US20030171538A1 - Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus - Google Patents

Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus Download PDF

Info

Publication number
US20030171538A1
US20030171538A1 US10/359,431 US35943103A US2003171538A1 US 20030171538 A1 US20030171538 A1 US 20030171538A1 US 35943103 A US35943103 A US 35943103A US 2003171538 A1 US2003171538 A1 US 2003171538A1
Authority
US
United States
Prior art keywords
leu
ser
arg
pro
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/359,431
Inventor
Francis Chisari
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scripps Research Institute
Original Assignee
Scripps Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Research Institute filed Critical Scripps Research Institute
Priority to US10/359,431 priority Critical patent/US20030171538A1/en
Publication of US20030171538A1 publication Critical patent/US20030171538A1/en
Priority to US11/221,470 priority patent/US7744898B2/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: SCRIPPS RESEARCH INSTITUTE
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Cytotoxic T lymphocytes 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.
  • HBV Hepatitis B Virus
  • HBV infection in adults typically leads to an 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.
  • a small percentage of individuals who are infected with HBV in adulthood develop fulminant hepatitis associated with a strong immune response with high lethality.
  • HBV surface antigen HBsAg
  • HBsAg HBV surface antigen
  • Synthetic HBsAg peptide-based vaccines have also been proposed. See, for example, U.S. Pat. Nos. 4,599,230 and 4,599,231.
  • the anti-HBsAg vaccines afford protection in only about 90% of immunized individuals. Those who are unimmunized, or immunized but unprotected, provide a significant reservoir of potential infection.
  • 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).
  • 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.
  • 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 sequence of the HBV polymerase protein.
  • the CTL inducing peptide will have the sequence of HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No.
  • HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No.
  • HBpol455-463 Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu
  • HBpol505-514 Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe
  • HBpol551-559 Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala
  • HBpol575-583 Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
  • HBpol655-663 Al-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile
  • HBpol748-757 Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg
  • HBpol758-766 Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu
  • HBpol773-782 Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val
  • HBpol803-811 (Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; and HBpol816-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5]; 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.
  • 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 which comprise a peptide of the invention formulated with an additional peptide, a liposome, an adjuvant and/or a pharmaceutically acceptable carrier.
  • 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 HBpol epitopes.
  • compositions 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 envelope or core.
  • 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 vaccine compositions comprise an immunogenically effective amount of a HBV polymerase peptide mentioned above which induces a MHC class I restricted cytotoxic T lymphocyte response, such as HLA-A2, -A1, -A3, A-11, and/or A24, and will typically further comprise an adjuvant, e.g., incomplete Freund's adjuvant or aluminum hydroxide.
  • the vaccine can further comprise components which elicit a protective antibody response to other HBV antigen, such as envelope (surface) antigen.
  • 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 polymerase antigen. The absence of such cells determines whether the individual of interest is susceptible to developing chronic HBV infection.
  • lymphocytes are peripheral blood lymphocytes and the individual of interest is suffering from an acute HBV infection.
  • FIG. 1 shows the CTL response to two polymerase peptides that contain the HLA-A2 motif in a patient using target cells pulsed with peptide that match only at HLA-A2.
  • FIG. 2 shows the ability of several polymerase 803-811 peptide specific clones to recognize endogenously synthesized polymerase.
  • FIG. 3 shows that the CTL response to polymerase peptide 803-811 can recognize cells pulsed with peptide and endogenously synthesized polymerase (Vpol), whereas the CTL response to polymerase peptide 61-69 only recognized cells pulsed with the 61-69 peptide.
  • FIG. 4 shows the aligned amino acid-sequences of 20 cloned HBV polymerase proteins; line 158 is a consensus sequence where capital letters represent 100% consensus, lower case letters represent >50% consensus, and “.” is ⁇ 50% consensus.
  • FIG. 5 shows HBV specific CTL response in patients with acute hepatitis (A-1-A-9), chronic hepatitis (C-1-C-9) and normal subjects (N-1-N-9).
  • PBMC were stimulated with the peptides indicated for 2 weeks and tested in a 4-h 51 Cr-release assay against JY target cells prepulsed overnight with the same peptide.
  • Peptide-specific cytotoxicity was measured by subtracting the 51 Cr-release by JY target cells not prepulsed with the peptide from the 51 Cr-release by JY target cells prepulsed with the peptide. Results shown represent percent specific lysis in a 4 hr 5 Cr-release assay at an E:T of 50:1.
  • FIG. 6 shows CD8 + cells recognize endogenously synthesized antigen in target cells sharing the HLA-A2 allele (Patient A-1). Epitope-specific lines were generated by stimulating PBMC with the individual peptide for three weeks with weekly restimulation. On day 15 of CD4+ (positive selection) and CD8+ (negative selection) enriched lines were generated from the original bulk culture by panning. FACS-analysis showed an average enrichment by a factor of 3. Results shown represent percent specific lysis in a 4 hr 51 Cr-release assay at an E:T of 30:1. Targets (JY-EBV) were either pulsed with the corresponding peptide overnight stably transfected with the polymerase expression vector.
  • FIG. 7 shows CTL-response to Pol455-463 GLSRYVARL [Seq ID No. 2].
  • Wild-type vaccinia virus (Wt) (X) or JY-EBV peptides without peptide (O) were used as a control.
  • FIG. 8 shows HLA-restriction of epitope Pol455-463.
  • Pol455-463-specific lines from patient A-1 and A-2, generated by stimulation with Pol455-463 peptide, were tested against allogeneic partly HLA-matched EBV-B cells prepulsed overnight with 10 ⁇ g/ml of the same peptide. Sharing HLA class I at other loci did not render target cells susceptible to lysis. Cytotoxicity was measured at E:T of 50:1 in a 4 hr 51 Cr-release assay.
  • FIG. 9 shows recognition of truncated, elongated (a) or variant peptides (b) by Pol455-463 specific CTL-lines, generated by weekly stimulation of PBMC from patient A-1 with peptide Pol455-463 for 4 weeks. Cytotoxicity was measured at E:T of 50:1 in a 4 hr 51 Cr-release assay against JY-EBV cells prepulsed with varying amounts of the same peptide overnight.
  • the present invention provides peptides derived from HBV polymerase proteins for use in compositions and methods for the treatment, prevention and diagnosis of HBV infection.
  • the peptides 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, including 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 sequence of the HBV polymerase protein (HBpol), particularly CTL epitopes within HBpol4-13, HBpol61-69, HBpol108-116, HBpol139-147, HBpol151-160, HBpol152-161, HBpol 455-463, HBpol505-514, HBpol551-559, HBpol575-583, HBpol655-663, HBpol748-757, HBpol758-766, HBpol773-782, HBpol803-811, or HBpol816-824, where the numbering is according to Galibert et al., supra.
  • HBpol HBV polymerase protein
  • 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 fewer than twenty-five, e.g., eight to seventeen amino acid residues derived from an HBc sequence. It may be desirable to optimize peptides of the invention to a length of eight to twelve amino acid residues, more preferably nine to eleven, commensurate in size with endogenously processed viral peptides that are bound to MHC class I molecules on the cell surface.
  • the peptides will have at least a majority of amino acids which are homologous to a corresponding portion of contiguous residues of the HBV pol sequences herein, and contain 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.
  • 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 glycosylation, 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.
  • the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide.
  • 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.
  • a cytotoxic T lymphocyte response is 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
  • lymphokines e.g., gamma interferon
  • products e.g., serine esterases
  • homologous denotes 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.
  • the peptides of the invention contain CTL-inducing epitopes derived from various epitopic regions of the HBV polymerase protein.
  • the peptides are from the region of HBpol 61-69 and include peptides derived from those sequence regions 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 HBpol 61-69 sequence, where HBpol 61-69 has the following sequence (for HBV subtype ayw):
  • the peptide embodiments of this HBpol 61-69 region and the other polymerase peptide regions described herein 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 HBpol, amino acids added to facilitate linking, other N- and C-terminal modifications, linked to carriers, etc., as further described herein.
  • the peptide HBpol 61-69 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • HBpol region peptides containing CTL epitopes of the invention comprises the peptide HBpol 455-463, and peptides derived from HBpol455-463 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 occurring HBpol455-463 sequence, where HBpol 455-463 has the sequence (for HBV subtype ayw):
  • the selected peptide can be flanked and/or modified at one or both termini as described herein.
  • the peptide HBpol 455-463 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • HBpol region peptides containing CTL epitopes of the invention comprises the peptide HBpol 773-782, and peptides derived from HBpol773-782 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 occurring HBpol773-782 sequence, where HBpol 773-782 has the sequence (for HBV subtype ayw):
  • HBpol 773-782 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • Other HBpol peptide embodiments of the invention are prepared from the region of HBpol803-811. 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 acids or more.
  • HBpol803-811 has the sequence (for HBV subtype ayw):
  • the selected peptide can be flanked and/or modified at one or both termini as described herein.
  • the peptide HBpol 803-811 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • HBpol peptide embodiments of the invention are prepared from the region of HBpol816-824.
  • 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 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 HBpol816-824 sequence, where HBpol816-824 has the sequence (for HBV subtype ayw):
  • the selected peptide can be flanked and/or modified at one or both termini as described herein.
  • the peptide HBpol 816-824 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • HBpol peptide embodiments of the invention are prepared from the regions of HBpol4-13, HBpol108-116, HBpol139-147, HBpol-151-160, HBpol152-161, HBpol505-514, HBpol551-559, HBpol575-583, HBpol655-663, HBpol748-757or HBpol758-766.
  • a peptide prepared from one of the aforementioned regions contains 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 acids or more.
  • HBpol4-13 Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu HBpol108-116 [Seq ID No. 13] Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arq HBpol139-147 [Seq ID No.
  • the selected peptide can be flanked and/or modified at one or both termini as described herein.
  • the peptide HBpol151-160 induces a CTL response which is mediated by at least the MHC class I molecule HLA-A1.
  • the peptides HBpol551-559 and HBpol655-663 induce a CTL response which is mediated by at least the MHC class I molecule HLA-A2.
  • the peptide HBpol575-583 induces a CTL response which is mediated by at least the MHC class I molecule HLA-A2.1.
  • the peptides HBpol108-116, HBpol139-147, HBpol152-161, and HBpol748-757 induce a CTL response which is mediated by at least the MHC class I molecule HLA-A3 (HBpol748-757 appearing to also be restricted by A24).
  • the peptides HBpol4-13, HBpol505-514, and HBpol758-766 induce CTL responses which are mediated by at least the MHC class I molecule HLA-A24.
  • amino acids can be added to the termini of an oligopeptide or peptide to provide 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.
  • the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2 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.
  • HBV peptides of the present invention or analogs or homologs 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.
  • 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.
  • 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 may be made CTL-inducing by substituting hydrophobic residues at the N-terminus of the peptide where the second residue is normally hydrophobic.
  • peptides employed in the subject invention need not be identical to peptides HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No.
  • HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol 455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No.
  • HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No.
  • HBpol748-757 Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg
  • HBpol758-766 Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu
  • HBpol773-782 Ile-Leu-Arc-Gly-Thr-15 Ser-Phe-Val-Tyr-Val
  • HBpol803-811 Ser-ou-Tyr-Ala-Asp-Ser-Pro-Ser-Val
  • N 81C-824 Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu [Seq ID No. 5]; or N 81C-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5], 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.
  • a common envelope determinant
  • Each strain also has two other envelope determinants, one of which is either “d” or “y”, and the second is either “w” or “r”.
  • HBV envelope region is also described in Galibert et al., Nature 281:646 (1979), each of the foregoing being incorporated herein by reference.
  • 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, the GenBank sequences also being incorporated herein by reference.
  • 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.
  • 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, Tyr.
  • the portion of the sequence which is intended to substantially mimic an HBV cytotoxic T lymphocyte 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.
  • 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.
  • 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.
  • 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.
  • residues can be identified by single amino acid substitutions, deletions, or insertions.
  • 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).
  • increased binding affinity of peptide analogues to its MHC molecule for presentation to a cytotoxic T-lymphocyte can also be achieved by such alterations.
  • 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 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 replacing the L-amino acids with D-amino acids.
  • the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide.
  • 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.
  • 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 HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No.
  • HBpol61-69 Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val
  • HBpol108-116 Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg
  • HBpol139-147 Arg-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg
  • HBpol151-160 His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr
  • HBpol152-161 Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys
  • HBpol 455-463 Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu
  • HBpol505-514 Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe
  • HBpol551-559 Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala
  • HBpol575-583 Phe-Leu-Leu-ser-Leu-Gly-Ile-His-Leu
  • HBpol655-663 Al-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile
  • HBpol748-757 Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg
  • HBpol758-766 Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu
  • HBpol773-782 Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val
  • HBpol803-811 Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val
  • HBpol816-824 Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
  • 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., 51 Cr) 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.
  • autologous labeled e.g., 51 Cr
  • target cells such as HLA matched macrophages, T cells, fibroblasts or B lymphoblastoid cells
  • 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.
  • 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., HBpol.
  • Cells having activity are also stimulated with an appropriate T cell mitogen, e.g., phytohemagglutinin (PHA).
  • PHA phytohemagglutinin
  • the restimulated cells are provided with irradiated allogeneic PBLs as an antigen nonspecific source of T cell help, and HBV antigen.
  • 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 allogeneic B-lymphoblastoid or other cells transfected or infected with appropriate antigen.
  • 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).
  • two or more peptides may define different or overlapping cytotoxic T lymphocyte epitopes from a particular region, e.g., the HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No.
  • HBpol61-69 Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val
  • HBpol108-116 Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg
  • HBpol139-147 Arg-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg
  • HBpol151-160 His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr
  • HBpol152-161 Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys
  • HBpol 455-463 Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu
  • HBpol505-514 Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe
  • HBpol551-559 Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala
  • HBpol575-583 Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
  • HBpol655-663 Al-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile
  • HBpol748-757 Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg
  • HBpol758-766 Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu
  • HBpol773-782 Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val
  • HBpol803-811 Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val
  • HBpol816-824 Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
  • peptides which peptides can be combined in a “cocktail” to provide enhanced immunogenicity for cytotoxic T lymphocyte responses.
  • 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 MHC restriction element different from the first.
  • Other CTL-inducing HBV peptides are oesdcribed in co-pending application U.S. Ser. Nos. 07/935,898 and 08/024,120, which are incorporated herein by reference.
  • 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.
  • Therapeutic 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.
  • 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 forming a homopolymer, a plurality of repeating epitopic units are presented.
  • the peptides differ, e.g., a cocktail representing 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.
  • 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.
  • 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.
