AU9240198A - Peptides for inducing cytotoxic T lymphocyte responses to Hepatitis B virus - Google Patents

Description

i i S F Ref: 311005D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECFICATION FOR A STANDARD PATENT

ORIGINAL

Name- an Adrs Name and Address of Applicant: Actual Inventor(s): 51* ii.

The Scrlpps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 UNITED STATES OF AMERICA Francis V Chisarl Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Peptides for Inducing Cytotoxic T Lymphocyte Responses to Hepatitis B Virus Address for Service: 1

L

Invention Title: S 3 The following statement is a full description best method of performing it known to melus:of this invention, including the i PEPTIDES FOR INDUCING CYTOTOXIC T LYMPHOCYTE RESPONSES TO HEPATITIS B VIRUS Government Support The U.S. Government may have certain rights in this invention pursuant to grants awarded by the National Institutes of Health.

15 Background of the Invention Cytotoxic T lymphocytes (CTLs) play an essential role in fighting cells infected with viruses, intracellular I bacteria and parasites, and tumor cells. They do so by direct 0i cytotoxicity and by providing specific and nonspecific help to 20 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 Speptides bound to HLA class I molecules to T cell receptors on CTLs. MHC class I molecules can also bind exogenous peptides S* and present them to CTLs without intracellular processing.

I At the present time it is difficult to accurately predict from the sequence of an antigenic protein how the A protein will be processed and which peptide portions will bind S 30 HLA class I molecules and be presented to CTLs. Binding m* otifs have been predicted for some HLA class I molecules ry based on sequence analysis of peptides eluted from these molecules (Falk et-al., Nature 351:290 (1991)). Further, of the peptides that are processed and do bind to HLA class I, which ones will contain CTL-recognizable epitopes is not vet predictable.

Hepatitis B Virus is a non-lytic virus which has currently infected approximately 250 million people worldwide. HBV infection in adults tvnically leads to an w d:id l a an 4 P ta~Rlla~e~oe~L esa~cs+r~ I rv~- l~ 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 Spercentage of individuals who are infected with HBV in adulthood develop fulminant hepatitis associated with a strong immune response with high lethality.

While there is no effective treatment for HBV infection, vaccines have been developed in recent years to prevent HBV infection. The vaccines employ either HBV surface antigen (HBsAg) purified from the plasma of chronic HBV Scarriers, or HBsAg produced by recombinant DNA technology.

S Synthetic HBsAg peptide-based vaccines have also been 15 proposed. See, for example, U.S. Patent Nos. 4,599,230 and 4,599,231. The anti-HBsAg vaccines, however, afford S protection in only about 90% of immunized individuals. Those i who are unimmunized, or immunized but unprotected, provide a significant reservoir of potential infection.

The contribution of CTLs to immunity to HBV antigens has been difficult to assess. Chisari et al. (Microbial Pathogen. 6:31 (1989)) have suggested that liver cell injury may be mediated by an HLA-Class I restricted, CD8' cytotoxic T cell response to HBV encoded antigens. Class I major S* 25 histocompatibility (MHC) -restricted cytotoxic T lymphocyte S* responses have been identified for a variety of other viruses, Ssuch as influenza. For example, Townsend et al., Cell 44:959 (1986) reported that epitopes of an influenza virus S nucleoprotein recognized by cvtotoxic T lymphocytes could be defined by synthetic peptides. In attempting to define the cytotoxic T lymphocyte response to HBV, it has been shown that Speripheral blood lymphocytes from patients with acute and chronic HBV may be able to kill autologous hepatocytes in i vitro, but the specificity of the cytolytic activity, its HLA I 35 restriction elements, and cellular phenotype were not established. See, Mondelli et al., J. Immunol. 129:2773 S(1982) and Mondelli et al., Clin. Ex. 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 HC class I-restricted, CD8 CytOtoxiC

T

lymphocytes.

As there is a large reservoir of individuals chronically infected'with HEV, it would be desirable to stimulate the immune response of these individuals to respond to appropriate HBV antigens and thereby eliminate their infi-aticn. It would also be desirable to prevent the evolution to a chronic irnV infection inl individuals suffering from an acute phase infection. Further, as the presently approved HEV 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 15 diversifying the immunogenicity of the vaccines. Quite surprisinglyr the present invention fulfills these and other .:related needs.

Smay ofthe invention The present invention provides peptides which induce NUC class I restricted cytotoxic T lymphocyte responses against HBV antigen. The peptides of interest are derived from the HBV envelope. In certain embodiments the CTL inducing peptide will have the sequence HBenvlS3-191 Phe-Leu- Le-h-r-l-e-h-l (Seq. ID No. HBenv248-257 Phe- (Seq. ID No. 3); HBenv2 49-257 Il-e-e-o-y-e-l-h-e (Seq. ID No.

H~env249-258 Ile-LueuLeus-Lu.IlePh-e-e (Seq.

ID No. 5) HBeflv250- 2 5 8 Le-e-e-y-euIePeLe-e 430 (Seq. ITD No. ff~env251-25g a-y-euIePeLulu Val (Seq. ID No. HBenv25l-260 Leu-LeuCys-ieu...le..PheLu- Leu-Val-Leu (Seq. ID No. ffBenv6-269 Leu-LeuAsp-r.Gn.

Gly-Met-Leu-Pro..al (Seq. ID No. HBenv335-3 4 3 Trp-Leu-Ser- Leu-Leu-Val..Pro-.Phe..Val (Seg. ID No. 10), HBenvl52.1 6 1 Ser- Ile-Leu-SerLysThr.Gly.AsPoVl (Seq.- ID No. 11); *HBenv177-185 Va-pudnAaGl-h-h-e-a (Seq. ID NO.

12); HB4-1 Phe-LeuCly..ly...hr.ProVl-Cs u(a.

*No. 13); or HBenv370-379 Ser-IJle-Val-Ser.Pro-Phe-IlePoLu IP7- 4 Leu (Seq. ID No. 14); or will have a sequence substantially homologous to one of the foregoing sequences. The peptide can be optionally flanked and/or modified at one or both of the Nand C-termini, as desired. Conservative substitutions, deletions and additions may be made at non-critical residue positions within the selected peptide without substantially adversely affecting its biological activity.

In the various peptide embodiments it will be understood that the peptides can be polymerized, each to itself to form larger homopolymers, or with different peptides to form heteropolymers. In some instances peptides will be combined in a composition as an admixture and will not be linked. The peptide can also be conjugated to a lipidcontaining molecules capable of enhancing a T lymphocyte response, or to a different peptide which induces a T-helper cell response, for example.

Compositions are provided which comprise a peptide of the invention formulated with an additional peptide, a S 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 S"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 25 pharmaceutical compositions are administered to infected individuals in amounts sufficient to stimulate immunogenically effective cytotoxic T cell responses against HBc epitopes.

S For treating these infections it may be particularly desirable to combine the peptides which induce MHC class I restricted cytotoxic T lymphocyte responses against HBV antigen with other peptides or proteins that induce immune response to other HBV antigens, such as HBV core. To treat individuals with chronic or carrier state infections the compositions may Sbe 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 envelope peptide mentioned above which induces a MHC class I restricted cytotoxic T lymphocyte response, such as HLA-A2, 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 HBV envelope antigen.

In yet other embodiments the invention relates to methods for diagnosis, where the peptides of the invention are used to determine the presence of lymphocytes in an individual which are capable of a cytotoxic T cell response to HBV envelope antigen.

io The absence of such cells determines whether the individual of interest is susceptible to S.developing chronic HBV infection. Typically the lymphocytes are peripheral blood lymphocytes and the individual of interest is suffering from an acute HBV infection.

According to a first embodiment of the invention, there is provided a CTL inducing peptide comprising from eight to thirteen amino acids, wherein at least a majority of the S is amino acids are homologous to a corresponding portion of HBenv having the sequence: HBenvl83-191 (Seq. ID No. 1).

According to a second embodiment of the invention, there is provided 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 HBenv having the sequence: HBenv248-260 (Seq. ID No. 2) Phe-Ile-Leu-Leu-Leu-Cys-Leu-Ile.-Phe-Leu-Leu-Val-Leu.

