of the cytolytic activity, its HLA
restriction elements, and cellular phenotype were not
established. See, Mondelli et al., J. Immunol. 129:2773
(1982) and Mondelli et al., Clin. Exp. Immunol. 6:311 (1987).
Moriyama et al., Science 248:361-364 (1990), have reported
WO 94/19011 2156416 PCTIUS94/02195
3
that the HBV major envelope antigen is expressed at the
hepatocyte surface in a form recognizable by envelope-specific
antibodies and by MHC class I-restricted, CD8+ cytotoxic T
lymphocytes.
As there is a large reservoir of individuals
chronically infected with HBV, it would be desirable to
stimulate the immune response of these individuals to respond
to appropriate HBV antigens and thereby eliminate their
infection. It would also be desirable to prevent the
evolution to a chronic HBV infection in individuals suffering
from an acute phase infection. Further, as the presently
approved HBV vaccines do not elicit protective immunity in
about 10% of immunized individuals, it would be desirable to
elicit more effective immunity, such as by increasing or
diversifying the immunogenicity of the vaccines. Quite
surprisingly, the present invention fulfills these and other
related needs.
Summary of the Invention
The present invention provides peptides which induce
MHC class I restricted cytotoxic T lymphocyte responses
against HBV antigen. The peptides of interest are derived
from the HBV envelope. In certain embodiments the CTL
inducing peptide will have the sequence HBenvl83-191 Phe-Leu-
Leu-Thr-Arg-Ile-Leu-Thr-Ile (Seq. ID No. 1); HBenv248-257 Phe-
Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu (Seq. ID No. 3);
HBenv249-257 Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu (Seq. ID No.
4); HBenv249-258 Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu (Seq.
ID No. 5); HBenv250-258 Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu
(Seq. ID No. 6); HBenv251-259 Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu-
Val (seq. ID No. 7), HBeriv251-260 Leu-Leu-Cys-Leu-Ile-Phe-Leu-
Leu-Val-Leu (Seq. ID No. 8), HBenv260-269 Leu-Leu-Asp-Tyr-Gln-
Gly-Met-Leu-Pro-Val (Seq. ID No. 9), HBenv335-343 Trp-Leu-Ser-
Leu-Leu-Val-Pro-Phe-Val (Seq. ID No. 10), HBenv152-161 Ser-
Ile-Leu-Ser-Lys-Thr-Gly-Asp-Pro-Val (Seq. ID No. 11);
HBenv177-185 Val-Leu-Gln-Ala-Gly-Phe-Phe-Leu-Leu (Seq. ID No.
12); HBenv204-212 Phe-Leu-Gly-Gly-Thr-Pro-Val-Cys-Leu (Seq. ID
No. 13); or HBenv370-379 Ser-Ile-Val-Ser-Pro-Phe-Ile-Pro-Leu-
WO 94/19011 PCT/US94/02195
4
Leu (Seq. ID No. 14); or will have a sequence substantially
homologous to one of the foregoing sequences. The peptide can
be optionally flanked and/or modified at one or both of the N-
and C-termini, as desired. Conservative substitutions,
deletions and additions may be made at non-critical residue
positions within the selected peptide without substantially
adversely affecting its biological activity.
In the various peptide embodiments it will be
understood that the peptides can be polymerized, each to
itself to form larger homopolymers, or with different peptides
to form heteropolymers. In some instances peptides will be
combined in a composition as an admixture and will not be
linked. The peptide can also be conjugated to a lipid-
containing molecules capable of enhancing a T lymphocyte
response, or to a different peptide which induces a T-helper
cell response, for example.
Compositions are provided which comprise a peptide of
the invention formulated with an additional peptide, a
liposome, an adjuvant and/or a pharmaceutically acceptable
carrier. Thus, pharmaceutical compositions can be used in
methods of treating acute HBV infection, particularly in an
effort to prevent the infection from progressing to a chronic
or carrier state. Methods for treating chronic HBV infection
and HBV carrier states are also provided, where the
pharmaceutical compositions are administered to infected
individuals in amounts sufficient to stimulate immunogenically
effective cytotoxic T cell responses against HBc epitopes.
For treating these infections it may be particularly desirable
to combine the peptides which induce MHC class I restricted
cytotoxic T lymphocyte responses against HBV antigen with
other peptides or proteins that induce immune response to
other HBV antigens, such as HBV core. To treat individuals
with chronic or carrier state infections the compositions may
be administered in repeated dosages over a prolonged period of
time, as necessary, to resolve or substantially mitigate the
infection and/or shedding of virus.
Vaccine compositions for preventing HBV infection,
particularly chronic HBV infection, are also provided. The
CA 02156416 2006-02-06
vaccine compositions comprise an immunogenically effective
amount of a HBV envelope peptide mentioned above which
induces a MHC class I restricted cytotoxic T lymphocyte
response, such as HLA-A2, and will typically further
5 comprise an adjuvant, e.g., incomplete Freund's adjuvant or
aluminum hydroxide. To achieve enhanced protection against
HBV, the vaccine can further comprise components which
elicit a protective antibody response to HBV envelope
antigen.
In yet other embodiments the invention relates to
methods for diagnosis, where the peptides of the invention
are used to determine the presence of lymphocytes in an
individual which are capable of a cytotoxic T cell response
to HBV envelope antigen. The absence of such cells
determines whether the individual of interest is susceptible
to developing chronic HBV infection. Typically the
lymphocytes are peripheral blood lymphocytes and the
individual of interest is suffering from an acute HBV
infection.
Various embodiments of this invention provide A
cytotoxic T lymphocyte (CTL) inducing peptide consisting of
nine to twelve amino acid residues, wherein said peptide
binds to an MHC molecule and induces a CTL response against
an HBV antigen or antigen mimetic, and wherein said peptide
comprises the following sequence: HBenv183-191 (SEQ ID
N0:1) Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile. Also provided
are conjugates of the aforementioned peptide in which the
conjugate contains a lipid containing molecule, a different
peptide to form a heteropolymer, another CTL inducing
peptide or a HTL inducing peptide. Multiple copies of a
peptide of this invention may be present as a homopolymer.
Various embodiments of this invention provide
compositions comprising a peptide/peptide conjugate of this
CA 02156416 2006-02-06
5a
invention and at least one additional peptide. The
additional peptide may be a CTL or a HTL inducing peptide.
Various embodiments of this invention provide a
composition comprising a peptide/peptide conjugate of this
invention and a liposome.
Various embodiments of this invention provide a
peptide/peptide conjugate of this invention and a
pharmaceutically acceptable carrier.
Various embodiments of this invention provide the
use of a peptide or a peptide conjugate of this invention in
the preparation of a medicament for the treatment or
prevention of hepatitis B infection.
Various embodiments of this invention provide the
use of a peptide or a peptide conjugate of this invention
for the treatment or prevention of hepatitis B infection.
Various embodiments of this invention provide a
nucleic acid encoding a peptide or a peptide conjugate of
this invention.
Various embodiments of this invention provide the
use of a nucleic acid of this invention in the preparation
of a medicament for the treatment or prevention of hepatitis
B infection.
various embodiments of this invention provide the
use of a nucleic acid of this invention for the treatment of
prevention of hepatitis B infection.
CA 02156416 2006-02-06
5b
Brief Description of the Drawinas
Fig. 1 shows that HBsAg335-343, WLSLLVPFV, is the
minimal optimal CTL epitope recognized by CTL stimulated
HBsAg329-348. A CTL clone from patient A-1 and a CTL cloned
line from patient A-3, generated by stimulation with
HBsAg329-348, were tested against JY target cells prepulsed
either with truncations (upper panels) or with overlapping
9-mers or 10-mers (lowers panels) covering HBsAg329-348.
Fig. 2 further confirms that an optimal epitope
within HBsA 329-348 for vitro ~' CTL induction is
HBsAg335-343.
Fig. 3 shows the HBV specific CTL respcnse in
patients with acute hepatitis B infection, chronic hepatitis B
infection, and normal subjects. PBMC from acute patients (A-1
to A-12), chronic patients (C-1 to C-6), and normal subjects
(N-1 to N-6) were stimulated with the following synthetic
peptides: 1-HBcAg18-27, 2=HBsAg201-210, 3=HBsAg251-259,
CA 02156416 2006-02-06
6
4=HBsAq260-269, 5-HBcAg335-343, 6=HBsAg338-347,
7=HBsAq348-357, 8=HBsAg378-387.
Fig. 4 shows the results of HLA-A2.1 competitive
bindinq inhibition assays, represented as % inhibition of
HBcAgl8-27 specific lysis in a 4 hour Slcr release assay.
Fig. 5 illustrates that the CTL response to
HBsAg335-343 and HBsAg348-357 are group specific and subtype
specific, respectively, and that the synthetic peptides
contain epitopes that are also generated by the endogenous
processinq of the larqe, middle and major HBV envelope
polypeptides within infected cells.
Description of the Specific Embodiments
The present invention provides peptides derived from
HBV envelope proteins for use in compositions and methods for
the treatment, prevention and diaqnosis of HBV infection. The
paptides stimulate MHC HLA-class I restricted cytotoxic T
lymphocyte responses against HBV infected cells. The
stimulated cytotoxic T lymphocytes are able to kill the
infected cells or inhibit viral replication and thus interrupt
or substantially prevent infection, includinq chronic HBV
infection. A peptide effective in eliciting a cytotoxic T
cell response may also be combined with an immunogen capable
of eliciting a T-helper response.
The peptides employed in the invention are derived
from the regions of HBenvl83-191 (Seq. ID No. 1), HBenv248-260
(Seq. ID No. 2), HBenv260-269 (Seq. ID No. 9), HBenv335-343
(Seq. ID No. 10), HBenv152-161 (Seq. ID No. 11), HBenv177-185
(Seq. ID No. 12), HBenv204-212 (Seq. ID No. 13), and
HBenv370-379 (Seq. ID No. 14), where the numbering is
according to Galibert et al., Nature 281:646 (1979).