  • 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.
  • disulfide/amide forming agents are known. See, for example, Immun. Rev. 62:185 (1982), which is incorporated herein by reference.
  • Other bifunctional coupling agents form a thioether rather-than a disulfide linkage.
  • Many of these 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-1-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.
  • 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. 6].
  • 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. 7], 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.
  • the peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc. 105:6442 (1983); Merrifield, Science 232:341-347 (1986); and Barany and Merrifield, The Peptides , Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284 (1979), each of which is incorporated herein by reference.
  • recombinant DNA technology may be 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.
  • 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.
  • fusion proteins which comprise one or more peptide sequences of the invention can be used to present the HBV cytotoxic T cell determinants.
  • a recombinant polymerase protein of the invention is prepared in which the HBpol amino acid sequence is altered so as to more effectively present epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response.
  • a polypeptide is used which incorporates several T cell epitopes.
  • coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the 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.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available.
  • 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.
  • 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; AB 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.
  • compositions 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.
  • 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 ⁇ 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.
  • 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.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of cytotoxic T-lymphocyte stimulatory peptides of the invention sufficient to effectively treat the patient.
  • 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.
  • the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CTL peptides 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.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration.
  • the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • 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.
  • an acceptable carrier preferably an aqueous carrier.
  • 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.
  • compositions may be packaged for use as is, or 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.
  • the pharmaceutical composition 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 vivo against viral antigens, e.g., tripalmitoyl-S-glycerylcysteinly-seryl-serine (P 3 CSS), which can effectively prime virus specific cytotoxic T lymphocytes when covalently attached to an appropriate peptide.
  • P 3 CSS tripalmitoyl-S-glycerylcysteinly-seryl-serine
  • Peptides of the invention can be coupled to P 3 CSS, for example, and the lipopeptide administered to an individual to specifically prime a cytotoxic T lymphocyte response to HBV.
  • the induction of neutralizing antibodies can also be primed with P 3 CSS 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.
  • 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, Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.
  • the peptides of the invention may also be administered via liposomes, which serve to target the peptides 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 dispersions, lamellar layers and the like.
  • 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 antigen, or with other therapeutic or immunogenic compositions.
  • a molecule which binds to e.g., a receptor, prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • 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/immunogenic peptide compositions.
  • Liposomes for 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.
  • lipids are generally guided by consideration of, e.g., liposome size and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, the mode of administration, the peptide being delivered, the stage of disease being treated, etc.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • 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%.
  • 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.
  • 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 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.
  • cytotoxic T lymphocyte responses can be primed by conjugating peptides of the invention to lipids, such as P 3 CSS.
  • lipids such as P 3 CSS.
  • 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 HBpol61-69 [Seq ID No.
  • HBpol 455-463 [Seq ID No. 2], Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol551-559 and HBpol655-663; HBpol 773-782 [Seq ID No. 3], Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 [Seq ID No. 4], Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 [Seq ID No.
  • Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu these will be administered to at least HLA-A2 individuals.
  • peptides from HBpol151-160 these will be administered to at least HLA-A1 individuals.
  • Vaccines comprising peptides from HBpol575-583 will be administered to at least HLA-A2.1 individuals.
  • Vaccines comprising peptides from HBpol575-583 will be administered to at least HLA-A2.1 individuals.
  • Vaccines comprising peptides from HBpol108-116, HBpol139-147, HBpol152-161, and HBpol748-757 will be administered to at least HLA-A3 individuals, and/or A24 individuals in the case of HBpol748-757.
  • the peptides HBpol4-13, HBpol505-514, and HBpol758-766 will be administered to at least HLA-A24 individuals.
  • HBV vaccines of the invention may be desirable to combine with vaccines which induce neutralizing antibody responses to HBV, particularly to HBV envelope and/or core antigens, such as recombinant HBV env- and/or nucleocapside-encoded antigens or vaccines prepared from purified plasma preparations obtained from HBV-infected individuals.
  • HBV vaccine preparations have been described, and are based primarily on HBsAg and polypeptide fragments thereof.
  • vaccines which can be formulated with the peptides of the present invention see generally, EP 154,902 and EP 291,586, and U.S. Pat. Nos.
  • the vaccines can be combined and administered concurrently, or as separate preparations.
  • the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia.
  • vaccinia This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the HBV peptides of the invention.
  • the recombinant vaccinia virus 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. Pat. No. 4,722,848, incorporated herein by reference.
  • BCG Bacille Calmette Guerin
  • BCG vectors are described in Stover et al. ( Nature 351:456-460 (1991)) which is incorporated herein by reference.
  • 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.
  • compositions and methods of the claimed invention may be employed for ex vivo therapy.
  • ex vivo therapy is meant that therapeutic or immunogenic manipulations are performed outside the body.
  • 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 peptide in the cell medium far in excess of levels which could be accomplished or tolerated by the patient.
  • the cells are returned to the host to treat the HBV infections;
  • the host's cells may also be exposed to vectors which carry genes encoding the peptides, as described above.
  • the cells Once transfected with the vectors, the cells may be propagated in vitro or returned to the patient.
  • the cells which are propagated in vitro may be returned to the patient after reaching a predetermined cell density.
  • ex vivo CTL responses to a HBV are induced by incubating in tissue culture a patient's CTL precursor cells (CTLP) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an HBV infected cell). To optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is typically maintained in an appropriate serum-free medium.
  • CTL precursor cells typically 1-4 weeks
  • Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp.
  • the appropriate APC are incubated with about 10-100 ⁇ M of peptide in serum-free media for 4 hours under appropriate culture conditions.
  • the peptide-loaded APC are then incubated with the responder cell populations in vitro for 5 to 10 days under optimized culture conditions.
  • Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the HBV polymerase antigen from which the peptide sequence was derived.
  • Specificity and MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides.
  • an amount of antigenic peptide is added to the APC or stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the APCs.
  • Resting or precursor CTLs are then incubated in culture with the appropriate APCs for a time period sufficient to activate the CTLs.
  • the CTLs are activated in an antigen-specific manner.
  • the ratio of resting or precursor CTLs to APCs may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the described treatment modality is used.
  • the CTL:APC ratio is in the range of about 30:1 to 300:1.
  • the CTL/APC may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CTL.
  • Activated CTL may be effectively separated from the APC using one of a variety of known methods.
  • monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the CTL (or a segment thereof) may be utilized to bind their appropriate complementary ligand.
  • Antibody-tagged molecules may then be extracted from the admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods..
  • Effective cytotoxic amounts of the activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 ⁇ 10 6 to about 1 ⁇ 10 12 , more preferably about 1 ⁇ 10 8 to about 1 ⁇ 10 11 , and even more preferably, about 1 ⁇ 10 9 to about 1 ⁇ 10 10 activated CD8+ cells are utilized for adult humans, compared to about 5 ⁇ 10 6 -5 ⁇ 10 7 cells used in mice.
  • Methods of reintroducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg, which are incorporated herein by reference.
  • administration of activated CTLs via intravenous infusion is typically appropriate.
  • the peptides may also find use as diagnostic reagents.
  • 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.
  • the peptides may also be used to predict which individuals will be at substantial risk for developing chronic HBV infection.
  • This Example describes the identification of an HLA-A2 restricted CTL response to two HBV polymerase peptides in a patient with acute viral hepatitis.
  • the epitopes are present in amino acid sequences HBpol 61-69 [Seq ID No. 1] Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val (GLYSSTVPV) (also designated peptide 927.32) and HBpol 803-811 [Seq ID No. 4] Ser-Leu-Ala-Asp-Ser-Pro-Ser-Val (SLYADSPSV) (also designated peptide 927.27).
  • both peptides HBpol 803-811 and HBpol 61-69 stimulated CTL responses in a patient (HLA-A2 + ) using target cells pulsed with peptide, whereas other peptides 927.24 (WILRGTSFR) [Seq ID No. 23] and 927.30 (DLNLNLNV) [Seq ID No. 24] and media controls did not stimulate the specific CTL response.
  • WILRGTSFR 927.24
  • DLNLNLNV 927.30
  • media controls did not stimulate the specific CTL response.
  • the ability of the HBpol 803-811 specific clones to recognize endogenously synthesized polymerase antigen (Vpol and EBO-pol) is shown in FIG. 2.
  • Be.27-1A1 and Be.27-1A5 Two clones, designated Be.27-1A1 and Be.27-1A5, were identified that recognized the HBpol 803-811 peptide. As shown in FIG. 3, CTL responses to HBpol 61-69 and HBpol 803-811 were shown with target cells pulsed with homologous peptide, but only the HBpol 803-811 clone showed a response to endogenously synthesized Vpol antigen.
  • This example demonstrates that acutely infected patients with clinically apparent viral hepatitis develop an HLA class I restricted CTL response to multiple epitopes in the conserved functional domains of the HBV polymerase protein, while persistently infected patients with chronic hepatitis and normal uninfected controls do not.
  • HLA-A2 positive patients with acute hepatitis B nine patients with chronic hepatitis B and ten healthy uninfected subjects were studied (Table II).
  • the diagnosis of acute hepatitis B was based on clinical and biochemical evidence of acute liver injury according to standard diagnostic criteria, together with serological evidence of acute HBV infection, i.e., hepatitis B surface antigen (HBsAg, hepatitis B e antigen (HBeAg) and IgM anti-HBc antibody (IgM HBc-Ab), and the absence of serologic evidence of hepatitis delta or hepatitis C virus infection.
  • HBsAg hepatitis B surface antigen
  • HeAg hepatitis B e antigen
  • IgM anti-HBc antibody IgM anti-HBc antibody
  • HBV polymerase The amino acid sequence of HBV polymerase was screened for 9-mers and 10-mers containing the HLA-A2 allele specific binding motif. This search yielded 220 candidate peptides. Out of this group 44 peptides were selected based on conservation in at least 4 of the 7 HBV adw sequences in the GenBank Database. Lyophilized peptides were reconstituted at 20 mg/ml in DMSO and diluted to 1 mg/ml with RPMI 1640 medium.
  • the binding affinity of the peptides to the class I molecule was determined by competitive binding assays using the radiolabeled peptide FLPSDYFPSV [Seq ID No. 25] representing HBc18-27.
  • the peptide was iodinated to a specific activity of 5-10 ⁇ 10 7 cpm/mol by the chloramine T method of Buus et al., Science 235: 1353 (1987), incorporated herein by reference.
  • Purified class I molecules (10 to 50 nM) were incubated at room temp.
  • peptides with various doses of the peptides, together with 5 to 10 nM of the labeled peptide and 1 ⁇ M human ⁇ 2-microglobulin in PBS, pH 7.0, 0.05% NP-40, 1 mM PMSF, 1.3 mM 1,10-phenanthroline, 73 ⁇ M pepstatin A, 8 mM EDTA, and 200 ⁇ M TLCK.
  • class I-peptide complexes were separated from free peptide by gel filtration on either a TSK2000 (7.8 mm ⁇ 15 cm) column eluted with PBS pH 6.5, 0.5% NP-40, 0.1% NaN 3 , or a Sephadex G-50 column (22 ml bed volume) eluted with the same buffer at pH 7.0.
  • Class I-bound and free radioactivity was measured and the doses of peptides yielding 50% inhibition of the binding of the labeled peptide (IC50) were calculated.
  • purified class I molecules were titered in the presence of a fixed amount of labeled peptide to determine the concentration necessary to bind 10 to 30% of the total radioactivity added. All subsequent inhibition assays were then performed using these class I concentrations. Each peptide was tested in two to four independent experiments.
  • PBMC from patients and normal donors were separated on Ficoll-Histopaque density gradients, washed three times in Hanks Balanced Salt Solution (HBSS), resuspended in RPMI 1640 supplemented with L-glutamine (2 mM), gentamicin (10 ⁇ g/ml), and 10% heat-inactivated human AB serum and plated in a 24-well plate at 4 ⁇ 10 6 cells/well.
  • HBSS Hanks Balanced Salt Solution
  • rHBcAG Biogen, Cambridge, Mass.
  • rHBcAg was either omitted or replaced by 10 ⁇ g/ml tetanus toxoid (Connaught Laboratories, Swiftwater, Pa.) since these individuals had not been previously exposed to HBV and did not benefit from rHBcAg-induced T cell help.
  • 1 ml of RPMI with 10% human AB serum and rIL-2 at 10 U/ml final concentration was added to each well.
  • the cultures were restimulated with peptide, rIL-2 and irradiated (3000 rad) autologous feeder cells and they were tested for cytotoxic activity on day 14. Selected cultures that displayed peptide specific cytolytic activity were separated into CD4+ and CD8+ populations by panning onto anti-CD4 coated flasks (Applied Immunosciences, Santa Clara, Calif.) and restimulated as described above.
  • CTL lines were established as described above and enriched in highly cytotoxic CD8+ CTLs by cloning at 10 and 3 cells per well in 96-well microwell plates in the presence of 0.5 ⁇ g/ml CD3-specific monoclonal antibody (Coulter Immunology, Hialeah, Fla.), rIL-2 (100 U/ml) and 10 5 irradiated (3000 rad) allogeneic PBMC.
  • HBV specific clones were established by cloning at 1 and 0.3 cells per well in the same way. Growing cultures were tested for cytotoxic activity against peptide-primed target cells on day 17 and cytotoxic lines and clones were expanded in a 24-well plate and restimulated every 7 to 10 days as described above.
  • target cells consisted of either 1) allogeneic HLA-matched and mismatched B-LCL (Amer. Soc. Histocompat. Immunogenetics, Boston, Mass.), incubated overnight with synthetic peptides at 10 ⁇ g/ml; 2) stable B-LCL transfectants that express BBsAg or HBpolAg produced by transfection of the EBV-transformed B-LCL with a panel of EBV-based expression vectors that contain the corresponding coding regions of the ayw subtype (Guilhot et al., J. Virol.
  • vaccinia viruses a recombinant vaccinia virus construct that encodes the HBV polymerase protein (Vpol) was produced by insertion of a 2766 fragment representing nucleotides 2290-1874 of the HBV genome (ayw subtype) into the Sma I site of the pSCII vector by standard techniques as described in Chakrabarti et al., Mol. Cell. Biol. 5: 3403 (1985), incorporated herein by reference.
  • Vaccinia-infected targets were prepared by infection of 10 6 cells at 50 PFU/cell on a rocking plate at room temp.
  • Spontaneous release was ⁇ 20% of maximal release in all assays.
  • the assay was considered positive if the specific 51 Cr release from target cells containing antigen was a 15% higher than the nonspecific 51 Cr release from antigen nonspecific 51 Cr release from antigen negative target cells and the nonspecific lysis was less than 15% of maximum.
  • HLA-A2 binding affinity of a peptide did not appear to be the only requirement for immunogenicity since the peptide (LLAQFTSAI) [Seq ID No. 26] with the highest binding affinity (9.600) did not elicit an immune response while one with a 600-times lower affinity (0.016) did.
  • LLAQFTSAI peptide with the highest binding affinity (9.600) did not elicit an immune response while one with a 600-times lower affinity (0.016) did.
  • PBMC were also stimulated with lower concentrations of this peptide (0.3, 1, 3 and 10 ⁇ g/ml) without inducing a CTL response, suggesting that nonresponsiveness to this and other high affinity peptides is probably due to other mechanisms.
  • the CTL response to these epitopes was mediated by CD8+ T cells since only the CD8+ fraction of each cell line recognized target cells that were either pulsed with the corresponding peptide or stably transfected with the polymerase expression vector.
  • each of the three responding cell lines was cloned by limiting dilution in the presence of anti-CD3, irradiated allogeneic PBL and IL-2. All of the derivative cytotoxic lines were highly enriched in CD8+ cells as determined by FACS analysis (0.5-1.0 ⁇ 10 6 cells were washed once in PBS with 5% BSA and 0.02% sodium azide, the pelleted cells were then stained with a fluorescent probe-conjugated anti-CD4 and anti-CD8 monoclonal antibody (Leu3a or Leu2a), and similarly labeled control antibody for 30 min.
  • FACS analysis 0.5-1.0 ⁇ 10 6 cells were washed once in PBS with 5% BSA and 0.02% sodium azide, the pelleted cells were then stained with a fluorescent probe-conjugated anti-CD4 and anti-CD8 monoclonal antibody (Leu3a or Leu2a), and similarly labeled control antibody for 30 min.
  • Target cells pulsed with no peptide or with an irrelevant peptide (Table V), which is an HLA-A2 restricted epitope in HCV-infected patients, were not lysed, now were cells infected by the control recombinant vaccinia virus that expresses the HBV envelope protein, indicating the specificity of the CTL.
  • GLSRYVARL-pulsed JY-EBV by CTL is peptide-dose dependent [Seq ID No.2] JY-EBV pulsed with 10 ⁇ M 1 ⁇ M 0.1 ⁇ M 0.01 ⁇ M Patient Line GLSRYVARL GLSRYVARL GLSRYVARL GLSRYVARL no peptide A-1 67-68 41% 26% 19% 13% 5% A-2 10 75% 56% 52% 25% 12% A-2 30 69% 40% 40% 19% 6%