According to a third embodiment of the invention, there is provided a method for treating or preventing hepatitis B infection, which comprises administering an effective 25 amount of a peptide to a host having an HLA-A2 haplotype, wherein the peptide is a CTL inducing peptide comprising the following sequence: HBenv183-191 (Seq. ID No. 1) Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile.

According to a fourth embodiment of the invention, there is provided a method for S 30 treating or preventing hepatitis B infection, which comprises administering an effective amount of a peptide to a host having an HLA-A2 haplotype, wherein the peptide is 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 HBenv having the sequence: HBenv248-260 (Seq- ID No. 2) Ph-i-e-uLuCsLuIePeLuLuVlLu or HBenv335-343 (Seq. ID No. S Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val.

According to a fifth embodiment of the invention, there is provided an immunogenic composition comprising the peptide according to the first or second embodiments of the invention, and a second immnunogenic peptide.

Brief Description of the Drawings Fig. I shows that HBsAg335-343, WLSLLVPFV, is the minimal optimal CTL epitope recognized by CIL stimulated HBsAg329-348. A CTL clone from patient A-I a nd a CTL cloned line from patient A-3, generated by stimulation with HBsAg329-348, were tested against JY target cells prepulsed either with truncations (upper panels) or with *overlapping 9-mers or lO-mers (lower panels) covering HBsAg329-348- Fig. 2 further confirms that an optimal epitope within H4BsAg329-348 for in vitro CTL induction is HBsAg335-343.

A Fig. 3 shows the HBV specific CTL response in patients with acute hepatitis B infection, chronic hepatitis B infection, and normal subjects. PBIVC from. acute patients (A-i to A-12), chronic patients (C-i to and normal subjects (N-I to N-6) were 2o stimulated with the following synthetic peptides: I-HBcAgL18-27, 2=HBsAg20I-21O, 3=llBsAu-25l-259, St2ii 4RBsAg26O-269, 5 -HBcAg335-343r 6liBSAg338-347, 7=HIBSAg348-357, 8=HBsAg378-.387.

Fig- 4 shows the results of HLA-A2.l competitive binding inhibition assays, represented as inlhibition of H~cAgl8-27 specific lysis in a 4 hour 5 1 Cr release assay.

Fig. 5 illustrates that the CTL response to H:BsAg35-343 and HBSAg348-357 are group specific and subtype specific, respectively, and that the synthetic peptides contain epitopes; that are also generated by the endogenous processing of the large, middle and major HBV envelope polyp eptides within infected cells.

Descrintlon of the Specific Embodiments The present invention provides peptides; derived from HBV envelope proteins for use in compositions and methods for *-the treatment, prevention and diagnosis of REV infection. The peptides stimulate NRC RLA-class I restricted cytotoxic

T

lymphocyte respo nses against HEV 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 cytotoxjc

T

cell response may also be combined with an immunogen capable 25 of eliciting a T-helper response.

The peptides employed in the invention are derived from the regions of HBenvls3-191 (Seq. ID No. HBenv248-260 (Seq. ID No. H~env260-26 9 (Seq. ID No. HBenv335-343 (Seq. ID No. 10), ffBenvl52-l6l (Seq. ID No-. 11), H~env177-185 (Seq. ID No. 12), HBenv2o4-21 2 (Seq. ID No. 13), and H~env37O-379 (Seq. ID No. 14), where, the numbering is according to Galibert at al., supra.

By HBV Cytotoxic T lymphocyte inducing "'peptide" or "oligopeptide" of the present invention is meant a chain of at least four REV 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 fever than about thirty-five, and preferably rrl2~Ba*~c~rrra~Eh Ifewer than twenty-five, 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, commensurate in size with endogenously processed viral peptides that are bound to MHC class I molecules on the cell surface. See generally, Schumacher et al., Nature 350:703-706 (1991); Van Bleek et al., Nature 348:213-216 (1990); Rotzschke et al., Nature 348:252-254 (1990); and Falk et al., Nature 351:290-296 (1991), 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 sequences identified herein, and containing a CTL-inducing epitope.

'The peptides can be prepared "synthetically," as described hereinbelow, or by recombinant DNA technology.

Although the peptide will preferably be substantially free of other naturally occurring HBV proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles. The term peptide is used interchangeably with polypeptide in the present S" specification to designate a series of amino acids connected one to the other by peptide bonds between the alpha-amino and 25 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 chaii oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypaptides as herein described.

Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. By biological activity is I- meant the ability to bind an appropriate MHC molecule and induce a cytotoxic T lymphocyte response against HBV antigen Sor antigen mimetic. By a cytotoxic T lymphocyte response is 4. meant a CD8' T lymphocyte response specific for dn JEBV antigen of interest, wher'ein CD8', MHC class I-restricted

T

lymphocytes are activated. The activated T lymphocytes secrete ivephokines gamma interferon) liberate products 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 501 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 por-tions of the reference sequence.

CTL-inducing HBV peptide embodiment of the invention from the nucleocapsid region comprises from six to *thirty-five amino acia-' and contains at least one HIArestricted CTL epitopir- site from the peptide region HBenvlS3-191 (Seq. ID No. 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 HBenvl8 3-191 region, where HBenvla3-191 has the sequence: HBenvl83-191 (Seq. ID No. 1) l~eLuLuTrAr-i-e-h-l The peptide embodiments of this HBenvl83-19l region can be optionally flanked and/or modified at one or both of the Uand C-termini, as desired, by amino acids from HBV sequences, including HBc, amino acids added to facilitate linking, other 30 o N- and C-terminal modifications, linked to carriers, etc., as further described herein. The peptide H7BenvlB3-.lgl induces a cytotoxic T lymphocyte response which is mediated by at least the MIIC class I molecule HLA-A2.

Other HBenv peptide embodiments of the invention are prepared from the region of HBenv248-260. Peptides, derived from this region contain at least one CTL-inducing HIA class !-restricted epitopic site, and will typically be at least seven amino acids, more usually nine, ten or eleven amino meant a CO8'~ T lymphocyte response specific for an IIBV antigen Of interest, wherein CD8', MXC class I-restricted

T

lymphocytes are activated. The activated T lymphocytes secrete lymphokines gamma interferon' liberate products serine esterases) that inhibit viral replication in infected autologous cells or transfected cells, with or without cell killing.

The terms "homologous", "substantially homologous", and "substantial homology" as used herein denote a sequence of amino acids having at least 50% identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.

A CTL-.inducjng HBV peptide embodiment of the invention from the nucleocapsid region comprises from six to thirty-five amino acids and contains at least one liLArestricted CTL epitopic site from the peptide region HBenvl83-191 (Seq- ID No. A majority of the amino acids 20 of the peptide will be identical or substantially homologous to the amino acids of the corresponding portions of the naturally occurring HBenvl83-191 region, where H-BenvlS3-191 has the sequence: HBenvl83-191 (Seq. ID No. 1) 25 Ph-e-e-h-rgIeLe-h-l The peptide embodiments of this HBenvlaj-191 region can be A optionally flanked and/or-modified at one or both of the Nand C-termini, as desired, by amino acids from HBV sequences, including H~c, amino acids added to facilitate linking, other.

46 30 and C-terminal modifications, linked to carriers, etc., as further described herein. The peptide HBenvla3-lgl induces a cotaxic T lyphye rsoewhcismdiated by at least the. MHiC class I molecule HLA-A2.

other li~env peptide embodiment* of the invention are 3_prepared from the region of HP4env248-260. Peptides derived from this recio cnan tlesoeC-inducing HLA class .I-restricted epitopic site and will typically-be at least seven amino acids, more, usually nine, ten or eleven amino 9 acids or more. A majority of the amino acids of the peptide will be identical or substantially homologous to the amino acids of the corresponding por-tions of the naturally occurring HBenv248-260 sequence, where.HBenv248-260 has the sequence .(for HBV subtype ayv): HBenv248-260 (Seg. ID No. 2) Pb-l-e-e-e-y-euIePeLuLuVlLu The peptide from the HBenv24S-260 region can be flanked and/or modified at neo botEh 'crmini as described herein.