By HBV cytotoxic T lymphocyte inducing "peptide" or
"oligopeptide" of the present invention is meant a chain of at
least four HBV amino acid sequence residues, preferably at
least six, more preferably eight or nine, sometimes ten to
twelve residues, and usually fewer than about fifty residues,
more usually fewer than about thirty-five, and preferably
CA 02156416 2006-02-06
7
fewer than twenty-five, e.q., eight to seventeen amino acid
residues derived from an HBc sequence. It may be desirable to
optimize peptides of the invention to a lenqth of eight to
twelve amino acid residues, commensurate in size with
endogenously processed viral peptides that are bound to MHC
class I molecules on the cell surface. See generally,
Schumacher et al., Nature 350:703-706 (1991); Van Bleek et
al., Nature 348:213-216 (1990); Rotzschke et al., Nature
348:252-254 (1990); and Falk et al., Nature 351:290-296
(1991). As set
forth in more detail below, usually the peptides will have at
least a majority of amino acids which are homoloqous to a
corresponding portion of contiguous residues of the HBV
sequences identified herein, and containing a CTL-inducing
epitope.
The peptides can be prepared "synthetically," as
described hereinbelow, or by recombinant DNA technology.
Although the peptide will preferably be substantially free of
other naturally occurring HBV proteins and fragments thereof,
in some embodiments the peptides can be synthetically
conjugated to native fragments or particles. The term peptide
is used interchangeably with polypeptide in the present
specification to designate a series of amino acids connected
one to the other by peptide bonds between the alpha-amino and
alpha-carboxy groups of adjacent amino acids. The
polypeptides or peptides can be a variety of lengths, either
in their neutral (uncharged) forms or in forms which are
salts, and either free of modifications such as qlycosylation,
side chain oxidation, or phosphorylation or containing these
modifications, subject to the condition that the modification
not destroy the biological activity of the polypeptides as
herein described.
Desirably, the peptide will be as small as possible
while still maintaining substantially all of the biological
activity of the large peptide. By biological activity is
meant the ability to bind an appropriate MHC molecule and
induce a cytotoxic T lymphocyte response against HBV antigen
or antigen mimetic. By a cytotoxic T lymphocyte response is
WO 94/19011 21~ ~ ~ 1.6 PCT/US94/02195
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meant a CD8+ T lymphocyte response specific for an HBV antigen
of interest, wherein CD8+, MHC class I-restricted T
lymphocytes are activated. The activated T lymphocytes
secrete lymphokines (e.g., gamma interferon) liberate products
(e.g., serine esterases) that inhibit viral replication in
infected autologous cells or transfected cells, with or
without cell killing.
The terms "homologous", "substantially homologous",
and "substantial homology" as used herein denote a sequence of
amino acids having at least 50% identity wherein one sequence
is compared to a reference sequence of amino acids. The
percentage of sequence identity or homology is calculated by
comparing one to another when aligned to corresponding
portions of the reference sequence.
A CTL-inducing HBV peptide embodiment of the
invention from the nucleocapsid region comprises from six to
thirty-five amino acids and contains at least one HLA-
restricted CTL epitopic site from the peptide region
HBenv183-191 (Seq. ID No. 1). A majority of the amino acids
of the peptide will be identical or substantially homologous
to the amino acids of the corresponding portions of the
naturally occurring HBenvl83-191 region, where HBenv183-191
has the sequence:
HBenv183-191 (Seq. ID No. 1)
Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile
The peptide embodiments of this HBenv183-191 region can be
optionally flanked and/or modified at one or both of the N-
and C-termini, as desired, by amino acids from HBV sequences,
including HBc, amino acids added to facilitate linking, other
N- and C-terminal modifications, linked to carriers, etc., as
further described herein. The peptide HBenv183-191 induces a
cytotoxic T lymphocyte response which is mediated by at least
the MHC class I molecule HLA-A2.
Other HBenv peptide embodiments of the invention are
prepared from the region of HBenv248-260. Peptides derived
from this'region contain at least one CTL-inducing HLA class
I-restricted epitopic site, and will typically be at least
seven amino acids, more usually nine, ten or eleven amino
WO 94/19011 2156416 PCT/US94/02195
9
acids or more. A majority of the amino acids of the peptide
will be identical or substantially homologous to the amino
acids of the corresponding portions of the naturally occurring
HBenv248-260 sequence, where HBenv248-260 has the sequence
(for HBV subtype ayw):
HBenv248-260 (Seq. ID No. 2)
Phe-Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu-Val-Leu.
The peptide from the HBenv248-260 region can be
flanked and/or modified at one or both termini as described
herein.
Representative CTL-inducing peptides prepared from
the region of HBenv248-260 include the following 9- and 10-mer
peptides:
HBenv248-257 (Seq. ID No. 3)
Phe-Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu;
HBenv249-257 (Seq. ID No. 4)
Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu;
HBenv249-258 (Seq. ID No. 5)
Ile-Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu;
HBenv250-258 (Seq. ID No. 6)
Leu-Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu;
HBenv251-259 (Seq. ID No. 7)
Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu-Val;
HBenv251-260 (Seq. ID No. 8)
Leu-Leu-Cys-Leu-Ile-Phe-Leu-Leu-Val-Leu;
The foregoing peptides contain a HLA-restricted CTL-
inducing epitope, typically at least HLA-A2 restricted, and
can be flanked and/or modified at one or both termini as
mentioned for peptide I above.
In a further embodiment, a peptide of the invention
comprises the 10-mer peptide HBenv260-269, and peptides
derived from HBenv260-269 which contain a CTL-inducing HLA
class I-restricted epitopic site(s) of at least seven
contiguous amino acids. A majority of the amino acids of the
peptide will be identical or substantially homologous to the
amino acids of the corresponding portions of the naturally
WO 94/19011 PCT/US94/02195
~15G416
occurring HBenv260-269 sequence, where HBenv260-269 has the
sequence:
HBenv260-269 (Seq. ID No. 9)
Leu-Leu-Asp-Tyr-Gln-Gly-Met-Leu-Pro-Val.
5 A peptide prepared from this region can be flanked
and/or modified at one or both termini as described herein.
The peptide HBenv260-269 (Seq. ID No. 4) induces a cytotoxic T
lymphocyte response which is mediated by at least the MHC
class I HLA-A2 molecule.
10 Yet other CTL-inducing peptides of the invention are
from the region of HBenv335-343 (Seq. ID No. 10), and includes
peptides derived from HBenv335-343 (Seq. ID No. 10) which
contain one or more CTL-inducing HLA class I-restricted
epitopic site(s) of at least seven contiguous amino acids. A
majority of the amino acids of the peptide will be identical
or substantially homologous to the amino acids of the
corresponding portions of the naturally occurring HBenv335-343
sequence, where HBenv335-343 has the sequence:
HBenv335-343 (Seq. ID No. 10)
Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val,
wherein the selected peptide can be flanked and/or modified at
one or both termini as described herein.
Yet other CTL-inducing peptides of the invention are
from the region of HBenv152-161 (Seq. ID No. 11), and includes
peptides derived from HBenv152-161 (Seq. ID No. 11) which
contain one or more CTL-inducing HLA class I-restricted
epitopic site(s) of at least seven contiguous amino acids. A
majority of the amino acids of the peptide will be identical
or substantially homologous to the amino acids of the
corresponding portions of the naturally occurring HBenvl52-161
sequence, where HBenv152-161 has the sequence (adw subtype)):
HBenv152-161 (Seq. ID No. 11)
Ser-Ile-Leu-Ser-Lys-Thr-Gly-Asp-Pro-Val,
wherein the selected peptide can be flanked and/or modified at
one or both termini as described herein.
Other CTL-inducing peptides of the invention are from
the region of HBenvl77-185 (Seq. ID No. 12), and includes
peptides derived from HBenv177-185 (Seq. ID No. 12) which
WO 94/19011 PCT/US94/02195
11
contain one or more CTL-inducing HLA class I-restricted
epitopic site(s) of at least seven contiguous amino acids. A
majority of the amino acids of the peptide will be identical
or substantially homologous to the amino acids of the
corresponding portions of the naturally occurring HBenv177-185
sequence, where HBenvl77-185 has the sequence (adw subtype):
HBenvl77-185 (Seq. ID No. 12)
Val-Leu-Gln-Ala-Gly-Phe-Phe-Leu-Leu,
wherein the selected peptide can be flanked and/or modified at
one or both termini as described herein.
Additional CTL-inducing peptides of the invention are
from the region of HBenv204-212 (Seq. ID No. 13), and includes
peptides derived from HBenv204-212 (Seq. ID No. 13) which
contain one or more CTL-inducing HLA class I-restricted
epitopic site(s) of at least seven contiguous amino acids. A
majority of the amino acids of the peptide will be identical
or substantially homologous to the amino acids of the
corresponding portions of the naturally occurring HBenv204-212
sequence, where HBenv204-212 has the sequence (adw subtype):
HBenv204-212 (Seq. ID No. 13)
Phe-Leu-Gly-Gly-Thr-Pro-Val-Cys-Leu,
wherein the selected peptide can be flanked and/or modified at
one or both termini as described herein.
Additional CTL-inducing peptides of the invention are
from the region of HBenv370-379 (Seq. ID No. 14), and includes
peptides derived from HBenv370-379 (Seq. ID No. 14) which
contain one or more CTL-inducing HLA class I-restricted
epitopic site(s) of at least seven contiguous amino acids. A
majority of the amino acids of the peptide will be identical
or substantially homologous to the amino acids of the
corresponding portions of the naturally occurring HBenv370-379
sequence, where HBenv370-379 has the sequence (adw subtype):
HBenv370-379 (Seq. ID No. 14)
Ser-Ile-Val-Ser-Pro-Phe-Ile-Pro-Leu-Leu,
wherein the selected peptide can be flanked and/or modified at
one or both termini as described herein.