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Peptides are used to define epitopes that stimulate HLA-restricted cytotoxic T lymphocyte activity against hepatitis B virus antigens. The peptides are derived from regions of HBV polymerase, and are particularly useful in treating or preventing HBV infection, including methods for stimulating the immune response of chronically infected individuals to respond to HBV antigens.

Description

    RELATED APPLICATIONS
  • The present application is a continuation-in-part application of U.S. Ser. No. 08/100,870, filed Aug. 2, 1993, which is a continuation-in-part of U.S. Ser. No. 07/935,898, which is a continuation-in-part of U.S. Ser. No. 07/749,540, the disclosures of which are incorporated herein by reference.[0001]
    • GOVERNMENT SUPPORT
  • [0002] 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. [0003]
  • 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., [0004] 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 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. [0005]
  • 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. Pat. 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. [0006]
  • The contribution of CTLs to immunity to HBV antigens has been difficult to assess. Chisari et al. ([0007] 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 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. [0008]
    • 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 sequence of the HBV polymerase protein. In certain embodiments the CTL inducing peptide will have the sequence of HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No. 2]; HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No. 20]; HBpol748-757 (Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg) [Seq ID No. 21]; HBpol758-766 (Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) [Seq ID No. 22]; HBpol773-782 (Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val) [Seq ID No. 3]; HBpol803-811 (Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; and HBpol816-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5]; 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. [0009]
  • 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. [0010]
  • 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 HBpol 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 envelope or 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. [0011]
  • Vaccine compositions for preventing HBV infection, particularly chronic HBV infection, are also provided. The vaccine compositions comprise an immunogenically effective amount of a HBV polymerase peptide mentioned above which induces a MHC class I restricted cytotoxic T lymphocyte response, such as HLA-A2, -A1, -A3, A-11, and/or A24, and will typically further 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 other HBV antigen, such as envelope (surface) antigen. [0012]
  • 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 polymerase 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. [0013]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the CTL response to two polymerase peptides that contain the HLA-A2 motif in a patient using target cells pulsed with peptide that match only at HLA-A2. [0014]
  • FIG. 2 shows the ability of several polymerase 803-811 peptide specific clones to recognize endogenously synthesized polymerase. [0015]
  • FIG. 3 shows that the CTL response to polymerase peptide 803-811 can recognize cells pulsed with peptide and endogenously synthesized polymerase (Vpol), whereas the CTL response to polymerase peptide 61-69 only recognized cells pulsed with the 61-69 peptide. [0016]
  • FIG. 4 shows the aligned amino acid-sequences of 20 cloned HBV polymerase proteins; [0017] line 158 is a consensus sequence where capital letters represent 100% consensus, lower case letters represent >50% consensus, and “.” is <50% consensus.
  • FIG. 5 shows HBV specific CTL response in patients with acute hepatitis (A-1-A-9), chronic hepatitis (C-1-C-9) and normal subjects (N-1-N-9). PBMC were stimulated with the peptides indicated for 2 weeks and tested in a 4-h [0018] 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 the peptide from the 51Cr-release by JY target cells prepulsed with the peptide. Results shown represent percent specific lysis in a 4 hr 5Cr-release assay at an E:T of 50:1.
  • FIG. 6 shows CD8[0019] + cells recognize endogenously synthesized antigen in target cells sharing the HLA-A2 allele (Patient A-1). Epitope-specific lines were generated by stimulating PBMC with the individual peptide for three weeks with weekly restimulation. On day 15 of CD4+ (positive selection) and CD8+ (negative selection) enriched lines were generated from the original bulk culture by panning. FACS-analysis showed an average enrichment by a factor of 3. Results shown represent percent specific lysis in a 4 hr 51Cr-release assay at an E:T of 30:1. Targets (JY-EBV) were either pulsed with the corresponding peptide overnight stably transfected with the polymerase expression vector.
  • FIG. 7 shows CTL-response to Pol455-463 GLSRYVARL [Seq ID No. 2]. Epitope-specific lines and clones, generated by stimulation with Pol455-463 peptide, were tested at varying E:T ratios against targets cells (JY-EBV), pulsed with the corresponding peptide ([0020]
    Figure US20030171538A1-20030911-P00001
    ) overnight or infected with recombinant vaccinia virus that express the HBV polymerase polypeptide (
    Figure US20030171538A1-20030911-P00002
    ), in a standard 4 hr 51Cr-release assay. Wild-type vaccinia virus (Wt) (X) or JY-EBV peptides without peptide (O) were used as a control.
  • FIG. 8 shows HLA-restriction of epitope Pol455-463. Pol455-463-specific lines from patient A-1 and A-2, generated by stimulation with Pol455-463 peptide, were tested against allogeneic partly HLA-matched EBV-B cells prepulsed overnight with 10 μg/ml of the same peptide. Sharing HLA class I at other loci did not render target cells susceptible to lysis. Cytotoxicity was measured at E:T of 50:1 in a 4 hr [0021] 51Cr-release assay.
  • FIG. 9 shows recognition of truncated, elongated (a) or variant peptides (b) by Pol455-463 specific CTL-lines, generated by weekly stimulation of PBMC from patient A-1 with peptide Pol455-463 for 4 weeks. Cytotoxicity was measured at E:T of 50:1 in a 4 hr [0022] 51Cr-release assay against JY-EBV cells prepulsed with varying amounts of the same peptide overnight.
    • DESCRIPTION OF THE SPECIFIC EMBODIMENTS
  • The present invention provides peptides derived from HBV polymerase proteins for use in compositions and methods for the treatment, prevention and diagnosis of HBV infection. The peptides 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, including 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. [0023]
  • The peptides employed in the invention are derived from the sequence of the HBV polymerase protein (HBpol), particularly CTL epitopes within HBpol4-13, HBpol61-69, HBpol108-116, HBpol139-147, HBpol151-160, HBpol152-161, HBpol 455-463, HBpol505-514, HBpol551-559, HBpol575-583, HBpol655-663, HBpol748-757, HBpol758-766, HBpol773-782, HBpol803-811, or HBpol816-824, where the numbering is according to Galibert et al., supra. [0024]
  • 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 fewer than twenty-five, e.g., eight to seventeen amino acid residues derived from an HBc sequence. It may be desirable to optimize peptides of the invention to a length of eight to twelve amino acid residues, more preferably nine to eleven, 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., [0025] 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), which are incorporated herein by reference. As set forth in more detail below, usually the peptides will have at least a majority of amino acids which are homologous to a corresponding portion of contiguous residues of the HBV pol sequences herein, and contain 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 glycosylation, 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. [0026]
  • 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 meant a CD8[0027] + 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. [0028]
  • The peptides of the invention contain CTL-inducing epitopes derived from various epitopic regions of the HBV polymerase protein. The peptides are from the region of HBpol[0029] 61-69 and include peptides derived from those sequence regions 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 HBpol61-69 sequence, where HBpol61-69 has the following sequence (for HBV subtype ayw):
  • (HBpol61-69) [Seq. ID No. 1]
  • Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val, and
  • The peptide embodiments of this HBpol[0030] 61-69 region and the other polymerase peptide regions described herein 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 HBpol, amino acids added to facilitate linking, other N- and C-terminal modifications, linked to carriers, etc., as further described herein. The peptide HBpol61-69induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2.
  • Other HBpol region peptides containing CTL epitopes of the invention comprises the peptide HBpol 455-463, and peptides derived from HBpol455-463 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 occurring HBpol455-463 sequence, where HBpol 455-463 has the sequence (for HBV subtype ayw): [0031]
  • (HBpol 455-463) [Seq ID No. 2]
  • Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu
  • wherein the selected peptide can be flanked and/or modified at one or both termini as described herein. The peptide HBpol 455-463 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2. [0032]
  • Yet other HBpol region peptides containing CTL epitopes of the invention comprises the peptide HBpol 773-782, and peptides derived from HBpol773-782 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 occurring HBpol773-782 sequence, where HBpol 773-782 has the sequence (for HBV subtype ayw): [0033]
  • (HBpol 773-782) [Seq ID No. 3]
  • Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val
  • wherein the selected peptide can be flanked and/or modified at one or both termini as described herein. The peptide HBpol 773-782 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2. Other HBpol peptide embodiments of the invention are prepared from the region of HBpol803-811. 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 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 HBpol803-811 sequence, where HBpol803-811 has the sequence (for HBV subtype ayw): [0034]
  • (HBpol803-811) [Seq ID No. 4]
  • Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val,
  • wherein the selected peptide can be flanked and/or modified at one or both termini as described herein. The peptide HBpol 803-811 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2. [0035]
  • Other HBpol peptide embodiments of the invention are prepared from the region of HBpol816-824. 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 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 HBpol816-824 sequence, where HBpol816-824 has the sequence (for HBV subtype ayw): [0036]
  • (HBpol816-824) [Seq ID No. 5]
  • Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu,
  • wherein the selected peptide can be flanked and/or modified at one or both termini as described herein. The peptide HBpol 816-824 induces a cytotoxic T lymphocyte response which is mediated by at least the MHC class I molecule HLA-A2. [0037]
  • Other HBpol peptide embodiments of the invention are prepared from the regions of HBpol4-13, HBpol108-116, HBpol139-147, HBpol-151-160, HBpol152-161, HBpol505-514, HBpol551-559, HBpol575-583, HBpol655-663, HBpol748-757or HBpol758-766. A peptide prepared from one of the [0038]
    Figure US20030171538A1-20030911-P00999
    aforementioned regions contains 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 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 HBpol sequence, where the HBpol regions have the sequences (for HBV subtype ayw):
    HBpol4-13 [Seq ID No. 12]
    Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu
    HBpol108-116 [Seq ID No. 13]
    Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arq
    HBpol139-147 [Seq ID No. 14]
    Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg
    HBpol151-160 [Seq ID No. 15]
    His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr
    HBpol152-161 [Seq ID No. 16]
    Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys
    HBpol505-514 [Seq ID No. 17]
    Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe
    HBpol551-559 [Seq ID No. 18]
    Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala
    HBpol575-583 [Seq ID No. 19]
    Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
    HBpol655-663 [Seq ID No. 20]
    Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile
    HBpol748-757 [Seq ID No. 21]
    Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg
    HBpo1758-766 [Seq ID No. 22]
    Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu
  • wherein the selected peptide can be flanked and/or modified at one or both termini as described herein. The peptide HBpol151-160 induces a CTL response which is mediated by at least the MHC class I molecule HLA-A1. The peptides HBpol551-559 and HBpol655-663 induce a CTL response which is mediated by at least the MHC class I molecule HLA-A2. The peptide HBpol575-583 induces a CTL response which is mediated by at least the MHC class I molecule HLA-A2.1. The peptides HBpol108-116, HBpol139-147, HBpol152-161, and HBpol748-757 induce a CTL response which is mediated by at least the MHC class I molecule HLA-A3 (HBpol748-757 appearing to also be restricted by A24). The peptides HBpol4-13, HBpol505-514, and HBpol758-766 induce CTL responses which are mediated by at least the MHC class I molecule HLA-A24. [0039]
  • As mentioned above, additional amino acids can be added to the termini of an oligopeptide or peptide to provide 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-NH[0040] 2 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 or homologs 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 N-terminus of the peptide where the second residue is normally hydrophobic. [0041]
  • The peptides employed in the subject invention need not be identical to peptides HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol 455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No. 2]; HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No. 20]; HBpol748-757 (Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg) [Seq ID No. 21]; HBpol758-766 (Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) [Seq ID No. 22]; HBpol773-782 (Ile-Leu-Arc-Gly-Thr-15 Ser-Phe-Val-Tyr-Val) [Seq ID No. 3]; HBpol803-811(Ser-ou-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; or N 81C-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5], 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. Pat. No. 4,935,235, and the complete sequence of the HBV envelope region is also described in Galibert et al., [0042] Nature 281:646 (1979), each of the foregoing being incorporated herein by reference. 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, the GenBank sequences also being incorporated herein by reference.
  • 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, Tyr. Usually, the portion of the sequence which is intended to substantially mimic an HBV cytotoxic T lymphocyte 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. [0043]
  • 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. [0044]
  • Peptides which tolerate multiple substitutions while retaining the desired biological activity may also be 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 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 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. [0045]
  • In addition to the exemplary peptides described 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 HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol 455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No. 2]; HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No. 20]; HBpol748-757 (Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg) [(Seq ID No. 21]; HBpol758-766 (Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) [Seq ID No. 22]; HBpol773-782 (Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val) [(Seq ID No. 3]; HBpol803-811 (Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; or HBpol816-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5]. 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., [0046] 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.
  • 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. [0047]
  • Carbone et al., [0048] 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., HBpol. 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 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, using the methods described herein, two or more peptides may define different or overlapping cytotoxic T lymphocyte epitopes from a particular region, e.g., the HBpol4-13 (Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu) [Seq ID No. 12]; HBpol61-69 (Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val) [Seq ID No. 1]; HBpol108-116 (Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg) [Seq ID No. 13]; HBpol139-147 (Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg) [Seq ID No. 14]; HBpol151-160 (His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr) [Seq ID No. 15]; HBpol152-161 (Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys) [Seq ID No. 16]; HBpol 455-463 (Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu) [Seq ID No. 2]; HBpol505-514 (Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe) [Seq ID No. 17]; HBpol551-559 (Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala) [Seq ID No. 18]; HBpol575-583 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 19]; HBpol655-663 (Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile) [Seq ID No. 20]; HBpol748-757 (Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg) [Seq ID No. 21]; HBpol758-766 (Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) [Seq ID No. 22]; HBpol773-782 (Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val) [Seq ID No. 3]; HBpol803-811 (Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val) [Seq ID No. 4]; or HBpol816-824 (Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu) [Seq ID No. 5] 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 MHC restriction element different from the first. Other CTL-inducing HBV peptides are oesdcribed in co-pending application U.S. Ser. Nos. 07/935,898 and 08/024,120, which are incorporated herein by reference. 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. Therapeutic 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. [0049]
    TABLE I
    HLA ALLELE FREQUENCIES AMONG
    PREVALENT ETHNIC GROUPS
    HLA Allele EUC NAC AFR JPN
    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
  • 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 forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, e.g., a cocktail representing 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 linkages, noncovalent linkages capable of forming intermolecular and intrastructural bonds are included. [0050]
  • 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, [0051] Immun. Rev. 62:185 (1982), which is incorporated herein by reference. Other bifunctional coupling agents form a thioether rather-than a disulfide linkage. Many of these 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-1-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-[0052] 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. 6]. 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. 7], 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. 8 ] (where Ile116 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 ID No. 9], 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. 10]. See, Ferrari et al., J. Clin. Invest. 88:214-222 (1991), and U.S. Pat. No. 4,882,145, each incorporated herein by reference.
  • The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, [0053] Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc. 105:6442 (1983); Merrifield, Science 232:341-347 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284 (1979), each of which is incorporated herein by reference.
  • Alternatively, recombinant DNA technology may be 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 Sambrook et al., [0054] Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (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 example, whose disclosures are each incorporated herein by reference. 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 polymerase protein of the invention is prepared in which the HBpol amino acid sequence is altered so as to more effectively present epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response. By 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, the phosphotriester method of Matteucci et al., [0055] 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. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the 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; AB 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. [0056]
  • 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 μg to about 2,000 mg of peptide for a 70 kg patient, with dosages of from about 10 μg to about 100 mg of peptide being more commonly used, followed by booster dosages from about 1 μg 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. [0057]
  • 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. [0058]
  • 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. [0059]
  • 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. [0060]
  • 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 μg to 1,000 mg, preferably about 5 μg 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. [0061]
  • 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 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. [0062]
  • 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 vivo against viral antigens, e.g., tripalmitoyl-S-glycerylcysteinly-seryl-serine (P[0063] 3CSS), 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), incorporated herein by reference. 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. [0064]
  • 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, [0065] Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.
  • The peptides of the invention may also be administered via liposomes, which serve to target the peptides 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 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 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/immunogenic peptide compositions. Liposomes for 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 blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., [0066] Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference. For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, the mode of administration, the peptide being delivered, the stage of disease being treated, etc.
  • For solid compositions, conventional nontoxic solid carriers may be used which include, for example, 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%. [0067]
  • 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. [0068]
  • 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 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 P[0069] 3CSS. 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 μg to about 500 mg per 70 kilogram patient, more commonly from about 50 μg 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 HBpol61-69 [Seq ID No. 1], Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 [Seq ID No. 2], Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol551-559 and HBpol655-663; HBpol 773-782 [Seq ID No. 3], Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 [Seq ID No. 4], Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 [Seq ID No. 5], Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu, these will be administered to at least HLA-A2 individuals. For peptides from HBpol151-160, these will be administered to at least HLA-A1 individuals. Vaccines comprising peptides from HBpol575-583 will be administered to at least HLA-A2.1 individuals. Vaccines comprising peptides from HBpol575-583 will be administered to at least HLA-A2.1 individuals. Vaccines comprising peptides from HBpol108-116, HBpol139-147, HBpol152-161, and HBpol748-757 will be administered to at least HLA-A3 individuals, and/or A24 individuals in the case of HBpol748-757. The peptides HBpol4-13, HBpol505-514, and HBpol758-766 will be administered to at least HLA-A24 individuals. [0070]
  • 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 and/or core antigens, such as recombinant HBV env- and/or nucleocapside-encoded antigens or vaccines prepared from purified plasma 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, EP 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, each being incorporated herein by reference. The vaccines can be combined and administered concurrently, or as separate preparations. [0071]
  • 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. Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (bacille Calmette Guerin). BCG vectors are described in Stover et al. ([0072] Nature 351:456-460 (1991)) which is incorporated herein by reference. 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 ex vivo therapy. By ex 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 peptide in the cell medium far in excess of levels which could be accomplished or tolerated by the patient. Following treatment to stimulate the CTLs, the cells are returned to the host to treat the HBV infections; The 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 vitro or returned to the patient. The cells which are propagated in vitro may be returned to the patient after reaching a predetermined cell density. [0073]