Representative CTL-inducing paptides prepared from the region of HBenv248-260 include the following 9- and lO-mer sap-.:peptides: HBenv248-257 (Seq. ID No. 3) Ph-l-e-e-e-CsLuIePeLu *so* HBenv249-257 (Seq. ID-No. 4) .*~*HBenv249-258 (Seq. ID No. Il*e-. .eu sLe-l-PeLu--u HBenv25O-258 (Seq. ID No. 6) Le-e*LuCs-e-lePe*e-~u **HBenv25l-259 (Seq. ID No. 7) HBenv25l-6 (Seq. ID No. 8) 25 u-Leu-Cys -Leu-Ile- Phe-Leu-Leu-Val -Leu; The foregoing peptides contain a HLA-restricted

CTL-

inducing epitope, typically at least HLA-A2 restricted, and can be flanked and/or modified at one or both termini as mentioned for peptide I above.

*In a further embodiment, a peptide of the invention comprises the 1O-mer peptide HBenv260-269, and peptides derived from HBenv26O-26g which contain a CTL-inducing

HLA

class P--restricted apitopic site(s) of at least seven contiguous amino acids. 'A majority of the amino acids of the peztlde will be identical or substantially homologous to the amino 'acids of the corresponding portion& of the naturally Vi occurring HBenv260-269 sequence, where HBenv26O-269 has the sequence: HBenv260-269 (Seq. ID No. 9) LEU-Leu-Asp-Tyr-Gln..Gly..Het..qeu-.Pro-Val.

A peptide prepared from this region can be flanked and/or modified at one or both termini as described herein.

The peptide HBenv260-269 (Seq. ED No. 4) induces a cytotoxic T lymphocyte response which is mediated by at least the NHC class I HLA-A2 molecule.

Yet other CTL-inducing peptides of the invention are from the region of HBenv335-343 (Seq. ID No. 10), and includes peptides derived from HBenv33S-343 (Seq. ID No. 10) which contain one or more CTL-inducing HLA class I-restricted sic~ 0 piopc it 's of at least seven contig-uous amino acids. A majority of thle amino acd fteppiewill b dnia C it or substantially homologous to the amino acids of the crepnigportions of the ntrlyoccurring HBanv335-343 sequence, where HBenv335-34,3-has the sequence: S. ~.HBenv335-343 (Seq. ID No. wherein the selected peptide can be flanked and/or modified at 0 SOone or both termini as described herein.

P.Yet other CTL-inducing peptides of the invention are from the region of HBenVl52-161 (Seq. ID No. 11), and includes S 25 peptides derived from.HBenvl52-16l (Seq. ID No. 11) which contain one or more CTL-inducing HL-hAclass I-restricted *Sepitopic site,(s) fatleast seven contiguous amino acids. A smajority of-the amino acids of the peptide will be identical 9 5or substantially homologous to the amino acids of the S 30 corresponding portions of the naturally occurring HBnvl52-161 sequence, where HB~avI52-161 has the sequence (adw subtype]): HBenvl52-161 (Sea. ID No. 11) erIe-e-srLy-Th-lyAp-r-Vl wherein the'selected peptide can be flanked and/ or modif ied at one or both termini as described, herein. 'Other.CTL-inducing p eptides o.f the inrvention are from the region-Of -HBbnvl77-185,_(seq. ID No. 12), and includes peptides deriVed from HBenVl777185 (Seq. ID No. 124hc 12)whch 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 HBenvl77-185 sequence, where H-Benvl77-185 has the sequence (adw subtype): HBenvl77-185 (Seq. ID No. 12) Val-Lu-Gln-Ala-Gly-Phe-Phe-eu-eu, wherein the selected pept~.de can be flanked and/or modified at one or both termini as described herein.

Additional CTL-inducing peptides of the invention are from the region of H~enV2Q4-212 (Seq. ID No. 13), and includes peptides derived from HBenv204-212 (Seq. ID No. 13) which contain one or more CTL-inducing HLA class I-restricted epitopic site(s) of at least seven contiguous amino acids. A *9 Smajority 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 H~anv2O4-212 94:sequence, where H~env2O4-212 has the sequence (adw subtype); HBenv204-212 (Seq. ID No. 13) Phe-Leu-Gly-Gly-Thr-Pro-Val-Cys-Leu, wherein the selected peptide can be flanked and/or modified at *.one or both termini as described he::ein.

J" AdtolCT-inducing peptides of the invention are 25 from the region of *HBenv370-379 (Seq. ID No. 14), and includes peptides derived from HBenv370-379 (Seq. ID No. 14) which contain one or more CTL-inducing HLA class I-restricted site(s) of at least seven- contiguous amino acids. A *a or 9o h m n c f th e t d i l b d n i a or. substantially homologous to the amino acids of the corresponding portions of the naturally occurring HBenv370-379 *9 sequence, where HBenv370-379 has the sequence (adw subtype): HBenv370-7 Sq ID No. 14) wherein the selected peptide can be flanked and/or modified at -one or ,both termini as described herein.

As,.mentioned above, additional amino acids can be added to: the termini of an -oligopeptide..or peptide to provide 41 12 for ease of linking peptides one to another, for coupling to a carrier, support or a larger peptide, for.reasons discussed herein, or for modifying the physical or chemical properties of the peptide or oligopeptide, and the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the c- or N-terminus of the peptide or oligopeptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-1Hz acylation, acetylation, or thioglycolic acid amidation, terminal-carboxy amidation, 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 15 present invention or analogs thereof which have cytotoxic T lymphocyte stimulating activity may be modified as necessary to provide certain other desired attributes, improved o pharmacological characteristics, while increasing or at least retaining substantially the biological activity of the 20 unmodified peptide. For instance, the peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, 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 S* 25 herein. The peptides may be modified to substantially enhance Sthe CTL inducing activity, such that the modified peptide Sanalogs have CTL activity greater than a peptide of the wildtype sequence. For example, it may be desirable to increase the hydrophobicity of the N-terminal of a peptide, 30 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 1 35 antigens, particularly those containing CTL inducina eoitopes f or which a host may not have significant CTL activity, may be made CTL-inducing by substitutin hyvdrohobic residues at the i «8a a^ 33 N-terminus of the peptide where the second residue is normally hydrophobic.

The Deptides emploDyed in the subject invention need not be identical to peptides HBenVI83-191 (Seq. ID Nc,. 1), HBenv248-257 (Seq. ID No. HBenv249-257 (Seq. ID No. 4), HBenv249-258 (Seq. ID No. HBenv250-258 (Seq. ID No. 6) HBenV25l-259 (Seq. ID No. kIBenv251-260 (Seq. ID No. 8), HBenv26O-269 (Seq. ID No. HBenv335-343 (Seqr. ID No. HBenvl52-161 (Seq. ID No. 11), HBenvl77-185 (Seq. ID No. 12), HBenv2O4-212 (Seq. ID No. 13), or HBenv370-379 (Seq. ID No.

14), so long as the subject compounds are able to provide for c'ftotoxic T lymphocytic activityaaisatlatoeoth four major subtypes of HBV. Although different strains of HBV exist, they each share at least one common envelope determinant, which is deinae all. Each strain also has two other envelope determinants, one of which is either I'd" or "yand the second is either or "r.Thus, there are tour possible subtypes of the virus: adw, ayw, adr, and ayr.

.,:The cloning, sequencing and expression of HBV are described in GB 2034323, EP 13828, U.S. 4,935,235, and the complete secruence of the EiBV envelope region is also described in Galibert et al., Nlature 281:646 (1979), each of the foregoing S **being incorporated herein by reference. Amino acid sequences are described in the GenBank-72 database for 20 different HBV 25 strains, including 7 of the adw subtype, 5 of the ayw subtype, 7 of the adr subtype, and 1 strain of teayr 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, 20 either conservative-or non-conservative, where such changes provide for certain advantages in their use. By conservative Ssubstitutions is meant replacing an amino acid residue with *another which is biologically and/or chemically similar, oiie-hydrophobic residue for another, or one polar residue for another. ;The substitutions include combinations such as Gly, Al;V-1, n: s,.1u. Asn, 11n; Ser, :hr; -vs, Arq; and-Phe,, Uy. :sually, the portion of the sequence which is intended to substantially mimic an HBV cytotoxic T lymphocyte 14 stimulating epitope will not differ by more than about 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, ease of linking or coupling, and the like. Where regions of the peptide sequences are found to be polymorphic among HBV subtypes, it may be desirable to vary one or more particular amino acids to more effectively mimic differing cytotoxic T-lymphocyte epitopes of different HBV strains or subtypes.