As mentioned above, additional amino acids can be
added to the termini of an oligopeptide or peptide to provide
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12
for ease of linking peptides one to another, for coupling to a
carrier, support or a larger peptide, for reasons discussed
herein, or for modifying the physical or chemical properties
of the peptide or oligopeptide, and the like. Amino acids
such as tyrosine, cysteine, lysine, glutamic or aspartic acid,
and the like, can be introduced at the C- or N-terminus of the
peptide or oligopeptide. In addition, the peptide or
oligopeptide sequences can differ from the natural sequence by
being modified by terminal-NH2 acylation, e.g., acetylation,
or thioglycolic acid amidation, terminal-carboxy amidation,
e.g., ammonia, methylamine, etc. In some instances these
modifications may provide sites for linking to a support or
other molecule.
It will be understood that the HBV peptides of the
present invention or analogs thereof which have cytotoxic T
lymphocyte stimulating activity may be modified as necessary
to provide certain other desired attributes, e.g., improved
pharmacological characteristics, while increasing or at least
retaining substantially the biological activity of the
unmodified peptide. For instance, the peptides can be
modified by extending, decreasing or substituting amino acids
in the peptide sequence by, e.g., the addition or deletion of
amino acids on either the amino terminal or carboxy terminal
end, or both, of peptides derived from the sequences disclosed
herein. The peptides may be modified to substantially enhance
the CTL inducing activity, such that the modified peptide
analogs have CTL activity greater than a peptide of the wild-
type sequence. For example, it may be desirable to increase
the hydrophobicity of the N-terminal of a peptide,
particularly where the second residue of the N-terminal is
hydrophobic and is implicated in binding to the HLA
restriction molecule. By increasing hydrophobicity at the
N-terminal, the efficiency of the presentation to T cells may
be increased. Peptides prepared from other disease associated
antigens, particularly those containing CTL inducing epitopes
for which a host may not have significant CTL activity, may be
made CTL-inducing by substituting hydrophobic residues at the
CA 02156416 2006-02-06
13
N-terminus of the peptide where the second residue is normally
hydrophobic.
The peptides employed in the subject invention need
not be identiCal to peptides HBenv183-191 (Seq. ID No. 1),
HBenv248-257 (Seq. ID No. 3), HBenv249-257 (Seq. ID No. 4),
HBenv249-258 (Seq. ID No. 5), HBenv250-258 (Seq. ID No. 6)
HBenv251-259 (Seq. ID No. 7), HBenv251-260 (Seq. ID No. 8),
HBenv260-269 (Seq. ID No. 9), HBenv335-343 (Seq. ID No. 10),
HBenvl52-161 (Seq. ID No. 11), HBenv177-185 (Seq. ID No. 12),
HBenv204-212 (Seq. ID No. 13), or HBenv370-379 (Seq. ID No.
14), so long as the subject compounds are able to provide for
cytotoxic T lymphocytic activity against at least one of the
four major subtypes of HBV. Although different strains of HBV
exist, they each share at least one common envelope
determinant, which is designated "a". Each strain also has
two other envelope determinants, one of which is either "d" or
"y", and the second is either "w" or "r". Thus, there are
four possible subtypes of the virus: adw, ayw, adr, and ayr.
The cloning, sequencing and expression of HBV are described in
GB 2034323, EP 13828, U.S. 4,935,235, and the complete
sequence of the HBV envelope region is also described in
Galibert et al., tJature 281:646 (1979).
Amino acid sequences
are described in the GenBank-72 database for 20 different HBV
strains, including 7 of the adw subtype, 5 of the ayw subtype,
7 of the adr subtype, and 1 strain of the ayr subtype.
Therefore, the peptides may be subject to various
changes, such as insertions, deletions, and substitutions,
either conservative or non-conservative, where such changes
provide for certain advantages in their use. By conservative
substitutions is meant replacing an amino acid residue with
another which is biologically and/or chemically similar, e.g.,
one hydrophobic residue for another, or one polar residue for
another. The substitutions include combinations such as Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg;
and Phe, -1"yr. _;sually, the portion of the sequence which is
intended to substantially mimic an HBV cytotoxic T lymphocyte
WO 94/19011 2156416 PCTIUS94/02195
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stimulating epitope will not differ by more than about 20%
from the sequence of at least one subtype of HBV, except where
additional amino acids may be added at either terminus for the
purpose of modifying the physical or chemical properties of
the peptide for, e.g., ease of linking or coupling, and the
like. Where regions of the peptide sequences are found to be
polymorphic among HBV subtypes, it may be desirable to vary
one or more particular amino acids to more effectively mimic
differing cytotoxic T-lymphocyte epitopes of different HBV
strains or subtypes.
Within the peptide sequences identified by the
present invention, including the representative peptides
listed above, there are residues (or those which are
substantially functionally equivalent) which allow the peptide
to retain their biological activity, i.e., the ability to
stimulate a class I-restricted cytotoxic T-lymphocytic
response against HBV infected cells or cells which express HBV
antigen. These residues can be identified by single amino
acid substitutions, deletions, or insertions. In addition,
the contributions made by the side chains of the residues can
be probed via a systematic scan with a specified amino acid
(e.g., Ala). Peptides which tolerate multiple substitutions
generally incorporate such substitutions as small, relatively
neutral molecules, e.g., Ala, Gly, Pro, or similar residues.
The number and types of residues which can be substituted,
added or subtracted will depend on the spacing necessary
between the essential epitopic points and certain
conformational and functional attributes which are sought
(e.g., hydrophobicity vs. hydrophilicity). If desired,
increased binding affinity of peptide analogues to its MHC
molecule for presentation to a cytotoxic T-lymphocyte can also
be achieved by such alterations. Generally, any spacer
substitutions, additions or deletions between epitopic and/or
conformationally important residues will employ amino acids or
moieties chosen to avoid steric and charge interference which
might disrupt binding.
Peptides which tolerate multiple substitutions while
retaining the desired biological activity may also be
WO 94/19011 2156416 PCT/US94/02195
synthesized as D-amino acid containing peptides. Such peptide
may be synthesized as "inverso" or "retro-inverso" forms, that
is, by replacing L-amino acids of a sequence with D-amino
acids, or by reversing the sequence of the amino acids and
5 replacing the L-amino acids with D-amino acids. As the
D-peptides are substantially more resistant to peptidases, and
therefore are more stable in serum and tissues compared to
their L-peptide counterparts, the stability of D-peptides
under physiological conditions may more than compensate for a
10 difference in affinity compared to the corresponding
L-peptide. Further, L-amino acid-containing peptides with or
without substitutions can be capped with a D-amino acid to
inhibit exopeptidase destruction of the antigenic peptide.
In addition to the exemplary peptides described
15 herein, the invention provides methods for identifying other
epitopic regions associated with said peptide regions capable
of inducing MHC-restricted cytotoxic T lymphocyte responses
against HBV. The methods comprise obtaining peripheral blood
lymphocytes (PBL) from infected or uninfected individuals and
exposing (stimulating) the cells with synthetic peptide or
polypeptide fragments derived from a peptide region of
HBenv183-191 (Seq. ID No. 1), HBenv248-260 (Seq. ID No. 2),
HBenv260-269 (Seq. ID No. 9), HBenv335-343 (Seq. ID No. 10),
HBenv152-161 (Seq. ID No. 11), HBenvl77-185 (Seq. ID No. 12),
HBenv204-212 (Seq. ID No. 13), or HBenv370-379 (Seq. ID No.
14). Pools of overlapping synthetic peptides, each typically
about 8 to 20 residues long, preferably 9-12 residues, can be
used to stimulate the cells. Active peptides can be selected
from pools which induce cytotoxic T lymphocyte activity. The
ability of the peptides to induce specific cytotoxic activity
is determined by incubating the stimulated PBL with autologous
labeled (e.g., 51Cr) target cells (such as HLA matched
macrophages, T cells, fibroblasts or B lymphoblastoid cells)
infected or transfected with the HBV subgenomic fragments
thereof, such that the targeted antigen is synthesized
endogenously by the cell (or the cell is pulsed with the
peptide of interest), and measuring specific release of label.
WO 94/19011 2156416 PCT/US94/02195
16
Once a peptide having an epitopic region which
stimulates a cytotoxic T lymphocyte response is identified,
the MHC restriction element of the response can be determined.
This involves incubating the stimulated PBL or short term
lines thereof with a panel of (labeled) target cells of known
HLA types which have been pulsed with the peptide of interest,
or appropriate controls. The HLA allele(s) of cells in the
panel which are lysed by the CTL are compared to cells not
lysed, and the HLA restriction element(s) for the cytotoxic T
lymphocyte response to the antigen of interest is identified.
Carbone et al., J. Exp. Med. 167:1767 (1988), have
reported that stimulation with peptides may induce cytotoxic T
lymphocytes with low affinity for corresponding endogenous
protein, such that repetitive peptide stimulation may yield
cytotoxic T lymphocytes that recognize peptide but not native
antigen. As the inability of stimulated cytotoxic T
lymphocytes to recognize native HBV proteins would be
undesirable in the development of HBV peptide therapeutics and
vaccine compositions, methods to circumvent this potential
limitation are used. A sequential restimulation of cytotoxic
T cells is employed in the present invention to identify and
select T cells with a higher affinity for naturally processed
antigen than for a synthetic peptide. Short term cytotoxic T
lymphocyte lines are established by restimulating activated
PBL. Cells stimulated with peptide are restimulated with
peptide and recombinant or native HBV antigen, e.g., HBsAg.
Cells having activity are also stimulated with an appropriate
T cell mitogen, e.g., phytohemagglutinin (PHA). The
restimulated cells are provided with irradiated allogeneic
PBLs as an antigen nonspecific source of T cell help, and HBV
antigen. To selectively expand the population of cytotoxic T
lymphocytes that recognize native HBV antigen and to establish
long term lines, PBL from a patient are first stimulated with
peptide and recombinant or native HBV antigen, followed by
restimulation with HLA-matched B lymphoblastoid cells that
stably express the corresponding HBV antigen polypeptide. The
cell lines are re-confirmed for the ability to recognize
endogenously synthesized antigen using autologous and
CA 02156416 2006-02-06
17
allogeneic B-lymphoblastoid or other cells transfected or
infected with appropriate antigen.