  • In one method, ex vivo CTL responses to a HBV are induced by incubating in tissue culture a patient's CTL precursor cells (CTLP) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an HBV infected cell). To optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is typically maintained in an appropriate serum-free medium. Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp. In one embodiment, the appropriate APC are incubated with about 10-100 μM of peptide in serum-free media for 4 hours under appropriate culture conditions. The peptide-loaded APC are then incubated with the responder cell populations in vitro for 5 to 10 days under optimized culture conditions. [0074]
  • Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the HBV polymerase antigen from which the peptide sequence was derived. Specificity and MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides. [0075]
  • The induction of CTL in vitro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC. Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary CTL responses. Since mutant cell lines do not exist for every human MHC allele, it may be advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest. The use of non-transformed, non-infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies. Typically, prior to incubation of the APCs with the CTLp to be activated, an amount of antigenic peptide is added to the APC or stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the APCs. Resting or precursor CTLs are then incubated in culture with the appropriate APCs for a time period sufficient to activate the CTLs. Preferably, the CTLs are activated in an antigen-specific manner. The ratio of resting or precursor CTLs to APCs may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the described treatment modality is used. Preferably, however, the CTL:APC ratio is in the range of about 30:1 to 300:1. The CTL/APC may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CTL. [0076]
  • Activated CTL may be effectively separated from the APC using one of a variety of known methods. For example, monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the CTL (or a segment thereof) may be utilized to bind their appropriate complementary ligand. Antibody-tagged molecules may then be extracted from the admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.. [0077]
  • Effective cytotoxic amounts of the activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1×10[0078] 6 to about 1×1012, more preferably about 1×108 to about 1×1011, and even more preferably, about 1×109 to about 1×1010 activated CD8+ cells are utilized for adult humans, compared to about 5×106-5×107 cells used in mice.
  • Methods of reintroducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg, which are incorporated herein by reference. For example, administration of activated CTLs via intravenous infusion is typically appropriate. [0079]
  • 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. [0080]
  • The following examples are offered by way of illustration, not by way of limitation. [0081]
    • EXAMPLE I HLA-Restricted CTL Response to HBV Polymerase Epitopes
  • This Example describes the identification of an HLA-A2 restricted CTL response to two HBV polymerase peptides in a patient with acute viral hepatitis. The epitopes are present in amino acid sequences HBpol[0082] 61-69 [Seq ID No. 1] Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val (GLYSSTVPV) (also designated peptide 927.32) and HBpol803-811[Seq ID No. 4] Ser-Leu-Ala-Asp-Ser-Pro-Ser-Val (SLYADSPSV) (also designated peptide 927.27).
  • The CTL induced by the HBpol peptides were identified in PBMCs from a patient with acute hepatitis according to the procedure set forth in Example VI of pending application U.S. Ser. No. 07/935,898, except that the PMBCs were stimulated with individual peptides rather than peptide mixtures. The resulting CTL lines and/or clones were then tested for the ability to kill HLA-A2 matched target cells that were either pulsed with the peptide or that expressed the corresponding endogenous polymerase antigen (Vpol or EBO-pol). Construction of the vaccinia based Vpol and Epstein-Barr virus based EBO-pol constructs was as described in Example II of U.S. Ser. No. 07/935,898. [0083]
  • As shown in FIG. 1, both peptides HBpol[0084] 803-811 and HBpol61-69 stimulated CTL responses in a patient (HLA-A2+) using target cells pulsed with peptide, whereas other peptides 927.24 (WILRGTSFR) [Seq ID No. 23] and 927.30 (DLNLNLNV) [Seq ID No. 24] and media controls did not stimulate the specific CTL response. The ability of the HBpol803-811 specific clones to recognize endogenously synthesized polymerase antigen (Vpol and EBO-pol) is shown in FIG. 2. Two clones, designated Be.27-1A1 and Be.27-1A5, were identified that recognized the HBpol803-811 peptide. As shown in FIG. 3, CTL responses to HBpol61-69 and HBpol803-811 were shown with target cells pulsed with homologous peptide, but only the HBpol803-811 clone showed a response to endogenously synthesized Vpol antigen.
    • EXAMPLE II
  • This example demonstrates that acutely infected patients with clinically apparent viral hepatitis develop an HLA class I restricted CTL response to multiple epitopes in the conserved functional domains of the HBV polymerase protein, while persistently infected patients with chronic hepatitis and normal uninfected controls do not. [0085]
  • Nine HLA-A2 positive patients with acute hepatitis B, nine patients with chronic hepatitis B and ten healthy uninfected subjects were studied (Table II). The diagnosis of acute hepatitis B was based on clinical and biochemical evidence of acute liver injury according to standard diagnostic criteria, together with serological evidence of acute HBV infection, i.e., hepatitis B surface antigen (HBsAg, hepatitis B e antigen (HBeAg) and IgM anti-HBc antibody (IgM HBc-Ab), and the absence of serologic evidence of hepatitis delta or hepatitis C virus infection. Six of the nine patients recovered completely with normalization of serum transaminases and clearance of HBsAg and HBeAg within four months of initial diagnosis; the remaining three patients were lost to follow up. All patients with chronic hepatitis B were repeatedly serologic 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 all HBV markers. [0086]
  • The amino acid sequence of HBV polymerase was screened for 9-mers and 10-mers containing the HLA-A2 allele specific binding motif. This search yielded 220 candidate peptides. Out of this [0087] group 44 peptides were selected based on conservation in at least 4 of the 7 HBV adw sequences in the GenBank Database. Lyophilized peptides were reconstituted at 20 mg/ml in DMSO and diluted to 1 mg/ml with RPMI 1640 medium.
    TABLE II
    Characteristics of Subjects Studied
    Subject Sex Diagnosis HLA class I haplotype
    A-1 Male Acute A2, A24, B51, B53, Cw1
    A-2 Male Acute A2, A63, B44, B54, Cw7
    A-3 Male Acute A2, A24, B27, B71/72, Cw1, Cw4
    A-4 Female Acute A2, A31, B51, B6, Cw3
    A-5 Male Acute A2, A30, B44, B35, Cw4, Cw7
    A-6 Female Acute A2, A69, B53, Cw4
    A-7 Female Acute A2, A74, B62, B57, Cw3, Cw6
    A-8 Male Acute A2, A68, B58, B27, Cw1, Cw6
    A-9 Male Acute A2, A30, B35, Cw5
    CH-1 Male Chronic A2, A23, B44
    CH-2 Male Chronic A2, A1, B8, B44, Cw7, Cw4
    CH-3 Male Chronic A2, A68, B59, B44, Cw5, Cw7
    CH-4 Male Chronic A2, B7801, B13, Cw7
    CH-5 Male Chronic A2, A30, B44, B13, Cw6
    CH-6 Male Chronic A2, A34, B8, B27, Cw7
    CH-7 Male Chronic A2, A33, B62, B67, Cw8
    CH-8 Male Chronic A2, A69, B41, B52
    CH-9 Male Chronic A2, A25, B18, Cw6
    N-1 Female Normal A2, A32, B18, B60, Cw3, Cw7
    N-2 Male Normal A2, B44, Cw7
    N-3 Male Normal A2, A1, B8, B18, Cw7
    N-4 Female Normal A2, B44, Cw63
    N-5 Male Normal A2, A23, B5, B58, Cw2, Cw6
    N-6 Male Normal A2, B35, B56, Cw1, Cw3
    N-7 Male Normal A2, A11, B8, B62, Cw4, Cw7
    N-8 Female Normal A2, A3, B7, B60, Cw3, Cw7
    N-9 Male Normal A2, A11, B35, B44, Cw4
    N-10 Male Normal A2, A3, B13, B35, Cw4
  • The binding affinity of the peptides to the class I molecule was determined by competitive binding assays using the radiolabeled peptide FLPSDYFPSV [Seq ID No. 25] representing HBc18-27. The peptide was iodinated to a specific activity of 5-10×10[0088] 7 cpm/mol by the chloramine T method of Buus et al., Science 235: 1353 (1987), incorporated herein by reference. Purified class I molecules (10 to 50 nM) were incubated at room temp. with various doses of the peptides, together with 5 to 10 nM of the labeled peptide and 1 μM human β2-microglobulin in PBS, pH 7.0, 0.05% NP-40, 1 mM PMSF, 1.3 mM 1,10-phenanthroline, 73 μM pepstatin A, 8 mM EDTA, and 200 μM TLCK. After 48 hrs., class I-peptide complexes were separated from free peptide by gel filtration on either a TSK2000 (7.8 mm×15 cm) column eluted with PBS pH 6.5, 0.5% NP-40, 0.1% NaN3, or a Sephadex G-50 column (22 ml bed volume) eluted with the same buffer at pH 7.0. Class I-bound and free radioactivity was measured and the doses of peptides yielding 50% inhibition of the binding of the labeled peptide (IC50) were calculated. Before conducting inhibition assays, purified class I molecules were titered in the presence of a fixed amount of labeled peptide to determine the concentration necessary to bind 10 to 30% of the total radioactivity added. All subsequent inhibition assays were then performed using these class I concentrations. Each peptide was tested in two to four independent experiments.
  • Fifteen of the peptides displayed an HLA-A2.1 binding affinity ratio greater than 0.01 (Table III) a threshold below which most peptides are not immunogenic. In addition two peptides which contain HLA-A2 restricted CTL epitopes were included for comparison, HBc18-27 and HBs335-343. [0089]
    TABLE III
    Characteristics of Peptides Tested
    Frequency in HBV subtypes
    Amino Acid Seq total adw ayw adr ayr Binding Acute HBV patients % 51Cr Release
    Peptide Sequence ID No. (20) (7) (5) (7) (1) affinity tested Responders Mean Range
     Pol527-53 LLAQFTSAI 26 19 6 5 7 1 9.6000 2 0
    *Pol575-583 FLLSLGIHL 19 19 6 5 7 1 0.5200 9 6 38 16-81
    *Pol816-824 SLYADSPSV 5 8 4 4 0 0 0.3500 9 3 30 25-32
     Pol502-510 KLHLYSHPI 27 19 6 5 7 1 0.2900 3 0
     Pol655-665 ALMPLYACI 28 19 6 5 7 1 0.2000 5 2 19 17-21
    *Pol551-559 YMDDVVLGA 18 18 5 5 7 1 0.1600 7 2 21 17-25
     Pol504-512 HLYSHPIIL 29 16 4 4 7 1 0.1300 2 0
    *Pol455-463 GLSRYVARL 2 11 7 4 0 0 0.1200 9 6 57 20-95
     Pol526-535 FLLAQFTSAI 30 19 6 5 7 1 0.0710 2 0
     Pol149-158 YLHTLWKAGI 31 20 7 5 7 1 0.0560 3 0
     Pol772-780 WILRGTSFV 32 16 6 5 4 1 0.0180 4 0
    *Pol773-782 ILRGTSFVYV 3 16 6 5 4 1 0.0160 7 3 18 16-21
     Pol765-774 LLGCAANWIL 33 16 6 5 4 1 0.0140 2 0
     Pol424-432 NLSWLSLDV 34 18 5 5 7 1 0.0130 3 0
    *Core18-27 FLPSDFPPSV 35 9 5 4 0 0 1.5000 9 4 64 54-78
     Env335-343 WLSLLVPFV 36 20 7 5 7 0.7200 9 6 66 21-88
  • To stimulate PBMC with the selected synthetic peptides and rHBcAg, PBMC from patients and normal donors were separated on Ficoll-Histopaque density gradients, washed three times in Hanks Balanced Salt Solution (HBSS), resuspended in RPMI 1640 supplemented with L-glutamine (2 mM), gentamicin (10 μg/ml), and 10% heat-inactivated human AB serum and plated in a 24-well plate at 4×10[0090] 6 cells/well. rHBcAG (Biogen, Cambridge, Mass.) was added to the cell cultures at 1 μg/ml and the synthetic peptides at 10 μg/ml. In some of the studies with healthy uninfected blood donors rHBcAg was either omitted or replaced by 10 μg/ml tetanus toxoid (Connaught Laboratories, Swiftwater, Pa.) since these individuals had not been previously exposed to HBV and did not benefit from rHBcAg-induced T cell help. On days 3 and 10, 1 ml of RPMI with 10% human AB serum and rIL-2 at 10 U/ml final concentration was added to each well. On day 7, the cultures were restimulated with peptide, rIL-2 and irradiated (3000 rad) autologous feeder cells and they were tested for cytotoxic activity on day 14. Selected cultures that displayed peptide specific cytolytic activity were separated into CD4+ and CD8+ populations by panning onto anti-CD4 coated flasks (Applied Immunosciences, Santa Clara, Calif.) and restimulated as described above.
  • CTL lines were established as described above and enriched in highly cytotoxic CD8+ CTLs by cloning at 10 and 3 cells per well in 96-well microwell plates in the presence of 0.5 μg/ml CD3-specific monoclonal antibody (Coulter Immunology, Hialeah, Fla.), rIL-2 (100 U/ml) and 10[0091] 5 irradiated (3000 rad) allogeneic PBMC. HBV specific clones were established by cloning at 1 and 0.3 cells per well in the same way. Growing cultures were tested for cytotoxic activity against peptide-primed target cells on day 17 and cytotoxic lines and clones were expanded in a 24-well plate and restimulated every 7 to 10 days as described above.
  • For the cytotoxicity assays, target cells consisted of either 1) allogeneic HLA-matched and mismatched B-LCL (Amer. Soc. Histocompat. Immunogenetics, Boston, Mass.), incubated overnight with synthetic peptides at 10 μg/ml; 2) stable B-LCL transfectants that express BBsAg or HBpolAg produced by transfection of the EBV-transformed B-LCL with a panel of EBV-based expression vectors that contain the corresponding coding regions of the ayw subtype (Guilhot et al., [0092] J. Virol. 66: 2670 (1992), incorporated herein by reference); or 3) B-LCL infected with recombinant vaccinia viruses (a recombinant vaccinia virus construct that encodes the HBV polymerase protein (Vpol) was produced by insertion of a 2766 fragment representing nucleotides 2290-1874 of the HBV genome (ayw subtype) into the Sma I site of the pSCII vector by standard techniques as described in Chakrabarti et al., Mol. Cell. Biol. 5: 3403 (1985), incorporated herein by reference. Vaccinia-infected targets were prepared by infection of 106 cells at 50 PFU/cell on a rocking plate at room temp. for 1 h followed by a single wash and overnight incubation at 37° C. Target cells were then labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 h and washed four times with HBSS. Cytolytic activity was determined in a standard 4-h 51Cr release assay using U-bottomed 96-well plates containing 5,000 targets/well. Stimulated PBMC from patients and normal controls were performed in duplicate. Percent cytotoxicity was determined from the formula 100×((experimental release−spontaneous release)/(maximum release−spontaneous release)). Maximum release was determined by lysis of targets by detergent (1% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release was <20% of maximal release in all assays. The assay was considered positive if the specific 51Cr release from target cells containing antigen was a 15% higher than the nonspecific 51Cr release from antigen nonspecific 51Cr release from antigen negative target cells and the nonspecific lysis was less than 15% of maximum.
  • As shown in Table III, eight of the nine acutely infected patients responded to at least one of the polymerase peptides and, as can be seen from Table III, six of the peptides were recognized by at least one patient, suggesting that they represented HLA-A2 restricted epitopes. The HLA binding ratio of 5 of 6 of these peptides was greater than 0.1, supporting a direct relationship between binding affinity and immunogenicity even among this group of high affinity peptides. [0093]
  • The HLA-A2 binding affinity of a peptide did not appear to be the only requirement for immunogenicity since the peptide (LLAQFTSAI) [Seq ID No. 26] with the highest binding affinity (9.600) did not elicit an immune response while one with a 600-times lower affinity (0.016) did. To exclude the possibility that this extremely high affinity peptide may have triggered potentially responsive CTL precursors to undergo apoptosis, PBMC were also stimulated with lower concentrations of this peptide (0.3, 1, 3 and 10 μg/ml) without inducing a CTL response, suggesting that nonresponsiveness to this and other high affinity peptides is probably due to other mechanisms. [0094]
  • The CTL responses of nine acutely infected patients who responded to one or more polymerase peptides are summarized in FIG. 5. Five of these patients also recognized the two control peptides, HBc18-27 and HBenv335-343, while one patient recognized only HBenv335-343, and one patient responded to neither. These results demonstrate the clonality and multispecificity of the CTL response against the polymerase protein during acute viral hepatitis. Importantly, nine of the 10 uninfected controls responded to any of the peptides used in this example (nine of these controls are shown in FIG. 5), suggesting that the CTL responses observed in the acutely infected patients represented in vitro secondary responses that were primed by exposure to infected cells in vivo. None of the nine patients with chronic hepatitis produced a response, suggesting that the vigor of the HBV specific CTL response has a role in determining which patients will clear the virus and which patients will not. [0095]
  • Having identified two HLA-A2 patients (A-1 and A-2) with acute hepatitis who responded strongly to HBpol575-583 and HBpol455-463 and HBpol816-824 (Table III), these patients and peptides were chosen for further analysis. After two weeks of in vitro stimulation, selected cultures that displayed peptide specific CTL responses were enriched for CD4+ and CD8+ subsets by panning using positive and negative selection, respectively, and they were restimulated with peptide and tested for recognition of endogenously processed polymerase antigen after one additional week of culture. As shown in FIG. 6, the CTL response to these epitopes was mediated by CD8+ T cells since only the CD8+ fraction of each cell line recognized target cells that were either pulsed with the corresponding peptide or stably transfected with the polymerase expression vector. These results suggest that the peptides represent the native epitopes that are produced by the cellular processing of the polymerase protein, and that they are presented in the context of class I HLA molecules. [0096]
  • To obtain pure CD8+ cell lines and to characterize the T cell response at the clonal level, each of the three responding cell lines was cloned by limiting dilution in the presence of anti-CD3, irradiated allogeneic PBL and IL-2. All of the derivative cytotoxic lines were highly enriched in CD8+ cells as determined by FACS analysis (0.5-1.0×10[0097] 6 cells were washed once in PBS with 5% BSA and 0.02% sodium azide, the pelleted cells were then stained with a fluorescent probe-conjugated anti-CD4 and anti-CD8 monoclonal antibody (Leu3a or Leu2a), and similarly labeled control antibody for 30 min. at 4° C., and after 3 washes in PBS with 5% BSA and 0.02% sodium azide, cells were analyzed with a FACScan flow cytometer). Furthermore, 5 of the 6 HBpol455-463 specific CTL clones derived in this manner also consisted of CD8+ cells.
  • Four highly cytotoxic long term CTL lines and two clones specific for HBpol455-463 pulsed targets were chosen for further analysis (FIG. 7). The strength of the cytotoxic activity was assessed by varying the amount of the peptide used to pulse the target cells and by varying the effector to target ratios. The CTL displayed peptide dose dependent cytotoxic activity that recognized targets pulsed with peptide concentrations as low as 10 nM (Table IV), and they efficiently lysed both peptide pulsed and vaccinia-pol infected targets at E:T ratios as low as 1.6:1 (FIG. 7). Target cells pulsed with no peptide or with an irrelevant peptide (Table V), which is an HLA-A2 restricted epitope in HCV-infected patients, were not lysed, now were cells infected by the control recombinant vaccinia virus that expresses the HBV envelope protein, indicating the specificity of the CTL. [0098]
    TABLE IV
    Recognition GLSRYVARL-pulsed JY-EBV by CTL
    is peptide-dose dependent [Seq ID No.2]
    JY-EBV pulsed with
    10 μM 1 μM 0.1 μM 0.01 μM
    Patient Line GLSRYVARL GLSRYVARL GLSRYVARL GLSRYVARL no peptide
    A-1 67-68 41% 26% 19% 13%  5%
    A-2
    10 75% 56% 52% 25% 12%
    A-2
    30 69% 40% 40% 19%  6%
  • [0099]
    TABLE V
    Induction of CTL with variant peptides to GLSRYVARL [Seq ID No.2]
    and an HCV epitope KLVALGINAV [Seq ID No.37]
    % Specific Cytotoxicity Against JY-EBV Preincubated with
    Peptide During GLSRYVARL GLPRYVARL SGLSRYVARL GLSRYVARLS KLVALGINAV
    CTL-Induction Seq ID 2 Seq ID 38 Seq ID 39 Seq ID 40 Seq ID 37
    GLSRYVARL 54% 18% 40% 41% 2%
    GLPRYVARL
     1%  0%  1%  0%
    SGLSRYVARL
     0%  0%  0%  0%
    GLSRYVARLS
     1%  1%  0%  0%
  • To identify the restriction element used by the HBpol455-463 specific CTL, cytotoxic lines and clones from patients A-1 and A-2 were tested against allogeneic EBV-B cell lines sharing individual HLA class I alleles with the effector cells. As shown in FIG. 8, not only was HLA-A2 the sole class I allele shared by these two patients, but their CTL only lyse peptide pulsed target cells that share this allele. Thus, HBpol455-463 specific CTLs from both patients are HLA-A2 restricted. [0100]
  • Peptides containing carboxy- and amino-terminal truncations and elongations of the HBpol455-463 sequence were synthesized to determine the optimal length and the precise termini of the epitope. As shown in FIG. 9A, truncation of Gly455 or Leu463 greatly reduced the HLA binding affinity of the peptides and totally abrogated their recognition by CTL induced by the original peptide HBpol455-463. Elongation of this peptide by adding a single Ser residue normally present upstream of the amino terminus or downstream of the carboxy-terminus of HBpol455-463 did not diminish its recognition by CTL (FIG. 9A), and may have even increased recognition, despite the fact that the HLA-A2 binding affinity of the extended peptides was reduced 4-10 fold relative to the original peptide (FIG. 9A). The Ser-extended peptides did not induce CTL, as shown in Table V. [0101]
  • Direct sequencing of the PCR products amplified from the serum of 5 of the 9 patients with acute hepatitis B by nested PCR demonstrated that the deduced HBV amino acid sequence was identical to GLSRYVARL [Seq ID No. 2] in these patients. The sequence is present in 7/7 and 4/5 adw and ayw subtype sequences in GenBank. The amino acid sequence of the remaining ayw isolate in the database is GVSRYVARL [Seq ID No. 41], while the sequence of 6/7 adr and 1/1 ayr isolates is GLPRYVARL [Seq ID No. 42] and the sequence of the remaining adr isolate is GLPRYVVCL [Seq ID No. 43]. [0102]
  • Peptides containing sequences of these different viral subtypes were tested for recognition by GLSRYVARL-stimulated [Seq ID No. 2] PBMC to assess cross-reactivity of the CTL response. None of the variants was efficiently recognized by the CTL. GLSRYVVCL [Seq ID No. 44] was not recognized, even at very high peptide concentration, despite the fact that its HLA-A2.1 binding affinity was greater than the prototype peptide GLSRYVARL [Seq ID No. 2]. Thus, Ser457, Ala461 and Arg462 may represent T cell receptor contact sites (epitope residues) in this peptide. A substitution in Ser457 in GLPRYVARL [Seq ID No. 42] variant yielded more than a 10-fold reduction in its recognition by the CTL, while decreasing the HLA binding affinity 2-fold. [0103]
  • The GVSRYVARL [Seq ID No. 41] variant which contained the substitution at Leu456, a presumptive HLA contact site (agretope residue), was poorly recognized by the CTL, commensurate with the 9-fold reduction in its HLA-A2 binding affinity. However, the amino- and carboxy-terminally extended peptides described above were well recognized by the CTL despite the fact that they displayed comparably reduced HLA-A2 binding affinities (FIG. 9A). This suggests that Leu456 not only serves as an agretope residue, but may also influence the T cell receptor binding affinity of the peptide. [0104]
  • All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. [0105]
  • From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various 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. [0106]