Within the peptide sequences identified by the present invention, including the representative peptides listed above, there are residues (or those which are substantially functionally equivalent) which allow the peptide 15 to retain their biological activity, the ability to stimulate a class I-restricted cytotoxic T-lymphocytic response against HBV infected cells or cells which express HBV S antigen. These residues can be identified by single amino acid substitutions, deletions, or insertions. In addition, 20 the contributions made by the side chains of the residues can be probed via a systematic scan with a specified amino acid Ala). Peptides which tolerate multiple substitutions generally incorporate such substitutions as small, relatively *0 neutral molecules, Ala, Gly, Pro, or similar residues.

S 25 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 hydrophobicity vs. hydrophilicity). If desired, 30 increased binding affinity of peptide analogues to its MHC molecule for presentation to a cytotoxic T-lymphocyte can also be achieved by such alterations. Generally, any spacer substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid steric and charge interference which *might disrupt binding.

Peptides which tolerate multiple substitutions while retaining the desired biological activity may also-be 4 won~ 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.

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 infectad or uninfected individuals and exposing (stimulating) the cells with synthetic peptide or polypeptide fragments derived from a peptide region of HBenvl83-191 (Seq. ID No. HBenv248-260 (Seq. ID No. 2), HBenv260-269 (Seq. ID No. HBenv335-343 (Seq. ID No. HBenvl52-161 (Seq. ID No. 11), HBenv177-1a5 (Seq. ID No. 12), 25 HBenv204-212 (Seq. ID No. 13), or HBenv370-379 (Seq. ID No.

14). Pools of overlapping synthetic peptides, each typically about 8 to 20 residues long, preferably 9-12 residues, can be used to stimulate the cells. Active peptides can be selected from pools which induce cytotoxic T lymphocyte activity. The 30 ability of the peptides to induce specific cytotoxic activity is determined by incubating the stimulated PBL with autologous labeled 5Cr) target cells (such as HLA matched Snacrophages, T cells, fibroblasts or B lymphoblastoid cells) infected or transfected with the HBV subgenomic fragments 35 thereof, such that the targeted antigen is synthesized endogenously by the cell (or the cell is pulsed with the S; peptide of interest), and measuring specific release of label.

I 16 Once a peptide having an epitopic region which stimulates a cytotoxic T lymphocyte response is identified, the MHC restriction element of the response can be determined.

This involves incubating the stimulated PBL or short term lines thereof with a panel of (labeled) target cells of known HLA types which have been pulsed with the peptide of interest, -or appropriate controls. The HLA allele(s) of cells in the panel which are lysed by the CTL are compared to cells not lysed, and the HLA restriction element(s) for the cytotoxic

T

lymphocyte response to the antigen of interest is identified.

Carbone et al., J. Exp. Med. 167:1767 (1988), have reported that stimulation with peptides may induce cytotoxic T lymphocytes with low affinity for corresponding endogenous protein, such that repetitive peptide stimulation may yield 15 cytotoxic T lymphocytes that recognize peptide but not native antigen. As the inability of stimulated cytotoxic T Slymphocytes to recognize native HBV proteins would be S* undesirable in the development of HBV peptide therapeutics and Svaccine 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, KBsAg.

.ells having activity are also stimulated with an appropriate T cell mitogen, phytohemagglutinin (PHA). The restimulated cells are provided with irradiated allogeneic 30 PBLs as an Pntigen 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 Sstably express:the corresponding HBV antigen polypeptide. The Scell lines are re-confirmed for the ability to recognize S; 'endogenously synthesized antigen using autologous and rtj

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*r S 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, the HBenv248-257 (Seq. ID No. HBenv249-257 (Seq. ID No. HBenv249-258 (Seq. ID No. HBenv250-258 (Seq. ID 15 No. 6) HBenv251-259 (Seq. ID No. and/or HBenv251-260 (Seq. ID No. 8) peptides, which peptides can be combined in a "cocktail" to provide enhanced immunogenicity for cytotoxic T lymphocyte responses. Moreover, peptides of one region can be combined with peptides of other HBV regions, from the same or different HBV protein, particularly when a second or subsequent peptide has a MHC restriction element different from the first. Other CTL-inducing HBV peptides are described in co-pending application USSN 07/935,898, which is 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.

:r i, j- 1

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i£ iiv j 17 allogeneic B-lymphoblastoid or other cells transfected or infected with appropriate antigen.- Having identified different peptides of the invention which contribute to inducing anti-HBV cytotoxic T lymphocyte S 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 epitnpes from a particular region, the HBenv248-257 (See. ID No. HBenv249-257 (Seq. ID No. HBenv249-258 (Sea. ID No. HBenv25O-258 (Seq. ID 15 No. 6) HBenv25l-259 (Seq. ID No. and/or HBenv251-260 *(Seq. ID No. 8) peptides, which peptides can be combined in a "cocktail" to provide enhanced immunogenicitv for cytotoxic T lymphocyte responses. Moreover, peptides of one region can be S* combined with peptides of other HBV regions, from the same or 20 different k{BV protein, particularly when a second or subsequent peptide has a MHfC restriction element different from the first. Other cTL-inducinq HBV peptides are described in co-pending application USSN 07/935,898, which is incorporated herein by reference., This composition of 25 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 *o.:population. For example, the different frequencies of J!LA alleles among prevalent ethnic groups (caucasian, asian and 3 0 african blacks) are shown 4n Table 7 below. Therapeutic or vaccine compositions of the invention may be formulated to provide potential therapy or imuiyto as high a percentage l of a population as possible.

is TABLE 1. lILA ALLELE FREQUENCIES AMONG PREVALENT ETHNIC GROUPS lILA Allele EUC NAC 5E PN 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 15.31 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.

S

4*t* t.

*t 4.

4* *4 *44* at 4 4* Ge 4*

C

C

o C Co

C

S.

Abbreviations: EUC, European Caucasian; HAC, North American Caucasian; AFR, African blacks, JPH, Japanese.

A28 represents the two alleles Aw68 and Aw69 The peptides of the invention can be combined via linkage to form polymers (multimers), or can be formulated in a composition without. linkage, as an admixture. Where the 20 same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, a cocktail representing different HBV subtypes, different epitopes within a subtype, different HIA restriction specificities, a peptide 25 which contains T helper epitopes, heteropolymers with repeating units are provided. in addition to covalent linkages, noncovalent linkages capable of formi-ng intermolecular and intrastrucrural bonds are included.

Linkages for homo- or hetero-polymers or for coupling 30 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 35 a disulfide link at one functional group end and a peptide link at the other, including N4-succidimidyl-3-(2-pyriiyldithio) 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 othp~r. -A variety of such disul fide/amide furming agents are known. See, for example, Immun. Rev. 6215(1982), which is incorporated herein by reference. Othpar bifunctional coupling agents form a thioether ratheir tha a disulfide linkace. pEany &F ts 19 thioether forming agents are commercially available and include reactive esters of 6-maleimidocauroic acid, 2 bromoacetic acid, 2-jodoacetic 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-maleimidometliyl) cyclohexane- 1-carboxylate (SMCC). It will be understood that linkage should not substantially.

interfere with either of the linked groups to function as described, as an HBVI cytotoxic T cell determinant, peptide analcas, 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 epitaves, apitopes which stimulate T cells eve. that cooperate in the induction of cytotoxic T cells to HBV.

The T-helper cells can be aither the T-helper 1 or T-helper 2 phenotype, for example. T-helper epitopes from HBV sequences have been identified at HBal-20, having the secuence: Met-As)-.

Phe-Leu-Pro (Seq. !D mo- 17). -other T-helper epitopes are provided by peptides from the region HBc50-69, having the *.seauence Pr-i-i-lrAaLuAa-GnAaieLuCsTp S.Gly-Glu-Leu-Met-Tyr-Eeu.Ala (Sea. ID No. 18), and from the **25 region of HBc100-139, including HBclOO-ll9 having the sequence *Leu-LuTp-PheH IleSerCysLuTlr-PheGlyAr..Glu-Thr *Val-Ile-Glu-Tyr-Leu (Sea. ID No. 19) (where Ile, 1 is Lau in the HiBV adw subtype), H~cI17-1-31 having the sequence Glu-Tyr- Le-a-e-h-l- a-rmIeAgTI.-r-r-l (Sea- 1D No- 20), and peptide HiBcl20-l39 having the secruence Val-Ser- Ph--l-a-r-l-r-h-roPoAaTrAgPoPoAn *SAla-Pro-Ile (Sea.. ID No. 21). Se, Ferrari-et. al., J. Cin.