Having identified different peptides of the invention
which contribute to inducing anti-HBV cytotoxic T lymphocyte
responses in one or more patients or HLA types, in some
instances it may be desirable to join two or more peptides in
a composition. The peptides in the composition can be
identical or different, and together they should provide
equivalent or greater biological activity than the parent
peptide(s). For example, usinq the methods described herein,
two or more peptides may define different or overlapping
cytotoxic T lymphocyte epitopes from a particular region,
e.g., the HBenv248-257 (Seq. ID No. 3), HBenv249-257 (Seq. ID
No. 4), HBenv249-258 (Seq. ID No. 5), HBenv250-258 (Seq. ID
No. 6) HBenv251-259 (Seq. ID No. 7), and/or HBenv251-260
(Seq. ID No. 8) peptides, which peptides can be combined in a
"cocktail" to provide enhanced immunogenicity for cytotoxic T
lymphocyte responses. Moreover, peptides of one region can be
combined with peptides of other HBV regions, from the same or
different HBV protein, particularly when a second or
subsequent peptide has a NDIC restriction element different
from the first. Othar CTL-inducing HBV peptides are described
in WO 93/03753, WO 95/03777, and U.S. 5,780,036.
This composition of
peptides can be used to effectively broaden the immunological
coverage provided by therapeutic, vaccine or diagnostic
methods and compositions of the invention among a diverse
population. For example, the different frequencies of HLA
alleles among prevalent ethnic groups (caucasian, asian and
african blacks) are shown in Table I below. :herapeutic or
vaccine compositions of the invention may be formulated to
provide potential therapy or immunity to as high a percentage
of a population as possible.
CA 02156416 2006-02-06
18
TABLE I. HLA ALLELE FRE UENCIES AMONG PREVALENT ETHNIC GROUPS
HLA Allele
A2 45.3 46.6 27.3 43.2
A29 7.4 8.1 12.3 0.4
A31 5.4 6.2 4.4 15.3
A32 8.8 7.1 3 0.1
A33 3.3 3.4 9 13.1
A28' 7.7 9.9 16.6 1.1
Abbreviations: EUC, European Caucasian; NAC, North American
Caucasian; AFR, African blacks, JPN, Japanese.
*A28 represents the two alleles Aw68 and Aw69
The peptides of the invention can be combined via
linkage to form polymers (multimers), or can be formulated in
a composition without linkage, as an admixture. Where the
same peptide is linked to itself, thereby forminq a
homopolymer, a plurality of repeatinq epitopic units are
presented. When the peptides differ, e.q., a cocktail
representinq different HBV subtypes, different epitopes within
a subtype, different HLA restriction specificities, a peptide
which contains T helper epitopes, heteropolymers with
repeating units are provided. In addition to covalent
linkaqes, noncovalent linkages capable of forming
intermolecular and intrastructural bonds are included.
Linkages for homo- or hetero-polymers or for coupling
to carriers can be provided in a variety of ways. For
example, cysteine residues can be added at both the amino- and
carboxy-termini, where the peptides are covalently bonded via
controlled oxidation of the cysteine residues. Also useful
are a large number of heterobifunctional agents which generate
a disulfide link at one functional group end and a peptide
link at the other, including N-succidimidyl-3-(2-pyridyl-
dithio) proprionate (SPDP). This reagent creates a disulfide
linkage between itself and a cysteine residue in one protein
and an amide linkage through the amino on a lysine or other
free amino group in the other. A variety of such
disulfide/amide forming agents are known. -See, for example,
Immun. Rev. 62:185 (1982). Other bifunctional coupling agents
form a thioether rather than a disulfide linkage. Many of these
CA 02156416 2006-02-06
19
thioether forming agents are commercially available and
include reactive esters of 6-maleimidocaproic acid, 2
bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)
cyclohexane- i-carboxylic acid and the like. The carboxyl
groups can be activated by combining them with succinimide or
1-hydroxy-2-nitro-4- sulfonic acid, sodium salt. A
particularly preferred coupling agent is succinimidyl
4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC). It
will be understood that linkage should not substantially
interfere with either of the linked groups to function as
described, e.g., as an HBV cytotoxic T cell determinant,
peptide analogs, or T helper determinant.
In another aspect the peptides of the invention can
be combined or coupled with other peptides which present HBV
T-helper cell epitopes, i.e., epitopes which stimulate T cells
that cooperate in the induction of cytotoxic T cells to HBV.
The T-helper cells can be either the T-helper 1 or T-helper 2
phenotype, for example. T-helper epitopes from HBV sequences
have been identified at HBcl-20, having the sequence: Met-Asp-
Ile-Asp-Pro-Tyr-Lys-Glu-Phe-Gly-Ala-Thr-Val-Glu-Leu-Leu-Ser-
Phe-Leu-Pro (Seq. ID No. 17). Other T-helper epitopes are
provided by peptides from the region HBc50-69, having the
sequence Pro-His-His-Tyr-Ala-Leu-Arg-Gln-Ala-Ile-Leu-Cys-Trp-
Gly-Glu-Leu-Met-Tyr-Leu-Ala (Seq. ID No. 18), and from the
region of HBc100-139, including HBc100-119 having the sequence
Leu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe-Gly-Arg-Glu-Thr-
Val-Ile-Glu-Tyr-Leu (Seq. ID No. 19) (where I1e116 is Leu in
the HBV adw subtype), HBc117-131 having the sequence Glu-Tyr-
Leu-Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala (Seq. :D
:0 No. :.0), and peptide HBc120-139 having the sequence Val-Ser-
Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-Tyr-Arg-Pro-Pro-Asn-
Ala-Pro-ile (Seq. ID No. 21). See, ?errari et al., J. Clin.
Invest. 88:214-222 (1991), and U.S. Pat. 4,882,145.
The peptides of the invention can be orepared :.n a
wide variety cf wavs. 3ecause of their -elativeiv short size.
the peptides can be synthesized in solution or on a solid
support in accorciance with conventionai techniques. Various
CA 02156416 2006-02-06
automatic synthesizers are commercially available and can be
used in accordance with known protocols. See, for example,
Stewart and Young, Solid Phase Peotide Synthesis, 2d. ed.,
Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc.
5 105:6442 (1983); Merrifield, Science 232:341-347 (1986); and
Barany and :ierrifield, The Peotides, Gross and Meienhofer,
eds., Academic Press, New York, pp. 1-284 (1979).
Alternatively, recombinant DNA technology may be
10 employed wherein a nucleotide sequence which encodes a peptide
of interest is inserted into an expression vector, transformed
or transfected into an appropriate host cell and cultivated
under conditions suitable for expression. These procedures
are generally known in the art, as described generally in
15 Sambrook et al., Molecular Clonina. A Laboratorv Manual, Cold
Spring Harbor Press, Cold Spring Harbor, New York (1982), and
Ausubel et al., (ed.) Current Protocols in Molecular Biology,
John Wiley and Sons, Inc., New York (1987), and U.S. Pat. Nos.
4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941, for
20 example,
Thus, fusion proteins which comprise one or more
peptide sequences of the invention can be used to present the
HBV cytotoxic T cell determinants. For example, a recombinant
envelope protein of the invention is prepared in which the
HBenv amino acid sequence is altered so as to more effectively
present epitopes of peptide regions described herein to
stimulate a cytotoxic T lymphocyte response. 3y this means a
polypeptide is used which incorporates several T cell
epitopes.
As the coding sequence for peptides of the length
contemplated herein can be synthesized by chemical techniques,
for example, zhe phosphotriester method of Matteucci et al.,
J. Am. Chem. Soc. 103:3185 (1981), modification can be made
simply by substituting the appropriate base(s) for those
encoding the native peptide sequence. 7he coding sequence can
then be nrovided with appropriate l4-nkers and ligated into
expression vectors commonly available in the art, and the
vectors used to transform suitable hosts to produce the
WO 94/19011 2156416 PCT/US94/02195
21
desired fusion protein. A number of such vectors and suitable
host systems are now available. For expression of the fusion
proteins, the coding sequence will be provided with operably
linked start and stop codons, promoter and terminator regions
and usually a replication system to provide an expression
vector for expression in the desired cellular host. For
example, promoter sequences compatible with bacterial hosts
are provided in plasmids containing convenient restriction
sites for insertion of the desired coding sequence. The
resulting expression vectors are transformed into suitable
bacterial hosts. Yeast or mammalian cell hosts may also be
used, employing suitable vectors and control sequences.
The peptides of the present invention and
pharmaceutical and vaccine compositions thereof are useful for
administration to mammals, particularly humans, to treat
and/or prevent HBV infection. As the peptides are used to
stimulate cytotoxic T-lymphocyte responses to HBV infected
cells, the compositions can be used to treat or prevent acute
and/or chronic HBV infection.
For pharmaceutical compositions, the peptides of the
invention as described above will be administered to an
individual already infected with HBV. Those in the incubation
phase or the acute phase of infection can be treated with the
immunogenic peptides separately or in conjunction with other
treatments, as appropriate. In therapeutic applications,
compositions are administered to a patient in an amount
sufficient to elicit an effective cytotoxic T lymphocyte
response to HBV and to cure or at least partially arrest its
symptoms and/or complications. An amount adequate to
accomplish this is defined as "therapeutically effective
dose." Amounts effective for this use will depend on, e.g.,
the peptide composition, the manner of administration, the
stage and severity of the disease being treated, the weight
and general state of health of the patient, and the judgment
of the prescribing physician, but generally range from about 1
g to about 2,000 mg of peptide for a 70 kg patient, with
dosages of from about 10 g to about 100 mg of peptide being
more commonly used, followed by booster dosages from about 1
WO 94/19011 PCT/US94/02195
-- 2156416 22
g to about 1 mg of peptide over weeks to months, depending on
a patient's CTL response, as determined by measuring HBV-
specific CTL activity in PBLs obtained from the patient. It
must be kept in mind that the peptides and compositions of the
present invention may generally be employed in serious disease
states, that is, life-threatening or potentially life
threatening situations. In such cases, in view of the
minimization of extraneous substances and the relative
nontoxic nature of the peptides, it is possible and may be
felt desirable by the treating physician to administer
substantial excesses of these peptide compositions.