  • 1 66 9 amino acids amino acid <Unknown> linear peptide 1 Gly Leu Tyr Ser Ser Thr Val Pro Val 1 5 9 amino acids amino acid <Unknown> linear peptide 2 Gly Leu Ser Arg Tyr Val Ala Arg Leu 1 5 10 amino acids amino acid <Unknown> linear peptide 3 Ile Leu Arg Gly Thr Ser Phe Val Tyr Val 1 5 10 9 amino acids amino acid <Unknown> linear peptide 4 Ser Leu Tyr Ala Asp Ser Pro Ser Val 1 5 9 amino acids amino acid <Unknown> linear peptide 5 Phe Leu Leu Ser Leu Gly Ile His Leu 1 5 20 amino acids amino acid <Unknown> linear peptide 6 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Le 1 5 10 15 Ser Phe Leu Pro 20 20 amino acids amino acid <Unknown> linear peptide 7 Pro His His Tyr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Le 1 5 10 15 Met Tyr Leu Ala 20 20 amino acids amino acid <Unknown> linear peptide 8 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Va 1 5 10 15 Ile Glu Tyr Leu 20 15 amino acids amino acid <Unknown> linear peptide 9 Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 1 5 10 15 20 amino acids amino acid <Unknown> linear peptide 10 Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pr 1 5 10 15 Asn Ala Pro Ile 20 845 amino acids amino acid <Unknown> linear protein Modified-site 1..845 /product= “OTHER” /note= “Xaa = any amino acid (<50% consensus)” 11 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Gly Thr Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala As 20 25 30 Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Le 35 40 45 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Le 50 55 60 Tyr Ser Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Se 65 70 75 80 Phe Pro Xaa Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gl 85 90 95 Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Il 100 105 110 Met Pro Ala Arg Phe Tyr Pro Asn Xaa Thr Lys Tyr Leu Pro Leu As 115 120 125 Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Xaa Val Asn His Tyr Ph 130 135 140 Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Ty 145 150 155 160 Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Se 165 170 175 Trp Glu Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Th 180 185 190 Arg His Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Se 195 200 205 Arg Ser Pro Val Gly Pro Cys Xaa Arg Ser Gln Leu Xaa Gln Ser Ar 210 215 220 Leu Gly Leu Gln Pro Gln Gln Gly Xaa Leu Ala Arg Arg Gln Gln Gl 225 230 235 240 Arg Ser Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Xa 245 250 255 Phe Gly Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Xaa Ala Se 260 265 270 Ser Ser Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala Ala Ty 275 280 285 Ser His Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Va 290 295 300 Glu Leu His Asn Xaa Pro Pro Asn Ser Ala Arg Ser Gln Ser Glu Gl 305 310 315 320 Pro Val Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cy 325 330 335 Ser Asp Tyr Cys Leu Xaa His Ile Val Asn Leu Leu Glu Asp Trp Gl 340 345 350 Pro Cys Thr Glu His Gly Glu His Xaa Ile Arg Ile Pro Arg Thr Pr 355 360 365 Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His As 370 375 380 Thr Thr Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gl 385 390 395 400 Xaa Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Se 405 410 415 Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Va 420 425 430 Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Hi 435 440 445 Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Se 450 455 460 Ser Asn Ser Arg Ile Ile Asn Xaa Gln His Gly Thr Met Gln Asn Le 465 470 475 480 His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Ty 485 490 495 Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Le 500 505 510 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Le 515 520 525 Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pr 530 535 540 His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Ly 545 550 555 560 Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Thr Asn Phe Le 565 570 575 Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gl 580 585 590 Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Le 595 600 605 Pro Gln Glu His Ile Val Gln Lys Ile Lys Gln Cys Phe Arg Lys Le 610 615 620 Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gl 625 630 635 640 Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Le 645 650 655 Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Se 660 665 670 Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pr 675 680 685 Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Th 690 695 700 Pro Thr Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Th 705 710 715 720 Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cy 725 730 735 Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Se 740 745 750 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cy 755 760 765 Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Se 770 775 780 Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ty 785 790 795 800 Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Se 805 810 815 Leu Tyr Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Va 820 825 830 His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 10 amino acids amino acid <Unknown> linear peptide 12 Ser Tyr Gln His Phe Arg Lys Leu Leu Leu 1 5 10 9 amino acids amino acid single linear peptide 13 Arg Leu Lys Leu Ile Met Pro Ala Arg 1 5 9 amino acids amino acid single linear peptide 14 Val Val Asn His Tyr Phe Gln Thr Arg 1 5 10 amino acids amino acid single linear peptide 15 His Thr Leu Trp Lys Ala Gly Ile Leu Tyr 1 5 10 10 amino acids amino acid single linear peptide 16 Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 1 5 10 10 amino acids amino acid single linear peptide 17 Leu Tyr Ser His Pro Ile Ile Leu Gly Phe 1 5 10 9 amino acids amino acid single linear peptide 18 Tyr Met Asp Asp Val Val Leu Gly Ala 1 5 9 amino acids amino acid single linear peptide 19 Phe Leu Leu Ser Leu Gly Ile His Leu 1 5 9 amino acids amino acid single linear peptide 20 Ala Leu Met Pro Leu Tyr Ala Cys Ile 1 5 10 amino acids amino acid single linear peptide 21 Gly Thr Asp Asn Ser Val Val Leu Ser Arg 1 5 10 9 amino acids amino acid single linear peptide 22 Lys Tyr Thr Ser Phe Pro Trp Leu Leu 1 5 9 amino acids amino acid single linear peptide 23 Trp Ile Leu Arg Gly Thr Ser Phe Arg 1 5 9 amino acids amino acid single linear peptide 24 Asp Leu Asn Leu Gly Asn Leu Asn Val 1 5 10 amino acids amino acid single linear peptide 25 Phe Leu Pro Ser Asp Tyr Phe Pro Ser Val 1 5 10 9 amino acids amino acid single linear peptide 26 Leu Leu Ala Gln Phe Thr Ser Ala Ile 1 5 9 amino acids amino acid single linear peptide 27 Lys Leu His Leu Tyr Ser His Pro Ile 1 5 9 amino acids amino acid single linear peptide 28 Ala Leu Met Pro Leu Tyr Ala Cys Ile 1 5 9 amino acids amino acid single linear peptide 29 His Leu Tyr Ser His Pro Ile Ile Leu 1 5 10 amino acids amino acid single linear peptide 30 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile 1 5 10 10 amino acids amino acid single linear peptide 31 Tyr Leu His Thr Leu Trp Lys Ala Gly Ile 1 5 10 9 amino acids amino acid single linear peptide 32 Trp Ile Leu Arg Gly Thr Ser Phe Val 1 5 10 amino acids amino acid single linear peptide 33 Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu 1 5 10 9 amino acids amino acid single linear peptide 34 Asn Leu Ser Trp Leu Ser Leu Asp Val 1 5 10 amino acids amino acid single linear peptide 35 Phe Leu Pro Ser Asp Phe Pro Pro Ser Val 1 5 10 9 amino acids amino acid single linear peptide 36 Trp Leu Ser Leu Leu Val Pro Phe Val 1 5 10 amino acids amino acid single linear peptide 37 Lys Leu Val Ala Leu Gly Ile Asn Ala Val 1 5 10 9 amino acids amino acid single linear peptide 38 Gly Leu Pro Arg Tyr Val Ala Arg Leu 1 5 10 amino acids amino acid single linear peptide 39 Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu 1 5 10 10 amino acids amino acid single linear peptide 40 Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser 1 5 10 9 amino acids amino acid single linear peptide 41 Gly Val Ser Arg Tyr Val Ala Arg Leu 1 5 9 amino acids amino acid single linear peptide 42 Gly Leu Pro Arg Tyr Val Ala Arg Leu 1 5 9 amino acids amino acid single linear peptide 43 Gly Leu Pro Arg Tyr Val Val Cys Leu 1 5 9 amino acids amino acid single linear peptide 44 Gly Leu Ser Arg Tyr Val Val Cys Leu 1 5 843 amino acids amino acid <Unknown> linear protein 45 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Met Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Ser Ala Ser Ser Th 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Ph 290 295 300 His Asn Ile Pro Pro Ser Ser Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn His Gln His Gly Ala Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Leu Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Ser Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Leu Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 amino acids amino acid <Unknown> linear protein Modified-site 16..18 /product= “OTHER” /note= “Xaa = unknown” 46 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Xa 1 5 10 15 Xaa Xaa Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala As 20 25 30 Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Le 35 40 45 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Le 50 55 60 Tyr Ser Ser Thr Val Pro Ile Phe Asn Pro Glu Trp Gln Thr Pro Se 65 70 75 80 Phe Pro His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gl 85 90 95 Tyr Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Il 100 105 110 Met Pro Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu As 115 120 125 Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Ph 130 135 140 Lys Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Ty 145 150 155 160 Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Se 165 170 175 Trp Glu Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Th 180 185 190 Arg His Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Se 195 200 205 Arg Ser Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Ar 210 215 220 Leu Gly Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gl 225 230 235 240 Arg Ser Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Se 245 250 255 Phe Gly Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Ser Ala Se 260 265 270 Ser Thr Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Ty 275 280 285 Ser His Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Va 290 295 300 Glu Leu His Asn Ile Pro Pro Ser Ser Ala Arg Pro Gln Ser Glu Gl 305 310 315 320 Pro Ile Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cy 325 330 335 Ser Asp Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gl 340 345 350 Pro Cys Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pr 355 360 365 Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His As 370 375 380 Thr Thr Glu Ser Thr Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gl 385 390 395 400 Ser Thr His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Se 405 410 415 Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Va 420 425 430 Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Hi 435 440 445 Leu Leu Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Val Cys Leu Se 450 455 460 Ser Thr Ser Lys Asn Ile Asn Tyr Gln His Gly Thr Met Gln Asp Le 465 470 475 480 His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Phe Leu Leu Ty 485 490 495 Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Le 500 505 510 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Le 515 520 525 Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pr 530 535 540 His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Ly 545 550 555 560 Ser Val Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Le 565 570 575 Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gl 580 585 590 Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Cys Trp Gly Thr Le 595 600 605 Pro Gln Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Le 610 615 620 Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gl 625 630 635 640 Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Le 645 650 655 Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Se 660 665 670 Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pr 675 680 685 Val Ala Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Th 690 695 700 Pro Thr Gly Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Th 705 710 715 720 Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cy 725 730 735 Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Se 740 745 750 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cy 755 760 765 Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Se 770 775 780 Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ty 785 790 795 800 Arg Pro Leu Leu His Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Se 805 810 815 Leu Tyr Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Va 820 825 830 His Phe Pro Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 843 amino acids amino acid <Unknown> linear protein 47 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Leu Asn Pro Glu Ser Gln Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Arg His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Asn Gln Gly Arg Se 225 230 235 240 Gly Arg Leu Arg Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Ser Ala Ser Ser Al 260 265 270 Ser Ser Cys Phe His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His Asn Ile Pro Pro Ser Ser Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn Tyr Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Leu Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Gln Gln Tyr Leu His Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Thr Phe Va 705 710 715 720 Val Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Asp Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Ser Leu Pro Phe Gln Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 843 amino acids amino acid <Unknown> linear protein 48 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Ile Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Trp Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Ser Ala Ser Ser Al 260 265 270 Ser Ser Cys Leu Tyr Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His Asn Ile Pro Pro Ser Cys Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Ser Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Glu Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn Tyr Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro His Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu His Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly Gln Ser Gly Met Arg Gly Thr Phe Va 705 710 715 720 Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu His Leu Pro Phe Arg Pro Thr Thr Gly Arg Ala Ser Leu Ty 805 810 815 Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Val Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 730 amino acids amino acid <Unknown> linear protein 49 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Thr Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ala Gly Ser Gly Arg Ile Asp Asn Arg Ala Ser Ser Th 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His Asn Ile Pro Pro Ser Ser Ala Arg Pro Gln Ser Glu Gly Pro Il 305 310 315 320 Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn Tyr Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Gly Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Cys Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Leu Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Ser Arg Met Arg Gly Pro Leu Tr 705 710 715 720 Leu Leu Cys Arg Ser Ile Leu Arg Asn Ser 725 730 842 amino acids amino acid <Unknown> linear protein 50 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu As 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Lys Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Val Pro Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Arg Ala Ser Ser Th 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His His Ile Ser Pro Ser Pro Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Phe Ser Ser Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Cys As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn His Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Gly Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu His Leu Tyr Pro Val Al 675 680 685 Arg Arg Thr Ala Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gl 690 695 700 Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Thr Phe Val Al 705 710 715 720 Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Ar 725 730 735 Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Val Le 740 745 750 Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala As 755 760 765 Trp Ile Leu Arg Gly Thr Tyr Phe Val Tyr Val Pro Ser Ala Leu As 770 775 780 Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ile Arg Pro Le 785 790 795 800 Leu His Leu Arg Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr Al 805 810 815 Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Phe Al 820 825 830 Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 842 amino acids amino acid <Unknown> linear protein 51 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu As 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Lys Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Val Pro Pro Thr Thr Arg Arg Ser Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Arg Ala Ser Ser Th 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His His Ile Ser Pro Ser Pro Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Phe Ser Ser Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn His Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Gly Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu His Leu Tyr Pro Val Al 675 680 685 Arg Arg Thr Ala Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gl 690 695 700 Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Thr Phe Val Al 705 710 715 720 Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Ar 725 730 735 Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Val Le 740 745 750 Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala As 755 760 765 Trp Ile Leu Arg Gly Thr Tyr Phe Val Tyr Val Pro Ser Ala Leu As 770 775 780 Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ile Arg Pro Le 785 790 795 800 Leu His Leu Arg Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr Al 805 810 815 Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Phe Al 820 825 830 Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 481 amino acids amino acid <Unknown> linear protein 52 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Gly Thr Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala As 20 25 30 Ala Asp Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Le 35 40 45 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Le 50 55 60 Tyr Ser Ser Thr Val Pro Ile Phe Asn Pro Glu Trp Gln Thr Pro Se 65 70 75 80 Phe Pro Lys Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gl 85 90 95 Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Il 100 105 110 Met Pro Ala Arg Phe Tyr Pro Thr His Thr Lys Tyr Leu Pro Leu As 115 120 125 Lys Gly Ile Lys Pro Tyr Tyr Pro Asp Gln Val Val Asn His Tyr Ph 130 135 140 Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Ty 145 150 155 160 Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Se 165 170 175 Trp Glu Gln Glu Leu Gln His Ser Gln Arg His Gly Asp Glu Ser Ph 180 185 190 Cys Ser Gln Ser Ser Gly Ile Pro Ser Arg Ser Ser Val Gly Pro Cy 195 200 205 Ile Arg Ser Gln Leu Asn Lys Ser Arg Leu Gly Leu Gln Pro His Gl 210 215 220 Gly Pro Leu Ala Ser Ser Gln Pro Gly Arg Ser Gly Ser Ile Arg Al 225 230 235 240 Arg Ala His Pro Ser Thr Arg Arg Tyr Phe Gly Val Glu Pro Ser Gl 245 250 255 Ser Gly His Ile Asp His Ser Val Asn Asn Ser Ser Ser Cys Leu Hi 260 265 270 Gln Ser Ala Val Arg Lys Ala Ala Tyr Ser His Leu Ser Thr Ser Ly 275 280 285 Arg Gln Ser Ser Ser Gly His Ala Val Glu Phe His Cys Leu Ala Pr 290 295 300 Ser Ser Ala Gly Ser Gln Ser Gln Gly Ser Val Ser Ser Cys Trp Tr 305 310 315 320 Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Ser Hi 325 330 335 Leu Val Asn Leu Arg Glu Asp Trp Gly Pro Cys Asp Asp His Gly Gl 340 345 350 His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Va 355 360 365 Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Va 370 375 380 Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pr 385 390 395 400 Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Se 405 410 415 Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Il 420 425 430 Pro Leu His Pro Ala Ala Met Pro His Leu Leu Ile Gly Ser Ser Gl 435 440 445 Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg Ile Asn As 450 455 460 Asn Gln Tyr Gly Thr Met Gln Asn Leu His Asp Ser Cys Ser Arg Gl 465 470 475 480 Leu 843 amino acids amino acid <Unknown> linear protein 53 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn His Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Pro Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 Asp Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Asn Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Val Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Ser Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Lys Arg Hi 180 185 190 Gly Asp Lys Ser Phe Cys Pro Gln Ser Pro Gly Ile Leu Pro Arg Se 195 200 205 Ser Val Gly Pro Cys Ile Gln Ser Gln Leu Arg Lys Ser Arg Leu Gl 210 215 220 Pro Gln Pro Thr Gln Gly Gln Leu Ala Gly Arg Pro Gln Gly Gly Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Ile His Pro Ser Pro Trp Gly Thr Val Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Thr His Ile Cys Ala Ser Ser Se 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Thr Ala Ala Tyr Ser Pr 275 280 285 Ile Ser Thr Ser Lys Gly His Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His His Phe Pro Pro Asn Ser Ser Arg Ser Gln Ser Gln Gly Ser Va 305 310 315 320 Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Gl 325 330 335 Tyr Cys Leu Ser His Ile Val Asn Leu Ile Glu Asp Trp Gly Pro Cy 340 345 350 Ala Glu His Gly Glu His Arg Ile Arg Thr Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Th 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser As 450 455 460 Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asp Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Met Leu Leu Tyr Lys Th 485 490 495 Tyr Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Tyr Ala Ala Val Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Trp Pro Gl 595 600 605 Asp His Ile Val Gln Asn Phe Lys Leu Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Met Thr Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ser Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Asn Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Arg Leu Pro Tyr Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 843 amino acids amino acid <Unknown> linear protein 54 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Gl 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Cys Phe Asn Pro Lys Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 Asp Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Asn Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Val Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Ser Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Lys Arg Hi 180 185 190 Gly Asp Lys Ser Phe Cys Pro Gln Ser Ser Gly Ile Leu Pro Arg Se 195 200 205 Ser Val Gly Pro Cys Ile Gln Ser Gln Leu Arg Lys Ser Arg Leu Gl 210 215 220 Pro Gln Pro Glu Gln Gly Gln Leu Ala Gly Arg Gln Gln Gly Gly Se 225 230 235 240 Gly Ser Ile Arg Ala Arg Val His Pro Ser Pro Trp Gly Thr Val Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly Pro Thr His Asn Cys Ala Ser Ser Se 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala Ala Tyr Ser Le 275 280 285 Ile Pro Thr Ser Lys Gly His Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His His Phe Pro Pro Asn Ser Ser Arg Ser Arg Ser Gln Gly Pro Va 305 310 315 320 Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Glu Pro Cys Ser Gl 325 330 335 Tyr Cys Leu Cys His Ile Val Asn Leu Ile Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Arg Ile Arg Thr Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Th 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser As 450 455 460 Ser Arg Ile Ile Asn Asn Gln His Arg Thr Met Gln Asn Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Met Leu Leu Tyr Lys Th 485 490 495 Tyr Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Met Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Tyr Ala Ala Val Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro His Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Gln Lys Ile Lys Met Trp Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Thr Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ser Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Arg Leu Leu Tyr Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 843 amino acids amino acid <Unknown> linear protein 55 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Ser Phe Asn Pro Gln Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 Asp Ile His Leu Gln Glu Asp Ile Ile Asn Lys Cys Lys Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Val Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Leu Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Lys Ser Phe Arg Pro Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Ile Gln Ser Gln Leu Arg Gln Ser Arg Leu Gl 210 215 220 Pro Gln Pro Thr Gln Gly Gln Leu Ala Gly Leu Gln Gln Gly Gly Se 225 230 235 240 Gly Ser Ile Arg Ala Gly Ile His Ser Thr Pro Trp Gly Thr Val Gl 245 250 255 Val Glu Pro Ser Ser Ser Gly His Thr His Asn Cys Ala Asn Ser Se 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Glu Ala Tyr Ser Pr 275 280 285 Val Ser Thr Ser Lys Arg His Ser Ser Ser Gly Asn Ala Val Glu Le 290 295 300 His His Val Pro Pro Asn Ser Ser Arg Ser Gln Ser Gln Gly Ser Va 305 310 315 320 Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Gl 325 330 335 His Cys Leu Phe His Ile Val Asn Leu Ile Asp Asp Trp Gly Pro Cy 340 345 350 Ala Glu His Gly Glu His Arg Ile Arg Thr Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Se 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Th 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asp Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser As 450 455 460 Ser Arg Ile Ile Asn His Gln His Arg Thr Met Gln Asn Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Met Leu Leu Tyr Lys Th 485 490 495 Tyr Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Tyr Ala Ala Val Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Gln Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Asn Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ser Pro Leu Pro Ile His Thr Val Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Arg Leu Pro Tyr Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 843 amino acids amino acid <Unknown> linear protein 56 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro His Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Pro Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Ser Phe Asn Pro Lys Trp Gln Thr Pro Ser Phe Pr 65 70 75 80 Asp Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Asn Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Val Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Arg Leu Val Leu Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Lys Ser Phe Arg Pro Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Ile Gln Ser Gln Leu Arg Gln Ser Arg Leu Gl 210 215 220 Pro Gln Pro Thr Gln Gly Gln Leu Ala Gly Leu Gln Gln Gly Gly Se 225 230 235 240 Gly Ser Ile Arg Ala Gly Ile His Ser Thr Pro Trp Gly Thr Val Gl 245 250 255 Val Glu Pro Ser Ser Ser Gly His Thr His Asn Cys Ala Asn Ser Se 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Glu Ala Tyr Ser Pr 275 280 285 Val Ser Thr Ser Lys Arg His Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His His Val Pro Pro Asn Ser Ser Arg Ser Gln Ser Gln Gly Ser Va 305 310 315 320 Leu Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Gl 325 330 335 His Cys Leu Phe His Ile Val Asn Leu Ile Glu Asp Trp Gly Pro Cy 340 345 350 Ala Glu His Gly Glu His Arg Ile Arg Thr Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Th 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asp Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser As 450 455 460 Ser Arg Ile Ile Asn His Gln His Arg Thr Met Gln Asn Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Met Leu Leu Tyr Lys Th 485 490 495 Tyr Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Tyr Ala Ala Val Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Gln Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Leu Lys Leu Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Thr Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ser Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu Arg Leu Pro Tyr Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Asp Ser Pro Ser Val Pro Ser Arg Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 amino acids amino acid <Unknown> linear protein 57 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Gly Thr Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala As 20 25 30 Ala Asp Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Le 35 40 45 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Le 50 55 60 Tyr Ser Ser Thr Ala Pro Ile Phe Asn Pro Glu Trp Gln Thr Pro Se 65 70 75 80 Phe Pro Lys Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gl 85 90 95 Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Il 100 105 110 Met Pro Ala Arg Phe Tyr Pro Thr His Thr Lys Tyr Leu Pro Leu As 115 120 125 Lys Gly Ile Lys Pro Tyr Tyr Pro Asp Gln Val Val Asn His Tyr Ph 130 135 140 Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Ty 145 150 155 160 Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Se 165 170 175 Trp Glu Gln Glu Leu Gln His Gly Arg Leu Val Ile Lys Thr Ser Gl 180 185 190 Arg His Gly Asp Glu Ser Phe Cys Ser Gln Pro Ser Gly Ile Leu Se 195 200 205 Arg Ser Ser Val Gly Pro Cys Ile Arg Ser Gln Leu Lys Gln Ser Ar 210 215 220 Leu Gly Leu Gln Pro His Gln Gly Pro Leu Ala Ser Ser Gln Pro Gl 225 230 235 240 Arg Ser Gly Ser Ile Arg Ala Arg Val His Pro Ser Thr Arg Arg Cy 245 250 255 Phe Gly Val Glu Pro Ser Gly Ser Gly His Val Asp Pro Ser Val As 260 265 270 Asn Ser Ser Ser Cys Leu Arg Gln Ser Ala Val Arg Lys Ala Ala Ty 275 280 285 Ser His Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Va 290 295 300 Glu Phe His Cys Leu Pro Pro Ser Ser Ala Arg Pro Gln Ser Gln Gl 305 310 315 320 Ser Val Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cy 325 330 335 Ser Glu Tyr Cys Leu Ser His Leu Val Asn Leu Arg Glu Asp Arg Gl 340 345 350 Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pr 355 360 365 Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His As 370 375 380 Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gl 385 390 395 400 Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Ile Pro Asn Leu Gln Se 405 410 415 Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Va 420 425 430 Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Hi 435 440 445 Leu Leu Ile Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Se 450 455 460 Ser Asn Ser Arg Ile Asn Asn Asn Gln Tyr Gly Thr Met Gln Asn Le 465 470 475 480 His Asp Ser Cys Ser Arg Gln Leu Tyr Val Ser Leu Met Leu Leu Ty 485 490 495 Lys Thr Tyr Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Val Le 500 505 510 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Le 515 520 525 Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pr 530 535 540 His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Ly 545 550 555 560 Ser Val Gln His Arg Glu Phe Leu Tyr Thr Ala Val Thr Asn Phe Le 565 570 575 Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gl 580 585 590 Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Le 595 600 605 Pro Gln Asp His Ile Val Gln Lys Ile Lys His Cys Phe Arg Lys Le 610 615 620 Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gl 625 630 635 640 Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Le 645 650 655 Met Pro Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Se 660 665 670 Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Met Asn Leu Tyr Pr 675 680 685 Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Th 690 695 700 Pro Thr Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Th 705 710 715 720 Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cy 725 730 735 Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Se 740 745 750 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cy 755 760 765 Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Se 770 775 780 Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Se 785 790 795 800 Arg Pro Leu Leu Arg Leu Pro Phe Gln Pro Thr Thr Gly Arg Thr Se 805 810 815 Leu Tyr Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Val Arg Va 820 825 830 His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 845 amino acids amino acid <Unknown> linear protein 58 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Gly Thr Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala As 20 25 30 Ala Asp Leu His Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Le 35 40 45 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Le 50 55 60 Tyr Ser Ser Thr Val Pro Ile Phe Asn Pro Glu Trp Gln Thr Pro Se 65 70 75 80 Phe Pro Lys Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gl 85 90 95 Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Il 100 105 110 Met Pro Ala Arg Phe Tyr Pro Thr His Thr Lys Tyr Leu Pro Leu As 115 120 125 Lys Gly Ile Lys Pro Tyr Tyr Pro Asp Gln Val Val Asn His Tyr Ph 130 135 140 Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Ty 145 150 155 160 Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Se 165 170 175 Trp Glu Gln Glu Leu Gln His Gly Arg Leu Val Ile Lys Thr Ser Gl 180 185 190 Arg His Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Se 195 200 205 Arg Ser Ser Val Gly Pro Cys Ile Arg Ser Gln Leu Lys Gln Ser Ar 210 215 220 Leu Gly Leu Gln Pro Arg Gln Gly Arg Leu Ala Ser Ser Gln Pro Se 225 230 235 240 Arg Ser Gly Ser Ile Arg Ala Lys Ala His Pro Ser Thr Arg Arg Ty 245 250 255 Phe Gly Val Glu Pro Ser Gly Ser Gly His Ile Asp His Ser Val As 260 265 270 Asn Ser Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala Ala Ty 275 280 285 Ser His Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Va 290 295 300 Glu Phe His Cys Leu Pro Pro Asn Ser Ala Gly Ser Gln Ser Gln Gl 305 310 315 320 Ser Val Ser Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cy 325 330 335 Ser Glu Tyr Cys Leu Ser His Leu Val Asn Leu Arg Glu Asp Trp Gl 340 345 350 Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pr 355 360 365 Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His As 370 375 380 Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gl 385 390 395 400 Ile Ser Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Se 405 410 415 Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Va 420 425 430 Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Hi 435 440 445 Leu Leu Ile Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Se 450 455 460 Ser Asn Ser Arg Ile Asn Asn Asn Gln Tyr Gly Thr Met Gln Asn Le 465 470 475 480 His Asp Ser Cys Ser Arg Gln Leu Tyr Val Ser Leu Met Leu Leu Ty 485 490 495 Lys Thr Tyr Gly Trp Lys Leu His Leu Tyr Ser His Pro Ile Val Le 500 505 510 Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Le 515 520 525 Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pr 530 535 540 His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Ly 545 550 555 560 Ser Val Gln His Arg Glu Ser Leu Tyr Thr Ala Val Thr Asn Phe Le 565 570 575 Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gl 580 585 590 Tyr Ser Leu Asn Phe Met Gly Tyr Ile Ile Gly Ser Trp Gly Thr Le 595 600 605 Pro Gln Asp His Ile Val Gln Lys Ile Lys His Cys Phe Arg Lys Le 610 615 620 Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gl 625 630 635 640 Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Le 645 650 655 Met Pro Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Se 660 665 670 Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Met Asn Leu Tyr Pr 675 680 685 Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Th 690 695 700 Pro Thr Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Th 705 710 715 720 Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cy 725 730 735 Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Se 740 745 750 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cy 755 760 765 Thr Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Se 770 775 780 Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Se 785 790 795 800 Arg Pro Leu Leu Arg Leu Pro Phe Gln Pro Thr Thr Gly Arg Thr Se 805 810 815 Leu Tyr Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Val Arg Va 820 825 830 His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 845 843 amino acids amino acid <Unknown> linear protein 59 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Asp Trp Lys Thr Pro Ser Phe Pr 65 70 75 80 His Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Tyr Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Leu Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu Tyr Ala Val Asn His Tyr Phe Lys Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Thr Arg Hi 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Se 195 200 205 Pro Val Gly Pro Cys Val Arg Ser Gln Leu Lys Gln Ser Arg Leu Gl 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Gly Lys Ser Gly Arg Se 225 230 235 240 Gly Ser Ile Trp Ser Arg Val His Pro Thr Thr Arg Arg Pro Phe Gl 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Thr Ala Ser Ser Th 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Thr Ala Tyr Ser Hi 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Le 290 295 300 His Asn Ile Pro Pro Ser Ser Ala Arg Ser Gln Ser Glu Gly Pro Il 305 310 315 320 Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser As 325 330 335 Tyr Cys Leu Thr His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cy 340 345 350 Thr Glu His Gly Glu His Asn Ile Arg Ile Pro Arg Thr Pro Ala Ar 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Th 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ser Th 385 390 395 400 His Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Th 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Al 420 425 430 Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro His Leu Le 435 440 445 Val Gly Ser Ser Gly Leu Pro Arg Tyr Val Ala Arg Leu Ser Ser Th 450 455 460 Ser Arg Asn Ile Asn Tyr Gln His Gly Thr Met Gln Asn Leu His As 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Th 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Ph 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gl 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cy 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Va 545 550 555 560 Gln His Leu Glu Ser Leu Phe Thr Ser Ile Thr Asn Phe Leu Leu Se 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Se 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gl 595 600 605 Glu His Ile Val Gln Lys Leu Lys Gln Cys Phe Arg Lys Leu Pro Va 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Le 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pr 645 650 655 Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Th 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pro Val Al 675 680 685 Arg Gln Arg Ser Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Th 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Arg Arg Met Arg Gly Thr Phe Va 705 710 715 720 Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Al 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Va 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Al 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Le 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pr 785 790 795 800 Leu Leu His Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Ty 805 810 815 Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Ph 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 832 amino acids amino acid <Unknown> linear protein 60 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Lys Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gl 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Ar 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Ar 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Phe His Pro Thr Al 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr As 245 250 255 Phe Ala Ser Lys Ser Ala Ser Cys Leu His Gln Ser Pro Val Arg Ly 260 265 270 Ala Ala Tyr Pro Ala Val Ser Thr Phe Glu Lys His Ser Ser Ser Gl 275 280 285 His Ala Val Glu Phe His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gl 290 295 300 Ser Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Se 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser Leu Ile Val Asn Leu Leu Gl 325 330 335 Asp Trp Gly Pro Cys Ala Glu His Gly Glu His His Ile Arg Ile Pr 340 345 350 Arg Thr Pro Ser Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys As 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Ph 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro As 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Se 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Al 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Al 435 440 445 Arg Leu Ser Ser Asn Ser Arg Ile Leu Asn Asn Gln His Gly Thr Me 450 455 460 Pro Asp Leu His Asp Tyr Cys Ser Arg Asn Leu Tyr Val Ser Leu Le 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pr 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pr 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Ar 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Le 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Th 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Ly 565 570 575 Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Cys Ty 580 585 590 Gly Ser Leu Pro Gln Glu His Ile Ile Gln Lys Ile Lys Glu Cys Ph 595 600 605 Arg Lys Leu Pro Ile Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Ar 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Ty 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Ph 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu As 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Al 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Val Met Gly His Gln Arg Me 690 695 700 Arg Gly Thr Phe Ser Ala Pro Leu Pro Ile His Thr Ala Glu Leu Le 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Ile Gly Th 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Le 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Ty 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Le 770 775 780 Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gl 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pr 805 810 815 Asp Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pr 820 825 830 832 amino acids amino acid <Unknown> linear protein 61 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu Tyr Leu Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gl 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Ar 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Ar 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Ile His Pro Thr Th 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Arg As 245 250 255 Val Ala Ser Lys Ser Ala Ser Cys Leu Tyr Gln Ser Pro Val Arg Ly 260 265 270 Ala Ala Tyr Pro Ala Val Ser Thr Phe Glu Lys His Ser Ser Ser Gl 275 280 285 His Ala Val Glu Leu His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gl 290 295 300 Ser Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Se 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser His Ile Val Asn Leu Leu Gl 325 330 335 Asp Trp Gly Pro Cys Ala Glu His Gly Glu His His Ile Arg Ile Pr 340 345 350 Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys As 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Ph 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro As 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Se 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Al 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Al 435 440 445 Arg Leu Ser Ser Asn Ser Arg Ile Phe Asn Tyr Gln His Gly Thr Me 450 455 460 Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Le 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pr 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pr 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Ar 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Le 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Th 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Ly 565 570 575 Arg Trp Gly Tyr Ser Leu His Phe Met Gly Tyr Val Ile Gly Cys Ty 580 585 590 Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile Lys Glu Cys Ph 595 600 605 Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Ar 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Ty 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Ph 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu As 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Al 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Val Met Gly His Gln Arg Me 690 695 700 Arg Gly Thr Phe Leu Ala Arg Leu Pro Ile His Thr Ala Glu Leu Le 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Leu Gly Th 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Tyr Pro Trp Le 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Ty 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Le 770 775 780 Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gl 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pr 805 810 815 Asp Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pr 820 825 830 832 amino acids amino acid <Unknown> linear protein 62 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Lys Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gl 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Ar 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Ar 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Phe His Pro Thr Al 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr As 245 250 255 Phe Ala Ser Lys Ser Ala Ser Cys Leu His Gln Ser Pro Val Arg Ly 260 265 270 Ala Ala Tyr Pro Ser Val Ser Thr Phe Glu Lys His Ser Ser Ser Gl 275 280 285 His Ala Val Glu Leu His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gl 290 295 300 Ser Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Se 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser Leu Ile Val Asn Leu Leu Gl 325 330 335 Asp Trp Gly Pro Cys Ala Glu His Gly Glu His His Ile Arg Ile Pr 340 345 350 Arg Thr Pro Ser Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys As 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Ph 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro As 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Se 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Al 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Al 435 440 445 Arg Leu Ser Ser Asn Ser Arg Ile Leu Asn His Gln His Gly Thr Me 450 455 460 Pro Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Le 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pr 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pr 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Ar 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Le 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Th 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Ly 565 570 575 Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Cys Ty 580 585 590 Gly Ser Leu Pro Gln Glu His Ile Ile Gln Lys Ile Lys Glu Cys Ph 595 600 605 Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Ar 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Ty 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Ph 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu As 660 665 670 Leu Tyr Pro Val Ala Gly Gln Arg Pro Gly Leu Cys Gln Val Phe Al 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His Gln Arg Me 690 695 700 Arg Gly Thr Phe Ser Ala Pro Leu Pro Ile His Thr Ala Glu Leu Le 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Ile Gly Th 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Le 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Ty 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Le 770 775 780 Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gl 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pr 805 810 815 Asp Leu Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pr 820 825 830 832 amino acids amino acid <Unknown> linear protein 63 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu Asp As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Phe Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Glu Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Lys Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Asp Leu Gln His Gly Ala Glu Ser Ile His Gln Gln Ser Ser Gl 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Ar 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Ar 210 215 220 Gln Gln Gly Trp Ser Trp Ser Ile Arg Ala Gly Thr His Pro Thr Al 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr Hi 245 250 255 Arg Ala Ser Lys Ser Ala Ser Cys Leu Tyr Gln Ser Pro Asp Arg Ly 260 265 270 Ala Thr Tyr Pro Ser Val Ser Thr Phe Glu Arg His Ser Ser Ser Gl 275 280 285 Arg Ala Val Glu Leu His Asn Phe Pro Pro Asn Ser Ala Arg Ser Gl 290 295 300 Ser Glu Arg Pro Ile Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Se 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser Leu Ile Val Asn Leu Leu Gl 325 330 335 Asp Trp Gly Pro Cys Asp Glu Tyr Gly Glu His His Ile Arg Ile Pr 340 345 350 Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys As 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Ph 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro As 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Se 405 410 415 Leu Asp Val Ser Ala Gly Phe Tyr His Leu Pro Leu His Pro Ala Al 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Val Ser Arg Tyr Val Al 435 440 445 Arg Leu Ser Ser Asn Ser Arg Asn Asn Asn Asn Gln Tyr Gly Thr Me 450 455 460 Gln Asn Leu His Asp Ser Cys Ser Arg Gln Leu Tyr Val Ser Leu Me 465 470 475 480 Leu Leu Tyr Gln Asn Phe Gly Trp Lys Leu His Leu Tyr Ser His Pr 485 490 495 Ile Val Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pr 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Ar 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Le 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Th 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Ly 565 570 575 Arg Trp Gly Tyr Ser Leu His Phe Met Gly Tyr Val Ile Gly Cys Ty 580 585 590 Gly Ser Leu Pro Gln Glu His Ile Ile Gln Lys Ile Lys Glu Cys Ph 595 600 605 Arg Lys Val Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Ar 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Ty 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Phe Lys Gln Ala Ph 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu As 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Al 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Gly Met Gly His Gln Arg Me 690 695 700 Arg Gly Thr Phe Ser Ala Pro Leu Pro Ile His Thr Ala Glu Leu Le 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Leu Gly Th 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Le 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Ty 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Le 770 775 780 Gly Leu Ser Arg Pro Leu Leu Cys Leu Pro Phe Arg Pro Thr Thr Gl 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pr 805 810 815 Asp Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pr 820 825 830 832 amino acids amino acid <Unknown> linear protein 64 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu His As 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Pro Asp Gln Gl 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Va 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Se 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pr 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Va 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pr 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gl 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Th 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Ar 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Gl 165 170 175 Gln Glu Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gl 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Ar 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Ar 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Phe His Pro Thr Al 225 230 235 240 Arg Arg Ser Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr Ty 245 250 255 Arg Ala Ser Lys Ser Ala Ser Cys Leu Tyr Gln Ser Pro Val Arg Ly 260 265 270 Ala Ala Tyr Pro Ser Val Ser Thr Phe Glu Lys His Ser Ser Ser Gl 275 280 285 His Ala Val Glu Leu His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gl 290 295 300 Ser Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Se 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser Leu Ile Val Asn Leu Arg Gl 325 330 335 Asp Trp Gly Pro Cys Thr Glu His Gly Glu His His Ile Arg Ile Pr 340 345 350 Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys As 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Ph 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro As 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Se 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Al 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Al 435 440 445 Arg Leu Ser Ser Asn Ser Arg Ile Phe Asn Asn Gln His Gly Thr Me 450 455 460 Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Le 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pr 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pr 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Ar 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Le 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Th 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Ly 565 570 575 Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Ile Ile Gly Ser Tr 580 585 590 Gly Thr Leu Pro Gln Asp His Ile Val Gln Lys Ile Lys Glu Cys Ph 595 600 605 Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Trp Gln Ar 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Ty 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Ph 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Met As 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Al 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Ile Gly Asn Gln Arg Me 690 695 700 Arg Gly Thr Ile Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Le 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Th 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Le 740 745 750 Leu Gly Cys Thr Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Ty 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Le 770 775 780 Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Gln Pro Thr Thr Gl 785 790 795 800 Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val Pro Ser His Leu Pr 805 810 815 Val Arg Val His Phe Ala Ser Pro Leu His Ile Ala Trp Arg Pro Pr 820 825 830 8 amino acids amino acid <Unknown> linear peptide 65 Leu Ser Arg Tyr Val Ala Arg Leu 1 5 8 amino acids amino acid <Unknown> linear peptide 66 Gly Leu Ser Arg Tyr Val Ala Arg 1 5