Invest. 88:214-222 (1991), and Pat- 4,882,145, each incorporated herein by reference.

peptides of the invention can be u~remaredia wide variety of :favs- 3ecause of their relati-l hr ie the vewtides ca n be synthesized in solution &rca a lid support- In accordance with -conventionai .techn cmes. various

W.

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C

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t0 S a.

4.

4 4* *4*1 a 4.

4* .54~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 ca. (1984) Tam et al., J. Am. Chem. Sac.

105:6442 (1983)~ Merrifield, Science 232:341-347 (1985); and Bairany ajid -:7errifieid, TIe Peoride, Gross and 1M-ejenhofer, ads. Academic Press, New York, pp. 1-284 (1979), each of which is incorporated herein by reference.

Alternatively, recombinant DNTA technology may be employed wherein a nucleotide seqruence which encodes a peptide.

of interest is insert-ed into an expression vector, transformed or transf-ected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art-, as described generally in 15 Sambrook et al., M!olecular cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), and Ausbel t al., fed.) Current Protocols in Molecular Bioloa, John Wiley and Sons, Inc., New York (1987), and U.S. Pat. Nos.

4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941, for 20 exampnle, whose disclosures are each incorporated herein by reference. Thus, f-usion rroteins which comsorise one or more peptide secuences of the invention can be used tz resent- the HBV cyt-otoxic T cell determinants. or exampmie, a recombinant envelTooe protein of the invention is prepared in whtich, the 25 HBenv amino acid seoruence is altered so as to more effectIVely present epitcpes of peptide regions described herein to stimulate a cvtotoxic .T lymphocyte response- By this means a polypeptide is used which incorporates several T cell epitones 306 As the codingr secuence for pntides of the length contemplated herein can be synthesized by chemical techniques.

for examule. zhe nhosmhotriester method of Matteucci et al., J. An. Citem. Soc. 103:3185 (1981), modification can be made simply by substitutina the appropriate base(s) fOr those encodin the native vntide sequence. he codina sauence can :then ba provided witch anuronriate linkers and licated into expression vectors commonly available in t-116'art, aznd the vetcors used to transform. suitable hosts to produce the !if1~ ft ft...

r4.

*5 ft a f fto 4 4* ft. f ft.

4 *5

S

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.

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., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York (1987), and U.S. Pat. Nos.

4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941, for 20 example, whose disclosures are each incorporated herein by reference. Thus, fusion proteins which comorise one or more peptide sequences of the invention can be used to present the HBV cytotxic T cell determinants. For example, a recombinant envelope protein of the invention is prepared in which the 25 HBenv amino acid sequence is altered so as to more effectively present epitopes of peptide regions described herein to stimulate a cytotoxic T lymphocyte response. By this means a polypeptide-is used which incorporates several T cell epitopes.

30 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., SJ. Am. Chem. Soc. 103:3185 (1981), modification can be made simply by substituting the appropriate base(s) for those 35 encoding the native peptide sequence. The codina sequence can then be provided with appropriate linkers and ligated into expression vectors commonly, available in the art, and the S vectors used to transform suitable hosts to produce the i; o. *s i' i 0 *t *a

L-

~i 21 desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.

The peptides of the present invention and pharmaceutical and vaccine compositions thereof are useful for S- 15 administration to mammals, particularly humans, to treat g and/or prevent HBV infection. As the peptides are used to f stimulate cytotoxic T-lymphocyte responses to HBV infected t cells, the compositions can be used to treat or prevent acute 3 and/or chronic HBV infection.

For pharmaceutical compositions, the peptides of the S''invention as described above will be administered to an individual already infected with HBV. Those in the incubation d4 phase or the acute phase of infection can be treated with the Simmunogenic peptides separately or in conjunction with other a: 25 treatments, as appropriate. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective cytotoxic T lymphocyte A response to HBV and to cure or at least partially arrest its symptoms and/or complications. An amount adequate to V o 30 accomplish this is defined as "therapeutically effective Sdose." 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 a-nd general state of health of the patient, and the judgment 35 of the prescribing physician, but generally range from about 1 S agto aSou t 2.000m of -peptide for a 70 k patient, with '8 t I dosa es. offrom about .10 g to about 100 mg of peptide being Smore commony used, followed by booster dosages from about 1

KI-

F

P

E

*r 01 9a 4 ig to about 1 mg of peptide over weeks to months, depending on a patient's CTL response, as determined by measuring HBVspecific 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 paptide compositions.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of cytotoxic T-lymphocyte stimulatory peptides of the invention sufficient to effectively treat the patient.

For therapeutic use, administration should begin at the first sign of HBV infection or shortly after diagnosis in 20 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 30 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.

S35 iWhere the susceptible individuals: are identified prior to or S during infection, for instance, as described herein, the i :composition can be targeted to them, minimizing need for S -administration to a larger population.

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i .a 23 The peptide compositions can also be used for the treatment of chronic hepatitis and to stimulate the immune system of carriers to substantially reduce or even eliminate virus-infected cells. Those with chronic hepatitis can be identified as testing positive for virus from about 3-6 months after infection. As individuals may develop chronic HBV infection because of an inadequate (or absent) cytotoxic T lymphocyte response during the acute phase of their infection, it is important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response. Thus, for treatment of chronic hepatitis, a representative dose is in the range of about 1 pg to 1,000 mg, preferably about 5 pg to 100 mg for a 70 kg patient per dose. Administration should 15 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.

,,e Immunizing doses followed by maintenance or booster doses at e; established intervals, from one to four weeks, may be 20 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 o.6 combine the CTL peptides with other peptides or proteins that •*06 induce immune response to other HBV antigens.

o6 25 The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local C t administration. Preferably, the pharmaceutical compositions I are administered parenterally, intravenously, subcutaneously, ntradermally, or intramuscularly. Thus, the i 30 invention provides compositions for parenteral administration which comprise a solution of the cytotoxic T-lymphocyte 4 a stimulatory peptides dissolved or suspended in an acceptable Scarrier, preferably an aqueous carrier. A variety of aqueous j carriers may be used, water, buffered water, 0.4% S -35 saline, 0.3% gycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known i terilization, techniques, or may be sterile filtered. The S resulting aqueous solutions may be packaged for use as is, or i ~*1 24 lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

In some embodiments it may be desirable to include in the pharmaceutical composition at least one component which primes CTL. Lipids have been identified which are capable of priming CTL in 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. See, *Deres et al., Nature 342:561-564 (1989), incorporated herein s nby reference. 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. Further, as the induction of neutralizing antibodies can also be primed with P 3 CSS conjugated to a peptide which displays an appropriate epitope, HBsAg epitopes, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to HBV infection.

S.The concentration of cytotoxic T-lymphocyte stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, 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 SI volumes, viscosities, etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution and g of peptide. ctual Smethods 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, aRg au S examplel 0 0 0** 0c..04

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4 9V Go

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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 HBVinfected 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, a receptor, prevalent among lymphoid cells, such as monoclonal antibodies which bind to the 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, liposome size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, Szoka et al., Ann.

Rev. Biophys. Bioenq. 9:467 (1980), U.S. Patent 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 35 according to, the mode of administration, the peptide being delivered, the stage of disease neing treated, arc.

For solid compositions, conventional nontoxic solid carriers nay be used which include, for example,

V--

i! .5 I I i- '-bi;~~lr pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the cytotoxic Tlymphocyte 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 The surfactant must, of course, be 15 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, lecithin for intranasal delivery.