Single or multiple administrations of the
compositions can be carried out with dose levels and pattern
being selected by the treating physician. In any event, the
pharmaceutical formulations should provide a quantity of
cytotoxic T-lymphocyte stimulatory peptides of the invention
sufficient to effectively treat the patient.
For therapeutic use, administration should begin at
the first sign of HBV infection or shortly after diagnosis in
cases of acute infection, and continue until at least symptoms
are substantially abated and for a period thereafter. In well
established and chronic cases, loading doses followed by
maintenance or booster doses may be required. The elicitation
of an effective cytotoxic T lymphocyte response to HBV during
treatment of acute hepatitis will minimize the possibility of
subsequent development of chronic hepatitis, HBV carrier
stage, and ensuing hepatocellular carcinoma.
Treatment of an infected individual with the
compositions of the invention may hasten resolution of the
infection in acutely infected individuals, about 90% of whom
are capable of resolving the infection naturally. For those
individuals susceptible (or predisposed) to developing chronic
infection the compositions are particularly useful in methods
for preventing the evolution from acute to chronic infection.
Where the susceptible individuals are identified prior to or
during infection, for instance, as described herein, the
composition can be targeted to them, minimizing need for
administration to a larger population.
WO 94/19011 2156416 PCTIUS94/02195
23
The peptide compositions can also be used for the
treatment of chronic hepatitis and to stimulate the immune
system of carriers to substantially reduce or even eliminate
virus-infected cells. Those with chronic hepatitis can be
identified as testing positive for virus from about 3-6 months
after infection. As individuals may develop chronic HBV
infection because of an inadequate (or absent) cytotoxic T
lymphocyte response during the acute phase of their infection,
it is important to provide an amount of immuno-potentiating
peptide in a formulation and mode of administration sufficient
to effectively stimulate a cytotoxic T cell response. Thus,
for treatment of chronic hepatitis, a representative dose is
in the range of about 1 g to 1,000 mg, preferably about 5 g
to 100 mg for a 70 kg patient per dose. Administration should
continue until at least clinical symptoms or laboratory
indicators indicate that the HBV infection has been eliminated
or substantially abated and for a period thereafter.
Immunizing doses followed by maintenance or booster doses at
established intervals, e.g., from one to four weeks, may be
required, possibly for a prolonged period of time, as
necessary to resolve the infection. For the treatment of'
chronic and carrier HBV infection it may also be desirable to
combine the CTL peptides with other peptides or proteins that
induce immune response to other HBV antigens.
The pharmaceutical compositions for therapeutic
treatment are intended for parenteral, topical, oral or local
administration. Preferably, the pharmaceutical compositions
are administered parenterally, e.g., intravenously,
subcutaneously, intradermally, or intramuscularly. Thus, the
invention provides compositions for parenteral administration
which comprise a solution of the cytotoxic T-lymphocyte
stimulatory peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.4%
saline, 0.3% glycine, hyaluronic acid and the like. These
compositions may be sterilized by conventional, well known
sterilization techniques, or may be sterile filtered. The
resulting aqueous solutions may be packaged for use as is, or
CA 02156416 2006-02-06
24
lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration. The compositions
may contain pharmaceutically acceptable auxiliary substances
as required to approximate physiological conditions, such as
pH adjusting and buffering agents, tonicity adjusting agents,
wetting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, etc.
In some embodiments it may be desirable to include in
the pharmaceutical composition at least one component which
primes CTL. Lipids have been identified which are capable of
priming CTL in vivQ against viral antigens, e.g.,
tripalmitoyl-S-glycerylcysteinly-seryl-serine (P3CSS), which
can effectively prime virus specific cytotoxic T lymphocytes
when covalently attached to an appropriate peptide. See,
Deres et al., Nature 342:561-564 (1989).
Peptides of the invention can be coupled to
P3CSS, for example, and the lipopeptide administered to an
individual to specifically prime a cytotoxic T lymphocyte
response to HBV. Further, as the induction of neutralizing
antibodies can also be primed with P3CSS conjugated to a
peptide which displays an appropriate epitope, e.g., HBsAg
epitopes, the two compositions can be combined to more
effectively elicit both humoral and cell-mediated responses to
HBV infection.
The concentration of cytotoxic T-lymphocyte
stimulatory peptides of the invention in the pharmaceutical
formulations can vary widely, i.e., from less than about 1%,
usually at or at least about 10% to as much as 20 to 50% or
more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular
mode of administration selected.
Thus, a typical pharmaceutical composition for
intravenous infusion could be made up to contain 250 ml of
sterile Ringer's solution, and 100 mg of peptide. Actual
methods for preparing parenterally administrable compounds
will be known or apparent to those skilled in the art and are
described in more detail in for example, Reminaton's
CA 02156416 2006-02-06
Pharmaceutical Science, 17th ed., Mack Publishing Company,
Easton, PA (1985).
The peptides of the invention may also be
administered via liposomes, which serve to target the peptides
5 to a particular tissue, such as lymphoid tissue or HBV-
infected hepatic cells. Liposomes can also be used to
increase the half-life of the peptide composition. Liposomes
useful in the present invention include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
10 dispersions, lamellar layers and the like. In these
preparations the peptide to be delivered is incorporated as
part of a liposome, alone or in conjunction with a molecule
which binds to, e.g., a receptor, prevalent among lymphoid
cells, such as monoclonal antibodies which bind to the CD45
15 antigen, or with other therapeutic or immunogenic
compositions. Thus, liposomes filled with a desired peptide
of the invention can be directed to the site of lymphoid or
hepatic cells, where the liposomes then deliver the selected
therapeutic/immunoqenic peptide compositions. Liposomes for
20 use in the invention are formed from standard vesicle-forming
lipids, which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The
selection of lipids is generally guided by consideration of,
e.g., liposome size and stability of the liposomes in the
25 blood stream. A variety of methods are available for
preparing liposomes, as described in, e.g., Szoka et al., Ann.
Rev. Bioohvs Bioenq. 9:467 (1980), U.S. Patent Nos.
4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting to the immune cells, a
ligand to be incorporated I-nto the -'-;posome can include, e.g.,
antibodies or fragments thereof specific for cell surface
determinants of the desired immune system cells. A liDosome
suspension containing a peptide may be administered
intravenously, iocallv, topically, 2tc. in a dose which varies
25 accordinc to, the mode of administration, .he neptide -teing
ielivered, zhe stage of disease being treatea, -mtc.
:or solid compositions, conventional :zontoxic solid
carriers may be used which include, ~or exampie,
WO 94/19011 2156416 PCT/US94/02195
26
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like. For oral
administration, a pharmaceutically acceptable nontoxic
composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed,
and generally 10-95% of active ingredient, that is, one or
more peptides of the invention, and more preferably at a
concentration of 25%-75%.
For aerosol administration, the cytotoxic T-
lymphocyte stimulatory peptides are preferably supplied in
finely divided form along with a surfactant and propellant.
Typical percentages of peptides are 0.01$-20$ by weight,
preferably 1%-10%. The surfactant must, of course, be
nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters
of fatty acids containing from 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic,
linolenic, olesteric and oleic acids with an aliphatic
polyhydric alcohol or its cyclic anhydride. Mixed esters,
such as mixed or natural glycerides may be employed. The
surfactant may constitute 0.1%-20$ by weight of the
composition, preferably 0.25-5%. The balance of the
composition is ordinarily propellant. A carrier can also be
included as desired, e.g., lecithin for intranasal delivery.
In another aspect the present invention is
directed to vaccines which contain as an active ingredient an
immunogenically effective amount of a cytotoxic T-lymphocyte
stimulating peptide as described herein. The peptide(s) may
be introduced into a host, including humans, linked to its own
carrier or as a homopolymer or heteropolymer of active peptide
units. Such a polymer has the advantage of increased
immunological reaction and, where different peptides are used
to make up the polymer, the additional ability to induce
antibodies and/or cytotoxic T cells that react with different
antigenic determinants of HBV. Useful carriers are well known
in the art, and include, e.g., keyhole limpet hemocyanin,
thyroglobulin, albumins such as human serum albumin, tetanus
2156416
WO 94/19011 PCT/US94/02195
27
toxoid, polyamino acids such as poly(D-lysine:D-glutamic
acid), and the like. The vaccines can also contain a
physiologically tolerable (acceptable) diluent such as water,
phosphate buffered saline, or saline, and further typically
include an adjuvant. Adjuvants such as incomplete Freund's
adjuvant, aluminum phosphate, aluminum hydroxide, or alum are
materials well known in the art. And, as mentioned above,
cytotoxic T lymphocyte responses can be primed by conjugating
peptides of the invention to lipids, such as P3CSS. Upon
immunization with a peptide composition as described herein,
via injection, aerosol, oral, transdermal or other route, the
immune system of the host responds to the vaccine by producing
large amounts of cytotoxic T-lymphocytes specific for HBV
antigen, and the host becomes at least partially immune to HBV
infection, or resistant to developing chronic HBV infection.
Vaccine compositions containing the peptides of the
invention are administered to a patient susceptible to or
otherwise at risk of HBV infection to enhance the patient's
own immune response capabilities. Such an amount is defined
to be a "immunogenically effective dose." In this use, the
precise amounts again depend on the patient's state of health
and weight, the mode of administration, the nature of the
formulation, etc., but generally range from about 1.0 g to
about 500 mg per 70 kilogram patient, more commonly from about
50 g to about 200 mg per 70 kg of body weight. The peptides
are administered to individuals of an appropriate HLA type,
e.g., for vaccine compositions of peptides from the region of
HBenv183-191 (Seq. ID No. 1), HBenv248-260 (Seq. ID No. 2),
HBenv260-269 (Seq. ID No. 9), HBenv335-343 (Seq. ID No. 10),
HBenv152-161 (Seq. ID No. 11), HBenv177-185 (Seq. ID No. 12),
HBenv204-212 (Seq. ID No. 13), and/or HBenv370-379 (Seq. ID
No. 14), these will be administered to at least HLA-A2
individuals.