Claims (16)

What is claimed is:
1. A CTL inducing peptide comprising from eight to thirteen amino acids, wherein at least a majority of the amino acids are homologous to a corresponding portion of HBpol having the sequence:
HBpol4-13 [Seq ID No. 12] Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu; HBpol108-116 [Seq ID No. 13] Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg; HBpol139-147 [Seq ID No. 14] Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg HBpol151-160 [Seq ID No. 15] His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr HBpol152-161 [Seq ID No. 16] Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys HBpol 455-463 [Seq ID No. 2] Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol505-514 [Seq ID No. 17] Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe; HBpol551-559 [Seq ID No. 18] Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala; HBpol575-583 [Seq ID No. 19] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu; HBpol655-663 [Seq ID No. 20] Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile; HBpol748-757 [Seq ID No. 21] Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg; HBpol758-766 [Seq ID No. 22] Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu); HBpol773-782 [Seq ID No. 3] Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val or HBpol816-824 [Seq ID No. 5] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
2. The CTL inducing peptide of claim 1, which comprises from nine to eleven amino acids.
3. The CTL inducing peptide of claim 1, which comprises:
HBpol4-13 [Seq ID No. 12] Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu HBpol108-116 [Seq ID No. 13] Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg; HBpol139-147 [Seq ID No. 14] Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg HBpol151-160 [Seq ID No. 15] His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr HBpol152-161 [Seq ID No. 16] Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys HBpol455-463 [Seq ID No. 2] Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol505-514 [Seq ID No. 17] Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe; HBpol551-559 [Seq ID No. 18] Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala; HBpol575-583 [Seq ID No. 19] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu; HBpol655-663 [Seq ID No. 20] Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile; HBpol748-757 [Seq ID No. 21] Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg; HBpol758-766 [Seq ID No. 22] Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu); HBpol773-782 [Seq ID No. 3] Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val or HBpol816-824 [Seq ID No. 5] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
4. The peptide of claim 1, suspended in pharmaceutically acceptable carrier which comprises a liposome.
5. The CTL inducing peptide of claim 1, wherein the peptide also contains a T helper epitope.
6. The peptide of claim 1, conjugated to a immunogenic lipid carrier.
7. A method for stimulating a cytotoxic T lymphocyte response to hepatitis B virus which comprises exposing cytotoxic T lymphocytes of a host to a peptide which contains a CTL epitope and which comprises from eight to thirteen amino acids, wherein at least a majority of the amino acids are homologous to a corresponding portion of HBpol having the sequence:
HBpol14-13 [Seq ID No. 12] Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu; HBpol108-116 [Seq ID No. 13] Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg; HBpol139-147 [Seq ID No. 14] Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg HBpol151-160 [Seq ID No. 15] His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr HBpol152-161 [Seq ID No. 16] Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys HBpol 455-463 [Seq ID No. 23] Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol505-514 [Seq ID No. 17] Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe; HBpol551-559 [Seq ID No. 18] Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala; HBpol575-583 [Seq ID No. 19] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu; HBpol655-663 [Seq ID No. 20] Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile HBpol748-757 [Seq ID No. 21] Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg HBpol758-766 [Seq ID No. 22] Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu) HBpol773-782 [Seq ID No. 3] Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val or HBpol816-824 [Seq ID No. 5] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu
8. The method of claim 7, wherein the CTL inducing peptide is:
HBpol4-13 [Seq ID No. 12] Ser-Tyr-Gln-His-Phe-Arg-Lys-Leu-Leu-Leu; HBpol108-116 [Seq ID No. 13] Arg-Leu-Lys-Leu-Ile-Met-Pro-Ala-Arg; HBpol139-147 [Seq ID No. 14] Val-Val-Asn-His-Tyr-Phe-Gln-Thr-Arg HBpol151-160 [Seq ID No. 15] His-Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr HBpol152-161 [Seq ID No. 16] Thr-Leu-Trp-Lys-Ala-Gly-Ile-Leu-Tyr-Lys HBpol 455-463 [Seq ID No. 2] Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol505-514 [Seq ID No. 17] Leu-Tyr-Ser-His-Pro-Ile-Ile-Leu-Gly-Phe; HBpol551-559 [Seq ID No. 18] Tyr-Met-Asp-Asp-Val-Val-Leu-Gly-Ala; HBpol575-583 [Seq ID No. 19] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu; HBpol655-663 [Seq ID No. 20] Ala-Leu-Met-Pro-Leu-Tyr-Ala-Cys-Ile; HBpol748-757 [Seq ID No. 21] Gly-Thr-Asp-Asn-Ser-Val-Val-Leu-Ser-Arg; HBpol758-766 [Seq ID No. 22] Lys-Tyr-Thr-Ser-Phe-Pro-Trp-Leu-Leu); HBpol773-782 [Seq ID No. 3] Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val or HBpol816-824 [Seq ID No. 5] Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu.
9. The method of claim 8, wherein the host cells exposed to the CTL-inducing peptides are HLA-A2.
10. The method of claim 8, wherein the cytotoxic T cells are removed from the host prior to being exposed to the CTL-inducing peptide.
11. The method according to claim 10, further comprising the step of returning the stimulated CTLs to the host.
12. The method of claim 8, wherein the host has chronic hepatitis B infection or is a hepatitis B carrier.
13. The method of claim 8, wherein the host has acute hepatitis B infection.
14. The method of claim 8, wherein the CTL inducing peptide is administered prophylactically to the host's cells.
15. The method of claim 8, wherein the CTL inducing peptide is administered to the host with a second peptide which elicits a T helper response to HBV.
16. The method of claim 15, wherein the CTL inducing peptide and the T helper inducing peptide are linked.
US10/359,431 1991-08-26 2003-02-05 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus Abandoned US20030171538A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/359,431 US20030171538A1 (en) 1991-08-26 2003-02-05 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US11/221,470 US7744898B2 (en) 1991-08-26 2005-09-08 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US74954091A 1991-08-26 1991-08-26
US93589892A 1992-08-26 1992-08-26
US10087093A 1993-08-02 1993-08-02
US08/591,502 US6607727B1 (en) 1991-08-26 1994-08-01 Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus
US10/359,431 US20030171538A1 (en) 1991-08-26 2003-02-05 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US08591502 Division 1994-08-01
PCT/US1994/008685 Division WO1995003777A1 (en) 1991-08-26 1994-08-01 Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus
US08/591,502 Division US6607727B1 (en) 1991-08-26 1994-08-01 Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/221,470 Continuation US7744898B2 (en) 1991-08-26 2005-09-08 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Publications (1)