S 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 S* 30 be introduced into a host, including humans, linked to its own carrier- oras 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 upthe 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, keyhole limpet hemocyanin, thyroglobulin, albumins suci as human serum albumin, tetanus 27 toxoid, polyamino acids such as poly(D-lysine:D-glutanic 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 conjugatihg" i peptides of the invention to lipids, such as P3CSS. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of cytotoxic T-lymphocytes specific for HBV antigen, and the host becomes at least partially immune to HBV 15 infection, or resistant to developing chronic HBV infection.

o Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or Sotherwise at risk of HBV infection to enhance the patient's oown 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 pg to about 500 mg per 70 kilogram patient, more commonly from about S 25 50 pg to about 200 mg per 70 kg of body weight. The peptides are administered to individuals of an appropriate HLA type, for vaccine compositions of peptides from the region of HBenv183-191 (Seq. ID No. HBenv248-2O (Seq. ID No. 2), *HBenv260-269 (Seq. ID No. HBenv335-343 (Seq. ID No. 30 HBenv152-161 (Seq. ID No. 11), HBenvl77-185 (Seq. ID No. 12), SHBenv204-212 (Seq. ID No. 13), and/or HBenv370-379 (Seq. ID No. 14), these will be administered to at least HLA-A2 I -individuals.

In some instances it may be desirable to combine the peptide vaccines of the invention with vaccines which induce neutralizing antibody-responsesto HBV, particularly to HBV Si envelope antigens, such as recoimbinantHBV env-encoded antigens or vaccines prepared from purified plasma 28 preparations obtained from HBV-infected individuals. A variety of HBV vaccine preparations have b&en 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 incorporae.d herein by reference. The vaccines can be combined and administered concurrently, or as separate preparations.

For therapeutic or immunization purposes, the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia. This approach involves the use Sof 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, U.S.

Patent No. 4,722,848, incorporated herein by reference.

Another vector is BCG (bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)) which is incorporated herein by reference. A wide variety of 25 other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., S. Salmonella typhi vectors and the like, will be apparent to 5.1 those skilled in the art from the description herein.

The compositions and methods of the claimed invention 30 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 patientl. Followin treatment to stimulate the CTLs, the cells are returled to the host to treat the HBV infection. -The

I

naie t trea men to s i 'h T s h el r 1 ~cc host's cells may also be exposed to vectors which carry gene.

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.

The peptides may also find use as diagnostic reagents. For example, a peptide of the inventionr -Be-dsdto 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 15 for developing chronic HBV infection.

The following examples are offered by way of illustration, not by way of limitation.

EXAMPLE I Identification of CTL-Specific HBenv Epitopes

C

*o C CoOS..

0 This Example describes the identification of HBenv peptides which stimulated HLA-restricted CTL responses specific for HBV envelope antigens.

All patients included in the study were HLA-A2 positive. Thirteen patients (A-l to A-13; Table II) were studied during an episode of acute hepatitis, 6 (C-l to C-6) were chronically infected by HBV, and 6 uninfected healthy volunteers (N-1 to N-6) served as normal controls. The patients and their HLA haplotypes, determined using PBMC in microcytotoxicity testing with HLA typing trays (One Lambda, Canoga Park, CA), are shown in Table II.

Diagnosis of acute hepatitis was based on standard diagnostic criteria. Diagnostic parameters included clinical :(jaundice) and biochemical evidence of liver injury (ALT activity at least 20 fold greater than the upper limits of Sormal), together with serological evidence of acute HBV infection (presence of HBV surface antigen (HBsAg) and IgG anti-HBc antibody) in the absence of scrological evidence of hepatitis delta and hepatitis C virus infection (Abbott Laboratories, North Chicago, IL). All patients were studied during the first 4 weeks after onset of jaundice, at which time their serum was positive for HBsAg and their ALT levels were markedly abnormal. Eleven of the 13 patients subsequently recovered from the illness, with-normallZation of serum transaminase and clearance of HBsAg within four months of initial diagnosis. One patient (A-ll, Table II) developed chronic active hepatitis and remained HBsAg positive 13 months after initial diagnosis. One patient (A-10) was lost to follow-up after the initial clinic visit. Patients with chronic hepatitis B were repeatedly serologically positive for 15 HBsAg for more than six months and displayed mildly to moderately elevated serum ALT activity. Normal controls had no clinical history of HBV infection and were serologically negative for HBV markers. All patients and

J

S.

S S *o 505 5 0r S. 00 S. S

S-

:31 Table 11.

Characteristics of Subjects Studied Subject Sex Diagnosis HLA class I haplotype A-1 Male Acute A2. A30, 835,8B44, CY!4, Cw7 A-2 Male Acute A2, A31, 8w58(5Y), B51. Cw3 A-3 Male Acute A2, 8w41, B~,C4 w A-4 Male Acute A2, A32, Bw4l.8Bw7lCw4,Cw 7 Male Acute A2, Al, B8, 8w58(5Y). Cw7 lee. A-6 Fmale te AAwG8, B35 Cw3, Cw4 A-7 Male Acute A2. Al. 8. Bw73, Cw3, Cw4 A-8 Female Acute A2. Aw69, 8w53, Cw4 Ma9le Acute A2. A24, B7, B27, Cw2, Cw7 :A-10 Male, Acute A2, A3. BW62. EBW7l, Cw3, Cw4 A-1I Male Acute A2. A24, B35, Cw4 A412 Male Acute A2, A3, A-13 Male Acute AZ. A3. B7, Bw6O, Cw3, Cw7 C.C -i Male Chronic A2, B27, 835. Cw2. Cw4 C-2 Male Chronic A2, Al, 88,844 C-3 Male Chronic A2, A24. B44, Bw67 C-4 Male Chronic A2, Awv69, 8w4l w52 Male Chronic A2,185, Bw62. Cw4 C_6 Male Chronic A2, A26, B35, Cw4 N-1 Male Normal A2, All 18 44, Cw4 N-2. Male Normal A2, Bw56, 835 N-3 Male Normal A2. All1, 8.Bw62. Cw4 N-4 Male Normal A2. A23,1B5, Bw58, Cw2, Cw6 N-S Male Noml A2 B44. Bw63

I

N6 Female Noml A2, All 1.5 9 C S 9594 *afl *54 S S

S.

S S 9* 5 '.55

S

09.5 9 95 55***

S

5* 5 5 .5 5*5955 5 4 32 normal controls were serologically negative for antibody to

HIV.

PBMC from patients and normal donors were separated on Ficoll-Hypaque density gradients (Sigma,-St. Louis, MO), washed three times in Hanks balanced salt solution (HBSS) (Gibco, Grand Island, NY), resuspended in EPI1640 medium (Gibco,_Grand Island, NY)_.supplemented with L-glutamine (2 mM) ,-gentanicin (10 pg/mI), penicillin_, E O U/i) streptomycin (SO pg/mi), and HEPPS (5 mM) containing 1O* heat inactivated.

hua ADl serum (comp lete medium) and plated in 24 well plates at 4 x 106 cells/well. The synthetic peptides were added, to the cell cultures at a.-final concentration of 10 yg/mI unless otherwise-noted. rH~cAg was added at 1 pg,'ml during -the first, week of stimulation. :At day 3, 1 ml of complete medium 15 supplemented with rIL2 (Hofftaan-La Roche, Nutley, at U/mi. final concentration was added in each well.. On day 7, the culture- were restimulated with peptide, rIL2 and' irradiated (3000 rads) autologous or HLA-A2 rmatched feeder cells, and the cultured PBMC were tested for. CTL activity on day 14. Selected cultures that displayed peptide specific cytolytic activity were expanded by weekly restimulation with 1--x 106. irradiated (6000 rads) allogeneicPBMC and 1,x 105 irradiated (18000 'rads) JY cells' (alloqeneic EBV-E transformed cell line,, HLA-A2 B7, CV7] (14) in 1.ml of complete medium 25 containing I jpg/ml peptide,, 20 U/mi 11.2 and 1 liq/ml phytohemaglutinin (PEA)' (Sigma, St-- Louis, MO).

For cytotoxicitv assays' target cells consisted either of a) autologous PEA stimulated blasts or alloqeneic.