In some instances it may be desirable to combine the
peptide vaccines of the invention with vaccines which induce
neutralizing antibody responses to HBV, particularly to HBV
envelope antigens, such as recombinant HBV env-encoded
antigens or vaccines prepared from purified plasma
CA 02156416 2006-02-06
28
preparations obtained from HBV-infected individuals. A
variety of HBV vaccine preparations have been described, and
are based primarily on HBsAg and polypeptide fragments
thereof. For examples of vaccines which can be formulated
with the peptides of the present invention, see generally, cP
154,902 and EP 291,586, and U.S. Pat. Nos. 4,565,697,
4,624,918, 4,599,230, 4,599,231, 4,803,164, 4,882,145,
4,977,092, 5,017,558 and 5,019,386.
The vaccines can be combined and
administered concurrently, or as separate preparations.
For therapeutic or immunization purposes, the
peptides of the invention can also be expressed by attenuated
viral hosts, such as vaccinia. This approach involves the use
of vaccinia virus as a vector to express nucleotide sequences
that encode the HBV peptides of the invention. Upon
introduction into an acutely or chronically HBV-infected host
or into a non-infected host, the recombinant vaccinia virus
expresses the HBV peptide and thereby elicits a host cytotoxic
T lymphocyte response to HBV. Vaccinia vectors and methods
useful in immunization protocols are described in, e.g., U.S.
Patent No. 4,722,848.
Another vector is BCG (bacille Calmette Guerin). BCG vectors
are described in Stover et al. (Nature 351:456-460 (1991)).
A wide variety of
other vectors useful for therapeutic administration or
immunization of the peptides of the invention, e.g.,
Salmonella typhi vectors and the like, will be apparent to
those skilled in the art from the description herein.
The compositions and methods of the claimed invention
may be employed for SX vivo therapy. 3y aX vivo therapy is
meant that therapeutic or immunogenic manipulations are
performed outside the body. For example, lymphocytes or other
target cells may be removed from a patient and treated with
high doses of the subject peptides, providing a stimulatory
concentration of pepti3e in the cell medium far in excess of
levels wnicn could be accomplished cr tolerated by the
patient. co1lowing treatment to stimulate the CTLs, the cells
are returned to the host to treat the HBV infection. The
WO 94/19011 2~ ~ ~ 416 PCT/US94/02195
29
host's cells may also be exposed to vectors which carry genes
encoding the peptides, as described above. Once transfected
with the vectors, the cells may be propagated in v t o or
returned to the patient. The cells which are propagated in
vitro may be returned to the patient after reaching a
predetermined cell density.
The peptides may also find use as diagnostic
reagents. For example, a peptide of the invention may be used
to determine the susceptibility of a particular individual to
a treatment regimen which employs the peptide or related
peptides, and thus may be helpful in modifying an existing
treatment protocol or in determining a prognosis for an
affected individual. In addition, the peptides may also be
used to predict which individuals will be at substantial risk
for developing chronic HBV infection.
The following examples are offered by way of
illustration, not by way of limitation.
EXAMPLE I
Identification of CTL-Specific HBenv Epitopes
This Example describes the identification of HBenv
peptides which stimulated HLA-restricted CTL responses
specific for HBV envelope antigens.
All patients included in the study were HLA-A2
positive. Thirteen patients (A-1 to A-13; Table II) were
studied during an episode of acute hepatitis, 6(C-i to C-6)
were chronically infected by HBV, and 6 uninfected healthy
volunteers (N-1 to N-6) served as normal controls. The
patients and their HLA haplotypes, determined using PBMC in
microcytotoxicity testing with HLA typing trays (One Lambda,
Canoga Park, CA), are shown in Table II.
Diagnosis of acute hepatitis was based on standard
diagnostic criteria. Diagnostic parameters included clinical
(jaundice) and biochemical evidence of liver injury (ALT
activity at least 20 fold greater than the upper limits of
normal), together with serological evidence of acute HBV
WO 94/19011 PCTIUS94/02195
2 15 630
infection (presence of HBV surface antigen (HBsAg) and IgG
anti-HBc antibody) in the absence of serological evidence of
hepatitis delta and hepatitis C virus infection (Abbott
Laboratories, North Chicago, IL). All patients were studied
during the first 4 weeks after onset of jaundice, at which
time their serum was positive for HBsAg and their ALT levels
were markedly abnormal. Eleven of the 13 patients
subsequently recovered from the illness, with normalization of
serum transaminase and clearance of HBsAg within four months
of initial diagnosis. One patient (A-11, Table II) developed
chronic active hepatitis and remained HBsAg positive 13 months
after initial diagnosis. One patient (A-10) was lost to
follow-up after the initial clinic visit. Patients with
chronic hepatitis B were repeatedly serologically positive for
HBsAg for more than six months and displayed mildly to
moderately elevated serum ALT activity. Normal controls had
no clinical history of HBV infection and were serologically
negative for HBV markers. All patients and
WO 94/19011 PCT/US94/02195
31
Table II.
Characteristics of Subjects Studied
Subject Sex Diagnosis HLA class I haplotype
A-1 Male Acute A2, A30, B35, B44, Cw4, Cw7
A-2 Male Acute A2, A31, 8w58(5Y), B51, Cw3
A-3 Male Acute A2, Bw41, Bw71, Cw4, Cw7
A-4 Male Acute A2, A32, Bw41, Bw71, Cw4, Cw7
A-5 Male Acute A2, Al, B8, 8w58(5Y), Cw7
A-6 Female Acute A2, Aw68, B35, Cw3, Cw4
A-7 Male Acute A2, A1, B8, Bw73, Cw3, Cw4
A-8 Female Acute A2, Aw69, Bw53, Cw4
A-9 Male Acute A2, A24, B7, B27, Cw2, Cw7
A-10 Male Acute A2, A3, Bw62, Bw71, Cw3, Cw4
A-11 Male Acute A2, A24, B35, Cw4
A-12 Male Acute A2, A3, Cw5
A-13 Male Acute A2, A3, B7, Bw60, Cw3, Cw7
C-1 Male Chronic A2, B27, B35, Cw2, Cw4
C-2 Male Chronic A2, A1, B8, B44
C-3 Male Chronic A2, A24, B44, Bw67
C-4 Male Chronic A2, Aw69, Bw41, Bw52
C-5 Male Chronic A2, B5, Bw62, Cw4
C-6 Male Chronic A2, A26, B35, Cw4
N-1 Male Normal A2, A11, B44, Cw4
N-2 Male Normal A2, Bw56, B35
N-3 Male Normal A2, A11, B8, Bw62, Cw4
N-4 Male Normal A2, A23, B5, Bw58, Cw2, Cw6
N-5 Male Normal A2, B44, Bw63
N-6 Female Normal A2, A11, Bw58
CA 02156416 2006-02-06
32
normal controls were serologically negative for antibody to
HIV.
PBMC from patients and normal donors were separated
on Ficoll-HypaqueTw density gradients (Sigma, St. Louis, MO).
washed three times in Hanks balanced salt solution (HBSS)
(Gibco, Grand Island, NY), resuspended in RPMI 1640 medium
(Gibco, Grand Island, NY) supplemented with L-glutamine (2
mM), gentamicin (10 g/ml), penicillin (50 U/ml), streptomycin
(50 g/ml), and HEPES (5 mM) containing 10t heat inactivated
human AB serum (complete medium) and plated in 24 well plates
at 4 x 106 cells/well. The synthetic peptides were added to
the cell cultures at a final concentration of 10 g/ml unless
otherwise noted. rHBcAg was added at 1 g/ml during the first
week of stimulation. At day 3, 1 ml of complete medium
supplemented with rIL2 (Hoffman-La Roche, Nutley, N.Y.) at 10
U/ml final concentration was added in each well. on day 7,
the cultures were restimulated with peptide, rIL2 and
irradiated (3000 rads) autologous or HLA-A2 matched feeder
cells, and the cultured PBMC were tested for CTL activity on
day 14. Selected cultures that displayed peptide specific
cytolytic activity were expanded by weekly restimulation with
1 x 106 irradiated (6000 rads) allogeneic PBMC and 1 x 105
irradiated (18000 rads) JY cells (allogeneic EBV-B transformed
cell line, HLA-A2.1, B7, Cw7) (14) in 1 ml of complete medium
containinq 1 q/ml peptide, 20 U/ml IL2 and 1 g/ml
phytohemagglutinin (PHA) (Sigma, St. Louis, MO).
For cytotoxicity assays, target cells consisted
either of a) autologous PHA stimulated blasts or allogeneic
HLA matched and mismatched EBV-transformed B lymphoblastoid
cell lines (B-LCL) incubated overnight with synthetic peptides
at 10 g/ml; b) stable B-LCL transfectants described above; or
c) B-LCL infected with recombinant vaccinia viruses (described
below). B-LCL were either purchased from The American Society
for Histocompatibility and Immunogenetics (Boston, MA) or
established from our own pool of patients and normal donors as
described in copending application 07/935,898. The cells were
maintained in RPMI 1-640 supplemented with L-glutamine (2 mM),
gentamicin (10 g/ml), penicillin (50 U/ml), streptomycin (50
WO 94/19011 PCT/US94/02195
33
g/ml), HEPES (5 mM), and 10% (vol/vol) heat inactivated FCS
(Gibco, Grand Island, NY). Short term lines of autologous
PBMC blasts were produced by stimulating peripheral blood PBMC
with PHA at 1 g/ml in the RPMI 1640 supplemented with L-
glutamine (2 mM), gentamicin (10 g/ml), penicillin (50 U/ml),
streptomycin (50 g/ml), HEPES (5 mM), 10% (vol/vol) heat
inactivated FCS, and 10 U/ml rIL2 for 7 days before use as
target cells. Vaccinia infected targets were prepared by
infection of i x 106 cells at 50 plaque-forming U/cell on a
rocking plate at room temperature for one hour followed by a
single wash and overnight incubation at 370C.