Publication Number Publication Date
US20030171538A1 true US20030171538A1 (en) 2003-09-11

Family

ID=29554392

Family Applications (3)

Application Number Title Priority Date Filing Date
US08/591,502 Expired - Fee Related US6607727B1 (en) 1991-08-26 1994-08-01 Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus
US10/359,431 Abandoned US20030171538A1 (en) 1991-08-26 2003-02-05 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US11/221,470 Expired - Fee Related US7744898B2 (en) 1991-08-26 2005-09-08 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US08/591,502 Expired - Fee Related US6607727B1 (en) 1991-08-26 1994-08-01 Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus

Family Applications After (1)

Application Number Title Priority Date Filing Date
US11/221,470 Expired - Fee Related US7744898B2 (en) 1991-08-26 2005-09-08 Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Country Status (1)

Country Link
US (3) US6607727B1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030021809A1 (en) * 1992-08-26 2003-01-30 Chisari Francis V. Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US20060051746A1 (en) * 1991-08-26 2006-03-09 Chisari Francis V Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
WO2019216603A1 (en) * 2018-05-09 2019-11-14 서울대학교산학협력단 Hepatitis b virus-derived polypeptide and anti-viral use thereof
WO2022035246A1 (en) * 2020-08-14 2022-02-17 서울대학교산학협력단 Immune adjuvant comprising hepatitis b virus-derived polypeptide

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110097352A9 (en) * 1992-01-29 2011-04-28 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions
US7611713B2 (en) * 1993-03-05 2009-11-03 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide compositions
US20070020327A1 (en) * 1998-11-10 2007-01-25 John Fikes Inducing cellular immune responses to prostate cancer antigens using peptide and nucleic acid compositions
US7785875B2 (en) 2004-07-03 2010-08-31 Mogam Biotechnology Research Institute Polynucleotide encoding HCV epitopes which can bind to various HLA supertypes, immunogenic composition comprising same and method of inducing an HCV-specific immune response using same
US20080112977A1 (en) * 2004-07-03 2008-05-15 Mogam Biotechnology Research Supertype Epitopes, Oligonucleotides Coding The Same Which Induce Effective Ctl Response Against Hcv And The Use Thereof
KR20070053229A (en) * 2004-08-13 2007-05-23 미게닉스 인코포레이티드 Compositions and methods for treating or preventing hepadnaviridae infection
CU23740A1 (en) * 2009-09-29 2011-12-28 Ct Ingenieria Genetica Biotech METHOD OF OBTAINING A FORMATION OF ANTIGENS FROM THE HEPATITIS B VIRUS
EP2545171B1 (en) * 2010-03-11 2017-12-20 OncoTherapy Science, Inc. Hjurp peptides and vaccines including the same
WO2012109404A1 (en) 2011-02-12 2012-08-16 Globeimmune, Inc. Yeast-based therapeutic for chronic hepatitis b infection
GB201223386D0 (en) * 2012-12-24 2013-02-06 Immune Targeting Systems Its Ltd Vaccine
US9850278B2 (en) 2013-04-25 2017-12-26 Carmel-Haifa University Economic Corp. Synthetic anti-inflammatory peptides and use thereof
EP3148566B1 (en) * 2014-06-02 2024-04-03 ISA Pharmaceuticals B.V. Synthetic long peptides (slp) for therapeutic vaccination against hepatitis b virus infection
WO2021110919A1 (en) * 2019-12-07 2021-06-10 Isa Pharmaceuticals Treatment of diseases related to hepatitis b virus
KR20220022021A (en) * 2020-08-14 2022-02-23 서울대학교산학협력단 A pharmaceutical composition for preventing or treating cancer comprising hepatitis b virus-derived polypeptide
EP4199907A1 (en) * 2020-09-21 2023-06-28 SRI International Targeted antigen delivery system and uses thereof

Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3645852A (en) * 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide
US4235877A (en) * 1979-06-27 1980-11-25 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
US4428941A (en) * 1979-08-03 1984-01-31 Institut Pasteur Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby
US4487715A (en) * 1982-07-09 1984-12-11 The Regents Of The University Of California Method of conjugating oligopeptides
US4599231A (en) * 1984-03-09 1986-07-08 Scripps Clinic And Research Foundation Synthetic hepatitis B virus vaccine including both T cell and B cell determinants
US4599230A (en) * 1984-03-09 1986-07-08 Scripps Clinic And Research Foundation Synthetic hepatitis B virus vaccine including both T cell and B cell determinants
US4659569A (en) * 1981-02-09 1987-04-21 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for the production of virus vaccine
US4818527A (en) * 1986-12-09 1989-04-04 Scripps Clinic And Research Foundation T cell epitopes of the hepatitis B virus nucleocapsid protein
US4882145A (en) * 1986-12-09 1989-11-21 Scripps Clinic And Research Foundation T cell epitopes of the hepatitis B virus nucleocapsid protein
US4935235A (en) * 1979-05-24 1990-06-19 The Regents Of The University Of California Non-passageable viruses
US5017558A (en) * 1980-01-14 1991-05-21 The Regents Of The University Of California Synthetic vaccine peptide epitomes of hepatitis B surface antigen
US5019386A (en) * 1984-07-31 1991-05-28 Institute Of Immunology Co., Ltd. Hepatitis B vaccine
US5039522A (en) * 1988-01-29 1991-08-13 New York Blood Center, Inc. Immunogens containing peptides with an attached hydrophobic tail for adsorption to hepatitis B virus surface antigen
US5133961A (en) * 1986-01-31 1992-07-28 Merck & Co., Inc. Vaccines comprising yeast-derived hepatits b virus polypeptides
US5143726A (en) * 1986-12-09 1992-09-01 The Scripps Research Institute T cell epitopes of the hepatitis B virus nucleocapsid protein
US5788969A (en) * 1993-02-26 1998-08-04 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses hepatitis B virus
US5840303A (en) * 1991-08-26 1998-11-24 The Scripps Research Foundation Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US5932224A (en) * 1991-08-26 1999-08-03 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US6037135A (en) * 1992-08-07 2000-03-14 Epimmune Inc. Methods for making HLA binding peptides and their uses
US6235288B1 (en) * 1992-08-26 2001-05-22 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2444713A1 (en) 1978-12-18 1980-07-18 Pasteur Institut PROCESS FOR PRODUCING DNA COMPRISING THE GENOME OF HEPATITIS B VIRUS AND VECTOR COMPRISING SAME
FI86437C (en) 1978-12-22 1992-08-25 Biogen Inc A FRUIT PROCEDURE FOR THE PREPARATION OF A POLYPEPTIDIDE WITH ANTIGENA EGENSKAPERNA HOS ETT HEPATITIS B- VIRUSANTIGEN.
FR2523976B1 (en) * 1982-03-26 1985-08-30 Pasteur Institut NEW PROTEIN PRODUCT, OBTAINING AND APPLICATION AS A MEDICINE, IN PARTICULAR AN IMMUNOREGULATOR AND ANTIALLERGIC
EP0105481A1 (en) 1982-09-30 1984-04-18 The Wellcome Foundation Limited Novel antigens and vaccines containing them
JPS60161999A (en) 1984-02-02 1985-08-23 Chemo Sero Therapeut Res Inst Peptide
US5158769A (en) * 1984-03-07 1992-10-27 New York Blood Center, Inc. Pre-S gene coded peptide hepatitis B immunogens, vaccines, diagnostics, and synthetic lipid vesicle carriers
DE3937412A1 (en) 1989-11-10 1991-05-16 Hoechst Ag SYNTHETIC VACCINE FOR THE SPECIFIC INDUCTION OF CYTOTOXIC T-LYMPHOZYTES
JPS6425800U (en) 1987-03-03 1989-02-13
EP0293201A3 (en) 1987-05-26 1990-02-28 The Wistar Institute Method of vaccination for hepatitis b virus
EP0326111A3 (en) 1988-01-29 1989-12-27 New York Blood Center, Inc. Peptide derivatives rendered immunogenic when administered with alum as an adjuvant
AU607306B2 (en) 1988-02-02 1991-02-28 Regents Of The University Of California, The Human platelet-derived growth factor receptor
US5100662A (en) * 1989-08-23 1992-03-31 The Liposome Company, Inc. Steroidal liposomes exhibiting enhanced stability
GB9016727D0 (en) 1990-07-31 1990-09-12 Clonit Spa Amino acid residue sequence of hepatitis b core antigen
CZ42794A3 (en) 1991-08-26 1994-11-16 Cytel Corp Hla-re-stringed ctl epitopes of hepatitis b virus
US6322789B1 (en) * 1991-08-26 2001-11-27 Epimmune, Inc. HLA-restricted hepatitis B virus CTL epitopes
US6607727B1 (en) * 1991-08-26 2003-08-19 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus
AU679901B2 (en) 1991-08-26 1997-07-17 Scripps Research Institute, The Peptides for inducing cytotoxic T-lymphocyte responses to hepatitis B virus
US6419931B1 (en) * 1991-08-26 2002-07-16 Epimmune Inc. Compositions and methods for eliciting CTL immunity
IL106610A0 (en) 1992-08-07 1993-12-08 Cytel Corp Hla binding peptides and their uses
SG49008A1 (en) 1993-03-05 1998-05-18 Cytel Corp Hla-a2.1 binding peptides and their uses
JP3795914B2 (en) 1993-04-27 2006-07-12 ユナイテッド・バイオメディカル・インコーポレイテッド Immunogenic LHRH peptide constructs for vaccines and synthetic universal immune stimulators
CA2168583C (en) 1993-08-02 2007-10-02 Francis V. Chisari Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus
WO1995004817A1 (en) 1993-08-06 1995-02-16 Cytel Corporation Methods for ex vivo therapy using peptide-loaded antigen presenting cells for the activation of ctl
US5985312A (en) * 1996-01-26 1999-11-16 Brown University Research Foundation Methods and compositions for enhancing the bioadhesive properties of polymers
US5800832A (en) * 1996-10-18 1998-09-01 Virotex Corporation Bioerodable film for delivery of pharmaceutical compounds to mucosal surfaces

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3645852A (en) * 1967-05-23 1972-02-29 Pharmacia Ab Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide
US4935235A (en) * 1979-05-24 1990-06-19 The Regents Of The University Of California Non-passageable viruses
US4235877A (en) * 1979-06-27 1980-11-25 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
US4428941A (en) * 1979-08-03 1984-01-31 Institut Pasteur Nucleotidic sequence coding the surface antigen of the hepatitis B virus, vector containing said nucleotidic sequence, process allowing the obtention thereof and antigen obtained thereby
US5017558A (en) * 1980-01-14 1991-05-21 The Regents Of The University Of California Synthetic vaccine peptide epitomes of hepatitis B surface antigen
US4659569A (en) * 1981-02-09 1987-04-21 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Process for the production of virus vaccine
US4487715A (en) * 1982-07-09 1984-12-11 The Regents Of The University Of California Method of conjugating oligopeptides
US4599230A (en) * 1984-03-09 1986-07-08 Scripps Clinic And Research Foundation Synthetic hepatitis B virus vaccine including both T cell and B cell determinants
US4599231A (en) * 1984-03-09 1986-07-08 Scripps Clinic And Research Foundation Synthetic hepatitis B virus vaccine including both T cell and B cell determinants
US5019386A (en) * 1984-07-31 1991-05-28 Institute Of Immunology Co., Ltd. Hepatitis B vaccine
US5133961A (en) * 1986-01-31 1992-07-28 Merck & Co., Inc. Vaccines comprising yeast-derived hepatits b virus polypeptides
US4882145A (en) * 1986-12-09 1989-11-21 Scripps Clinic And Research Foundation T cell epitopes of the hepatitis B virus nucleocapsid protein
US4818527A (en) * 1986-12-09 1989-04-04 Scripps Clinic And Research Foundation T cell epitopes of the hepatitis B virus nucleocapsid protein
US5143726A (en) * 1986-12-09 1992-09-01 The Scripps Research Institute T cell epitopes of the hepatitis B virus nucleocapsid protein
US5039522A (en) * 1988-01-29 1991-08-13 New York Blood Center, Inc. Immunogens containing peptides with an attached hydrophobic tail for adsorption to hepatitis B virus surface antigen
US5840303A (en) * 1991-08-26 1998-11-24 The Scripps Research Foundation Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US5932224A (en) * 1991-08-26 1999-08-03 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US6037135A (en) * 1992-08-07 2000-03-14 Epimmune Inc. Methods for making HLA binding peptides and their uses
US6235288B1 (en) * 1992-08-26 2001-05-22 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US5788969A (en) * 1993-02-26 1998-08-04 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses hepatitis B virus

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060051746A1 (en) * 1991-08-26 2006-03-09 Chisari Francis V Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US7744898B2 (en) 1991-08-26 2010-06-29 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US20030021809A1 (en) * 1992-08-26 2003-01-30 Chisari Francis V. Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US20040241143A1 (en) * 1992-08-26 2004-12-02 Chisari Francis V. Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US6919203B2 (en) 1992-08-26 2005-07-19 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US20050232935A1 (en) * 1992-08-26 2005-10-20 Chisari Francis V Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US7368118B2 (en) 1992-08-26 2008-05-06 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
WO2019216603A1 (en) * 2018-05-09 2019-11-14 서울대학교산학협력단 Hepatitis b virus-derived polypeptide and anti-viral use thereof
KR20190128999A (en) * 2018-05-09 2019-11-19 서울대학교산학협력단 Hepatitis b virus-derived polypeptide and use thereof
KR102234027B1 (en) 2018-05-09 2021-03-31 서울대학교산학협력단 Hepatitis b virus-derived polypeptide and use thereof
US11692011B2 (en) 2018-05-09 2023-07-04 Seoul National University R&Db Foundation Hepatitis B virus-derived polypeptide and anti-viral use thereof
WO2022035246A1 (en) * 2020-08-14 2022-02-17 서울대학교산학협력단 Immune adjuvant comprising hepatitis b virus-derived polypeptide

Also Published As

Publication number Publication date
US6607727B1 (en) 2003-08-19
US7744898B2 (en) 2010-06-29
US20060051746A1 (en) 2006-03-09

Similar Documents

Publication Publication Date Title
US7744898B2 (en) Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US5780036A (en) Peptides for inducing cytotoxic T lymphocyte responses to hepattis B virus
EP0726758B1 (en) Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus
US7368118B2 (en) Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
AU695261B2 (en) Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
JP3694300B2 (en) Peptides that induce the response of cytotoxic T lymphocytes to hepatitis B virus
US5840303A (en) Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
CA2156416C (en) Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus
CA2594184A1 (en) Peptides for inducing cytotoxic t lymphocyte responses to hepatitis b virus
AU9240198A (en) Peptides for inducing cytotoxic T lymphocyte responses to Hepatitis B virus

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:SCRIPPS RESEARCH INSTITUTE;REEL/FRAME:021726/0872

Effective date: 20030818