HLA matched and mismatched* EBV-transformed B lymiphcblastoid 30-, cell lines.(B-LCL) incubated overnight' with synthetic peptides at 10 pg/ml; b) stable' B-LCL-transfectants descrbed-eabove; or c) ELCL-infected with recombinant vaccinia viruses (described below)-' B-LCL vere either purchased from The I inIerican Society for Histocoimpatibility and .Imimmognetics' (Boston, 'HA) or established from our own -pool of patients-and normal donors as described in copending~application 7g35,89 8_ The-cells were -maintained -in RPHT- 1640 supPlemented-with L-glutamine (2 mM), qet _cin:1 (10 pga) eiiln(5O' U/ml)~

V

3~3 jig/ml), RIEPES (5 mM)F, and lot (vol/vol) heat inactivated ECS (Gibco, Grand Island, NY). Short term lines of autologous PBMC blasts were produced by stimulating peripheral blood PBHC with PHA at 1 p/mI in the 1RPHI 1640 supplemented with L- (2 genrtamicin (10 [Eg/ml) penicillin (50 U/mi), streptomycin (50 gi/m), HEPES (5 mM), 101 (vol/vol) heat inactivated FCS, and !0 UlmI rIL2 for 7 days before use as target cells_. Vaccinia infected targets7 were prepared by infection of.I x 10~ cells at 50 plaaue-forming U/cell on a rocking plate at room temperaure for one hour followed by a sincie wash and overnight incubation at 371C_ Target cells were labeled with 100 j±Ci of -~Cr (Amiersham, Arlington Heights. IL) for one hour and washed three times With HBSS. cytolytic activity was determined in a standard 4 hour 51 Cr release assay using U-bottom 96 well act" plates containing 5000 targets per well. All assays were performed in duplicate.~ Percent cytotoxicity was determined *from the formula: 100 x L(expermnareas spontaneous arelease)/ (maximum release -spontaneous release)]. Maximum release was determined by lysis of target's by detergent (It Trito n X- 100, Sigma). Spontaneous release was less than .of maximal release in all assays.

(A Ta w 1~o HLA-A2 positive nati _rs with =cr en ts (A!adA-3) wer-e initiallv selected for analvsis of the C1__ rsnone toHBsAgq329-348 (ASAPFSWT.SLLVPFVQWFVG Sc--I o 2. 1, which contains 2overlapping iiIA A2..1 allele specific binding motifs- (WLSLLVPFV and. I&VPFVQWFi) Z One of these patients was knowin from pr:evious experiments- to display, .HL A2resricedCTL re6nse-to .2 10- residue- H-BV 0 incleocansid eniiore (_HBcAgl8-2.7) that -also represents an HLA A2.1 allele. -specific. binding noti (FLPSOFFP5Y). This patient was -considered' a potential .responder to o ohof the motfsin ~sg32-38.Anather patient known to be a.

nonresxnonder-to H~cA18-27, -was studiied foric 3-commvarison.- *5 HLAa29-348 sDeific _CIL -Line; -wer~e aenerared from pBC-f bbth atnsb si lati n wit--the rieptide as descrie aoe Xtr2-3 ees-fstimulation. 'both patients displayed a st-rong cymttoxic. response against a K fl.

a 4*49 99** 9b 0 *0 0 9* .9 44 4 .4 4*e9 o @9 9* S pg/ml), HEPES,(5 m1M), and 10% (vol/vol) heat inactivated FCS (Gibco, Grand Island, NY). Short term lines of autologous PBMC blasts were produced by stimulating peripheral blood PBMC withPHA at 1 gg/ml in the RPMI-1640 supplemented with Lglutamine (2 mM), gentamicin (10 jig/ml), penicillin (50 U/ml), streptomycin (50 g±g/ml), HEPES (5 mM), 10% (vol/vol) heat inactivat ed F CS, and 10 U/ml rIL2_ for 71 days before use as target cells- Vaccinia infected& targetswere prepared by infection of I x 106, cells at 50 plaqrue-forming U/cell on a rocking plate at room temperature for one hour followed by a single wash and overnight incubation at 37 0

C.

Targat cells were labeled with 100 juCi of 51 Cr (Nmersham, Arlington Heights, IL) for one hour and washed three times-with HBSS. Cytolytic activity was determined in a Is5- standard 4 hour 5 1 cr release assay using U-bottom. 96 well plates containing 5000 targets per well. All assays were performed in duplicate. Percent cyto-taxicity was determined from the formula: 100 x [(experimental release spontaneous release)/(maximum release spontaneous release)). M~aximumn release was determined by lysis of targets by detergent (1% Triton.X-l00, Sigma). Spontaneous release was less than of maximal release in all assays.

Two HLA-A2 positive Ipat Iients with acute hepatitis (A-1 and A-3) were initially selected for analysis of the CTL 25 response to HBSAgj29-34B (ASAkFSWLSLLVPFVQWFVG (Seq. ID 14o.

which contains 2 overlapping liLA A2.1 allele specific binding motifs,(WLSLLVPFV and LLVPFVQWFV). One of these patients (A-3)..was known from previous experiments to display an HLA A2 restricted CTL response to a 10 residue HBV ~0 nupleocapsid etoe(cg8-27),that also represents an liLA A2.1 allele specific binding motif (FLPSDFFPsv). This patient was considered'a potential- responder to one or both of the motifs in HBSAg329-348.1 Anothbr patient known to be a nonresuonder to HBcAgl8-27, was studied for comparison.

HBsAa32 9-248 specific lines -were generated from PBmc cf. both patients-by sr imulation. with the nepride as .,described above. After- 2-3 weeds ot. stimulation. bjoth aint iplydastrong cyto-toxic-response aqaifist a *9*t9* 9. *4 0 S 999*04

F

to 0 too: *0400 34 homozygous HLA A2.! positive EBV cell line (JY) prepulsed with the HBsAg329-348 peptide. Patient A-I's HBsAg329-348, specific cell line was selected for cloning.

^TL lines were cloned originally at 1, 10, and 100 cells per well and then subcloned at 0.3 or I cell per well in 96 well mfirotiter plates. The cells were plated in the presence of peptide (1 pg/nil), PHA (1 pg/mI), rIL-2 (20 U/mi), irradiated (6000 rads) allogene-PfMC (105 cells/well), and irradiated (18000 rads) JY cells (104 cells/well). HBV specific clones were restimulated in a 24 well plate as describe: above except that the peptide was omitted and irradiated JY cells, transflected with a plasmid that confers stable expression of the HBV large envelope antigen (EBOpreSI, reference 10), were added at 105 cells per well.

15 1 Using patient A-i's HBsAg329-348 specific cell line four clones were derived from cells plated at 1 cell per well (clone B13, B16, B17) or 0.3 cells per well (clone B3). Clone B3 was tested against a panel of allogeneic target cells partially matched with the effectors at the level of liLA class 1 alleles. Using allogeneic target cells partially matched at liLA class I with patient A-1, the cytolytic activity of clone B3 was f ound to be HLA-A2 restricted,.due to the presence of 2 HIA-A2.1 binding motifs in-the peptide. An HBsAg329-348 specific polyclonal CTL line derived from patient A-3 was also determined to be HLA-A2 restricted in the same manner. since the HLA-A2 subtypes of the patients were not determined; it is not known if the CTL response to the peptides is restricted only by thie HLA-A2 .1 allele or whiether it extends to other HLA-A2 subtypesl as well.

To determine~ a -minimum, optimal HLA -A2 restricted -CTL epitope within RBsAg329-348, a panel of amino-terminal truncations and overlapping nine-mers and ten-mers derived from the HBSAg29-348 sequence were produced to map the HLA-A2 _.restricted-CTL epitope(s) present in this 20 residue peptide, which contains 2, overlapping id eal HLA-A2.1,binding motifs.

The HLA-A2 restricted CTL clone B17 from patient A-1. and a polyclonal CTL line 1B9,from patient A-3, derived byrepetitive stimulation-of the initial cell line described 4 0*9* 9999 9 *99* 9 99 99 .99.

*9 9 S 99 e. 9 9.

9.

9* 9 *990*.9

S

~94 0 above and HBsAg329-348, were used as etfector cells to establish the fine specificity of the CTIE response to HBsAg329-348. Target cells were produced by incubating an HLA-A2.l positive B cell line (JY) either with the original 20-mer or with the truncated peptides.

The results, shown in Fig. 1, indicated that only the first of the 2 HLA-A2.1 binding motifs (HBsAg335-343) is recognized by the CTL.- Furthermore, the data demonstrate that this peptide (WISLLVPFvV) Is the minimal, optimal HLA-A2 restricted epitope recognized by HBsAg329-348 stimulated CTL, since omission of the extreme amino-terminal or the extreme carboxy-terminal residue from HBsAg335-343 abolishes recognition by the CTL.