Target cells were labeled with 100 Ci of 51Cr
(Amersham, Arlington Heights, IL) for one hour and washed
three times with HBSS. Cytolytic activity was determined in a
standard 4 hour 51Cr release assay using U-bottom 96 well
plates containing 5000 targets per well. All assays were
performed in duplicate. Percent cytotoxicity was determined
from the formula: 100 x[(experimental release - spontaneous
release)/(maximum release - spontaneous release)]. Maximum
release was determined by lysis of targets by detergent (1%
Triton X-100, Sigma). Spontaneous release was less than 25%
of maximal release in all assays.
Two HLA-A2 positive patients with acute hepatitis
(A-1 and A-3) were initially selected for analysis of the CTL
response to HBsAg329-348 (ASARFSWLSLLVPFVQWFVG (Seq. ID No.
22)), which contains 2 overlapping HLA A2.1 allele specific
binding motifs (WLSLLVPFV and LLVPFVQWFV). One of these
patients (A-3) was known from previous experiments to display
an HLA A2 restricted CTL response to a 10 residue HBV
nucleocapsid epitope (HBcAg18-27) that also represents an HLA
A2.1 allele specific binding motif (FLPSDFFPSV). This patient
was considered a potential responder to one or both of the
motifs in HBsAg329-348. Another patient (A-i), known to be a
nonresponder to HBcAg18-27, was studied for comparison.
HBsAg329-348 specific CTL lines were generated from
PBMC of both patients by stimulation with the peptide as
described above. After 2-3 weeds of stimulation, both
patients displayed a strong cytotoxic response against a
PCTIUS94/02195
WO 94/19011 2156416
34
homozygous HLA A2.1 positive EBV cell line (JY) prepulsed with
the HBsAg329-348 peptide. Patient A-1's HBsAg329-348 specific
cell line was selected for cloning.
CTL lines were cloned originally at 1, 10, and 100
cells per well and then subcloned at 0.3 or 1 cell per well in
96 well microtiter plates. The cells were plated in the
presence of peptide (1 g/ml), PHA (1 g/ml), rIL-2 (20 U/ml),
irradiated (6000 rads) allogeneic PBMC (105 cells/well), and
irradiated (18000 rads) JY cells (104 cells/well). HBV
specific clones were restimulated in a 24 well plate as
described above except that the peptide was omitted and
irradiated JY cells, transfected with a plasmid that confers
stable expression of the HBV large envelope antigen (EBO-
preSl, reference 10), were added at 105 cells per well.
Using patient A-1's HBsAg329-348 specific cell line
four clones were derived from cells plated at 1 cell per well
(clone B13, B16, B17) or 0.3 cells per well (clone B3). Clone
B3 was tested against a panel of allogeneic target cells
partially matched with the effectors at the level of HLA class
I alleles. Using allogeneic target cells partially matched at
HLA class I with patient A-1, the cytolytic activity of clone
B3 was found to be HLA-A2 restricted, due to the presence of 2
HLA-A2.1 binding motifs in the peptide. An HBsAg329-348
specific polyclonal CTL line derived from patient A-3 was also
determined to be HLA-A2 restricted in the same manner. Since
the HLA-A2 subtypes of the patients were not determined, it is
not known if the CTL response to the peptides is restricted
only by the HLA-A2.1 allele or whether it extends to other
HLA-A2 subtypes as well.
To determine a minimum, optimal HLA-A2 restricted CTL
epitope within HBsAg329-348, a panel of amino-terminal
truncations and overlapping nine-mers and ten-mers derived
from the HBsAg329-348 sequence were produced to map the HLA-A2
restricted CTL epitope(s) present in this 20 residue peptide,
which contains 2 overlapping ideal HLA-A2.1 binding motifs.
The HLA-A2 restricted CTL clone B17 from patient A-1, and a
polyclonal CTL line 1B9 from patient A-3, derived by
repetitive stimulation of the initial cell line described
WO 94/19011 2156416 PCT/US94/02195
above and HBsAg329-348, were used as effector cells to
establish the fine specificity of the CTL response to
HBsAg329-348. Target cells were produced by incubating an
HLA-A2.1 positive B cell line (JY) either with the original
5 20-mer or with the truncated peptides.
The results, shown in Fig. 1, indicated that only the
first of the 2 HLA-A2.1 binding motifs (HBsAg335-343) is
recognized by the CTL. Furthermore, the data demonstrate that
this peptide (WLSLLVPFV) is the minimal, optimal HLA-A2
10 restricted epitope recognized by HBsAg329-348 stimulated CTL,
since omission of the extreme amino-terminal or the extreme
carboxy-terminal residue from HBsAg335-343 abolishes
recognition by the CTL.
The superiority of HBsAg335-343 at the effector level
15 was reiterated when the peptides were used to stimulate a CTL
response in PBMC from patient A-1. Synthetic peptides
representing assorted HBsAg329-348 subunits were used at 10 M
to stimulate PBMC of patient A-i. After 2 weeks of
stimulation the cytotoxicity of these lines was tested at E:T
20 of 60:1 against JY target cells prepulsed with 10 M of the
same peptide and JY target cells prepulsed with 10 pM
HBsAg335-343. Results shown in Fig. 2 represent % lysis in a
4 hour 51Cr release assay. As can be seen in Fig. 2, although
HBsAg335-343 and its extended variants proved capable of
25 inducing a CTL response, omission of the extreme amino- and
carboxy-terminal amino acids completely abolished the ability
of the peptides to stimulate a CTL response, thereby
reinforcing the conclusion that HBsAg335-343 is the minimal
optimal HLA-A2 restricted epitope between residues 329-348 of
30 HBsAg.
EXAMPLE II
CTL Response to Seven HLA-A2.1 Binding Motifs in HBVenv
35 Seven ideal HLA-A2.1 allele specific binding motifs,
defined as peptides between 9-10 residues in length that
contain a leucine in the second position and a valine as the
carboxy-terminal residue, are present in the HBsAg region of
WO 94/19011 PCT/US94/02195
215641" 6 36
the HBV envelope protein (Table III). Based on the results
obtained in Example I, the ability of these seven envelope
peptides, plus the known HLA-A2 restricted HBV nucleocapsid
epitope (HBcAg18-27), to stimulate a CTL response in 12 HLA-A2
positive patients with acute hepatitis B, was examined. For
comparison, six HLA-A2 positive patients with chronic
hepatitis and 6 uninfected normal controls were tested for
responsiveness to the same panel of peptides.
Table III. HBV-derived HLA-A2.1 binding motifs and peptides
Peptide Sequence Seq. ID No.
1 HBcAg18-27 FLPSDFFPSV 23
2 HBsAg201-210 SLNFLGGTTV 24
3 HBsAg251-259 LLCLIFLLV 7
4 HBsAg260-269 LLDYQGMLPV 9
5 HBsAg335-343 WLSLLVPFV 10
6 HBsAg338-347 LLVPFVQWFV 25
7 HBsAg348-357 GLSPTVWLSV 26
8 HBsAg378-387 LLPIFFCLWV 27
PBMC from acute patients (A-i to A-12), chronic
patients (C-i to C-6), and normal subjects (N-1 to N-6) were
stimulated with the following synthetic peptides:
1=HBcAg18-27, 2=HBsAg201-210, 3=HBsAg251-259, 4=HBsAg260-269,
5=HBcAg335-343, 6=HBsAg338-347, 7=HBsAg348-357,
8=HBsAg378-387, for two weeks as described in Example I, and
tested in a 4 hour 51Cr release assay against JY target cells
prepulsed overnight with the same peptide. Peptide specific
cytotoxicity was measured by subtracting the 51Cr release by
JY target cells not prepulsed with peptide from the 51Cr
release by JY target cells prepulsed with the peptide.
Results shown (Fig. 3) represent % specific lysis in a 4 hour
51Cr release assay.
WO 94/19011 215 64 1 G PCTIUS94/02195
37
As can be seen in Fig. 3, nine of the twelve HLA-A2
positive patients with acute hepatitis responded to at least
one of the peptides in the panel. In contrast, none of the
six HLA-A2 positive uninfected normal controls responded to
any of the peptides following the same in vitro stimulation
strategy, suggesting that responsiveness to these peptides by
the patients reflects in vivo priming by the corresponding
HBV-derived epitopes.
Importantly, eight of the nine responders recognized
multiple epitopes within the panel, indicating that the CTL
response to HBV during acute hepatitis is both polyclonal and
multispecific. Furthermore, there was substantial variation
in the spectrum of epitopes recognized among the nine
responders, with certain epitopes being recognized more
frequently than others. For example, HBcAg18-27 and
HBsAg335-343 were recognized individually by seven and eight
of the nine responders, respectively, and when combined they
were recognized by all nine of the responders. In contrast,
HBsAg348-357, HBsAg251-259 and HBsAg260-269 were recognized by
only 3/9, 2/5 and 3/6 of the responders in whom they were
tested.
Nucleotide sequence analysis of circulating virion
DNA in acutely infected patients showed that all patients,
including the CTL nonresponders, were infected by viruses that
expressed the precise amino acid sequence present in the
prototype HBsAg335-343 peptide used to stimulate expansion of
CTL in vitro. Since residues 335-343 are known to be
conserved in all the published HBV sequences derived from all
4 HBV subtypes, as published in the GenBank-72 database, as
well as in the 10 patients studied herein, it may be concluded
that HBsAg335-343 is an HBV group specific CTL epitope. The
same was not true for HBsAg348-357, however, since only seven
of the ten patients were found to be infected by viruses that
encode the prototype sequence used for in vitro stimulation
(GLSPTVWLSV). The remaining three patients (A-9, A-10, A-13)
displayed a variant sequence in which the carboxy-terminal
valine was substituted by an alanine at position 357. Among
the patients infected by the prototype virus, CTL responders
WO 94/19011 2156416 PCT/US94/02195
38
and nonresponders to HBsAg348-357 were observed, just as for
the response to HBsAg335-343. On the other hand, none of the
3 patients infected by the variant virus displayed a CTL
response to the prototype peptide.
It is noteworthy that all nine responders
subsequently became HBsAg negative and their liver disease
completely resolved. In contrast, all six patients with
chronic hepatitis, who failed to clear the virus, also failed
to mount a peripheral blood CTL response to any of these
epitopes. Three of the acutely infected patients (A-10, A-11,
A-12) also failed to respond to any of these peptides.
Furthermore, one of the nonresponders (A-il) developed chronic
active hepatitis and was still HBsAg positive 13 months after
his acute illness. These combined data strongly suggest a
relationship between the CTL response and viral clearance.
However, nonresponder patient A-12 seroconverted to HBsAg
negativity between 1-4 months after disease onset.
As indicated in Table II and Fig. 3, four of the nine
responders shared only the HLA A2 allele with the JY target
cell line used in this study (HLA-A2, B7, Cw7), demonstrating
that the response to all of the peptides was HLA-A2 restricted
in these individuals. Since the remaining responders also
share the HLA B7 and/or Cw7 alleles present in the JY target
cells in addition to A2, it is possible, although unlikely,
that these alleles could also serve as restriction elements
for these epitopes in these patients.
The molecular basis for the differential
immunogenicity of the seven HBV envelope peptides was not
immediately evident from a comparison of their sequences.
Potential differences in the relative binding affinity of the
peptides to HLA-A2.1 were examined by determining the ability
of the seven envelope peptides to compete with the binding of
an unrelated HLA-A2 restricted nucleocapsid epitope
(HBcAg18-27) to a homozygous HLA-A2.1 positive B cell line
(JY).
The competitive binding inhibition assay used an
HBcAg18-27 specific CTL clone from patient A-4 as a source of
effector cells. Blocking peptides (1, 10, 100 M) were added
WO 94/19011 2156416 PCT/US94/02195
39
to a mixture of 51Cr-labelled, HLA-A2.1 positive JY target
cells and effector cells (E:T=40:1, 3000 target cells/well)
for 40 minutes before the addition of a subsaturating
concentration (0.03 M) of the target peptide, HBcAg18-27. The
binding ability of each peptide was assessed by calculating
the degree to which it blocked the lysis of target cells in a
4 hour 51Cr release assay.
As shown in Fig. 4, all four immunogenic peptides and
two of the three nonimmunogenic peptides bound to the HLA-A2.1
molecule, but with widely (more than 100-fold) variable
efficiencies that did not correlate with their relative
immunogenicity. Importantly, the only peptide that did not
bind to HLA-A2.1 in this assay (HBsAg337-348) was
nonimmunogenic. For the other two nonimmunogenic peptides,
however, the HLA-A2.1 binding affinity was as high or higher
than some of the immunogenic peptides. Thus, although the
capacity of a peptide to bind to this class I molecule is
required for immunogenicity, it does not guarantee it. This
suggests that the additional factors at the level of antigen
processing and the T cell repertoire may play a role in
determining which HLA-A2.1 binding peptides within a viral
protein are able to induce a CTL response.
EXAMPLE III
Peptide Specific CTL Recognize Endogenous HBenv Antigen
The ability of HBsAg335-343 and HBsAg348-357 specific
CTL to recognize endogenously synthesized antigen was examined
by measuring their ability to lyse target cells that had been
infected with two groups of recombinant vaccinia viruses which
encode the large, middle and major envelope polypeptides
derived from cloned HBV genomes of either the ayw or the adw
subtypes of HBV.
Recombinant vaccinia viruses expressing the HBV
large, middle and major envelope polypeptides (adw subtype)
and a corresponding control wild type vaccinia were obtained
(Smith et al., Nature 302:490 (1983); Cheng et al., J. Virol.
CA 02156416 2006-02-06
60:337 (1986); and Cheng and Moss, J. Virol. 61:1286 (1987)).
An
independent series of recombinant vaccinia viruses expressing
the same three HBV envelope polypeptides of the HBV ayw
5 subtype was derived as follows. For expression of the HBsAg,
an XhoI/Sphl restriction fragment containing nucleotides 1409-
2514 of the HBV sequence was cloned into a vaccinia virus
expression vector downstream from the 7.5 K early/late
promoter. For the presi expressing vaccinia virus, a Bgl
10 II/Sph I fragment containing nucleotides 937-2514 was used.
For cloning the preS2 coding sequence, first a short adapter
oligonucleotide was synthesized which started at nucleotide
1267 (e.g., six base-paris upstream from the preS2 start
codon) and spanned the Eco RI site at position 1280. After
15 cloning this oligonucleotide into the vaccinia virus
expression vector, the coding sequence was completed by
recloning the Eco RI/SphI HBV fragment (nucleotides 1280-2514)
into this intermediate construct. Generation of recombinant
vaccinia viruses was done according to standard procedures as
20 described in Smith et al., sunra. Stable transfectants that
expressed the HBV envelope proteins (ayw subtype) were
produced by transfection of B-LCL with a panel of EBV based
expression vectors that contain the corresponding HBV (ayw
subtype) coding regions, as described in Guilhot at al., J.
25 Virol. 66:2670 (1992), incorporated herein by reference.
An HBsAg335-343 specific CTL line (patient A-1) and an
HBsAg348-357 specific CTL line (patient A-4) were generated by
stimulation with peptide sequences WLSLLVPFV and GLSPTVWLSV,
respectively. CTL were incubated with 51Cr-labelled JY target
20 cells that had been preincubated either with media, with the
inducing peptide or (in the case of HBSAg348-357) with a
variant peptide (GLSPTVWLSA). CTL were also incubated with
51Cr-labelled JY target cells that had been infected with a
panel of 6 recombinant vaccinia viruses that express the HBV
35 major (V-HBs), middle (V-pres21, and large (V-oreSl) envelope
polvpeptides derived from avw and adw HBV genomes. :aild-type
vaccinia viruses (V-wt) were used as controls. "he
HBSAg335-343 specific CTL line (right panel) was used at an
CA 02156416 2006-02-06
41
E:T=40:1. The HBsAg348-357 specific CTL line (left panel) was
used at an E:T=3:1. Results shown represent % lysis in a 4
hour 51Cr release assay.
Both HBsAq335-343 and HBsAq348-357 specific CTL from
patients A-1 and A-4 were able to lyse recombinant vaccinia
virus infected tarqet cells that synthesize all three of the
HBV envelope proteins (Fig. 5). This indicates that both of
these synthetic peptides represent epitopes that are generated
by the endogenous processinq of the larqe, middle and major
HBV envelope polypeptides within infected cells.
Importantly, HBsAq335-343 specific CTL could lyse
targets that were infected by both sets of recombinant
vaccinia viruses with equal efficiency, with the HBsAq348-357
specific CTL lysed with ayw infected target cell panel much
more efficiently than the adw targets.
The results described in the foregoing Examples
illustrate that the CTL response to HBV in-man appears to be
quite polyvalent, presumably to afford more efficient
protection against this serious viral infection. Furthermore
the data indicate that the peptide stimulation strategy
employed herein is both efficient and effective for the
identification and analysis of the polyvalent response,
restricted as it is by the polymorphic HLA class I locus. As
additional HLA allele specific binding motifs are identified,
HBV-derived peptides containing these motifs can be used for
ja vitro stimulation of CTL precursors.
5
From the foregoing it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
10 modifications may be made without deviating from the spirit
and scope of the invention. Accordingly, the invention is not
limited except as by the appended claims.
WO 94/19011 PCT/US94/02195
2156416
42
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Chisari, Francis V.
(ii) TITLE OF INVENTION: PEPTIDES FOR INDUCING CYTOTOXIC T
LYMPHOCYTE RESPONSES TO HEPATITIS B VIRUS
(iii) NUMBER OF SEQUENCES: 27
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Townsend and Townsend
(B) STREET: One Market Plaza, Steuart Street Tower
(C) CITY: San Francisco
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(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Parmelee, Steven W.
(B) REGISTRATION NUMBER: 31,990
(C) REFERENCE/DOCKET NUMBER: 14740-4
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 206-467-9600
(B) TELEFAX: 415-543-5043
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Phe Leu Leu Thr Arg Ile Leu Thr Ile
1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 94/19011 2 15 " 4 16 PCT/US94/02195
43
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ile Leu Leu Leu Cys Leu Ile Phe Leu
1 5
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
PCT/US94/02195
WO 94/19011 2156416
44
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Leu Leu Cys Leu Ile Phe Leu Leu
1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Leu Leu Cys Leu Ile Phe Leu Leu Val
1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Leu Leu Cys Leu Ile Phe Leu Leu Val Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Leu Leu Asp Tyr Gln Gly Met Leu Pro Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
2156416
WO 94/19011 PCT/US94/02195
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Trp Leu Ser Leu Leu Val Pro Phe Val
1 5
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ser Ile Leu Ser Lys Thr Gly Asp Pro Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Val Leu Gln Ala Gly Phe Phe Leu Leu
1 5
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Phe Leu Gly Giy Thr Pro Val Cys Leu
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 94/19011 ~ PCT/US94/02195
RD 416
46
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ser Ile Val Ser Pro Phe Ile Pro Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ser Trp Leu Ser Leu Leu Val Pro Phe Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Trp Leu Ser Leu Leu Val Pro Phe Val Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15
Ser Phe Leu Pro
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 94/19011 _ 2156416 PCT/US94/02195
47
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Pro His His Tyr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu
1 5 10 15
Met Tyr Leu Ala
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val
1 5 10 15
Ile Glu Tyr Leu
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro
1 5 10 15
Asn Ala Pro Ile
(2) INFORMATION FOR SEQ ID NO:22:
WO 94/19011 PCTIUS94/02195
48
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln
1 5 10 15
Trp Phe Val Gly
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ser Leu Asn Phe Leu Gly Gly Thr Thr Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Leu Leu Val Pro Phe Val Gln Trp Phe Val
1 5 10
WO 94/19011 2156 416 PCT/US94/02195
49
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Gly Leu Ser Pro Thr Val Trp Leu Ser Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Leu Leu Pro Ile Phe Phe Cys Leu Trp Val
1 5 10