The superiority of HBsAg335-343 at the effector level 1s was reiterated when the peptides were used to stimulate a CTL response in PBI(C from patient A-i. Synthetic peptides representing assorted HBsAg329-348 subunits were used at 10 PHK to stimulate PEMC of patient A-1. After 2 weeks of stimulation the cytotoxicity of these lines was tested at E:T of 60:1 against JY target cells prepulsed with 10 jiM of the same peptide and JY targat cells prepulsed with 10 AM HBsAg335-343. Results shown' in Fig. 2 represent lysis in a 4 hour 51 Cr release assay. As can be seen in Fig. although *HBsAg335-343 and its extended variants proved capable of 25 inducing a CTL response, omission of the extreme amino- and carboxy-terminal amino acids completely abolished the ability *of the peptides to stimulate a CTL response, thereby reinforcing the conclusion that HBSAg335-343 is the minimal optimal HLA -A2 restricted epitope between residues 329-348 of 30 HBsAg.

09 94 .99 .9 gi 9 5 CTL Response to Sevy 1 5Seven ideal HI2 *defi1ned as. peptides. bati .contain a leucine ini thu carbovy-terminal res'Idue EXAMPLE II n HLA-A2.lrBinding Motifs in HBVenv ~-A2.1i allele specific binding motifs, Ween 9-10 residues in length that a 'second position and a valine as the e, are present in the HBsAg region of

ES..

SEES

Er ES ES S

WE

S S E* 9 S

ES

SEE.

S .5 8 CS S EWE SE

S

55 S. 3 the HBV envelope protein (Table III). Based on the results obtained in Example 1, the ability of these seven envelope pePtides, plus the known HIA-A2 restricted HBV nucleocapsid epitope (HBcAgl8-27), to stimulate a CTL response in 12 HLA-A2 -posit ive patients with acute hepatitis B, was examined. For comparison, six HLA-A2 positive patients with chronic hepatitis and 6 uninfected normal controls were tested for responsiveness to the same panel of peptides.

Table ITT. HBV-derived HLA-A2.1 binding motifs and pentides Peptide Sequience Seq. ID No.

15 1 HBcAg1G-27 FLPSDFFPSV 23 2 HBsAg2Ol-210 SLNFLGGTTV 24 3 HBsAg25l-259 LLCLIFLLV 7 20 4 HBsAg260-269 LLDYQGMLPV 9 5 HBsAg335-343 WLSLLVPFV 6 HBsAg338-347 LLVPFVQWFV 7 HBsAg348-357 GLSPTVWLSV 26 SHBsAg378-387 LLPIFFCLWV 27 PBMC from acute patients (A-i to A-12), chronic patients (C-1 to and normal subjects (N-1 to N-6) we-re 35 stimulated with the following synthetic peptides: l=HBcAgl8-27, 2=HBSAg2Ol-210, 3=HBSAg25l-259, 4=HBsAg26O-269,

-I

t4 i was&..35-43 6=HBsAg338-347, 71=HBsAg346-3~ 8=HBsAg37B-B7 for two weeks as described ir tested ia4hour 51 Cr release assay against 410 prepulsed overnight with' he sam ptide. I cytotoxicity-was measured by subtracting the JY target cells -notE prepulsead with peptide f2 release _byJY target cellsp repulsed with thE Results shown (Fig. representA* specific 5 Cr release assay,.

IExample 1, and JY target cells ?eptide specific 51 Cr release by .,om the 51 Cr Lysis in A 4 hour .4.

0 abo 4 soC 4944 Sf 1 As can be seen in Fig. 3, nine of the twelve HL-k-A2 positive patients with acute hepatitis responded to at least one of the peptides in the panel. In contrast," .one of the six HLA-A2 positive uninfected normal controls responded to any of the peptides following the same in vitro stimulation strategy, suggesting that responsiveness to these-peptides by the patients reflects in vivo priming by thiicorresponding HBV-derived epitones.

Importantly, eight of the nine responders recognized multiple epitopes within the panel, indicating that the CTL response to HBV during acute hepatitis is both polyclonal and multisoecif ic. Furthermore, there was substantial variation in the spectrum of epitopes recognized among the nine responders, with certain epitopes being recognized more frequently than others. For example, H~cAg18-27 and HBsAg2335-343 were recognized individually by seven and eight of the nine responders, respectively, and when combined they were recognized by all nine of the responders. In contrast, HBsAg348-357, HBsAg25l-259 and H~sAg26O-269 were recognized by only 3/9, 2/5 and 3/6 of the responders in whom they were tested.

Nucleotide sequence analysis of circulating virion DNA in acutely infected patients showed that all patients, including the CTL nonresoonders, were infected by viruses that expressed the precise amino acid sequence present in the prototype HBsAg335- 343:peptide used to stimulate expansion of cTL -in vitro. since residues 335-343 are known to be conserved in all the Published HBV seanuences derived from all 4 HBV subtypes, as published-in the GenBank-7.2 database, as "OWell as in the 10 patients studied herein, it may be concluded thatHBsAg335-343 is an IHV group specific CTL epitope. The same was not true for HbsAg348-357, however, since only seven of the ten patients were foundto be infected by viruses that encode'the vrototype se I uence used for in vitro stimulation .(GI-sPTVWLSV). The remaining three patients A-bO, A-13) displayed a viriant-.:eauence in 'which the carboxv-terminal valine was'substituted, bvwan a lanine at. position .157. Among rhe patients infected by the-prototyp&,virus, C'rL-respond Iers I f I7 T i rI 38 g e g.

1 *e 664.6 so to' 0 *60000 03 3

U*

4 6 *3 0 and nonresponders to HBsAg348-357 were observed, just as for the response to HBsAg335-343. On the other hand, none of the 3 patients infected by the variant virus displayed a CTL response to the prototype peptide.

It is noteworthy that all nine responders subsequently became HBsAg negative and their liver disease completely resolved. In contrast, all six patients with chronic hepatitis, who failed to clear the virus, also'failed to mount a peripheral blood CTL response to any of these epitopes. Three of the acutely infected patients (A-10, A-1i, A-12) also failed to respond to any of these peptides.

Furthermore, one of the nonresponders (A-ll) developed chronic active hepatitis and was still HBsAg positive 13 months after his acute illness. These combined data strongly suggest a 15 relationship between the CTL response and viral clearance.

However, nonresponder patient A-12 seroconverted to HBsAg negativity between 1-4 months after disease onset.

As indicated in Table II and Fig. 3, four of the ninL responders shared only the HLA A2 allele with the JY target 20 cell line used in this study (HLA-A2, B7, Cw7), demonstrating that the response to all of the peptides was HLA-A2 restricted in these individuals. Since the remaining responders also share the HLA B7 and/or Cw7 alleles present in the JY target cells in addition to A2, it is possible, although unlikely, 25 that these alleles could also serve as restriction elements for these epitopes in these patients.

The molecular basis for the differential immunogenicity of the seven HBV envelope peptides was not immediately evident from a comparison of their sequences.

30 Potential differences in the relative binding affinity of the peptides to HLA-A2.1 were examined by determining the ability of the seven envelope peptides to compete with the binding of an unrelated HLA-A2 restricted nucleocapsid epitope S(HBcAg18-27) to a homozygous HLA-A2.1 positive B cell line

(JY).

as -t 8 i ii

I:

I

The competitive binding inhibition HBcAgl -27 specific ,CTL clone from patient A S effector cells. Blocking peptides lo, 1 assay used an -4 as a source of 00 gM) were added

I

00** a.

a *5 S S e.

S..

S

.Ca**S@ to a mixture of 5 1 Cr-labelled, HLA-A2.l positive JY target cells and effector cells (E:T=40:1, 3000 target calls/well) for 40 minutes before the addition of a subsaturating concentration (0.03 tiM) of the target peptide, HBcAg 1 8- 2 7 The binding ability of each peptide was assessed by calculating the degree to which it blocked the lysis of target ceLls in a 4 hour S:LCr release assay.

As shown in Fig. 4, all four immunogenic peptides--andtwo of the three nonimmunogenic peptides bound to the HLA-A2.1 molecule, but with widely (more than 100-fold) variable efficiencies that did not correlate with their relative i-munogenicity. Importantly, the only peptide that did not bind to HLA-A2.1 in this assay (HBsAg337-348) was nonimmunogenic. For the other two nonimmunogenic peptides, however, the HL.A-A2.1 binding affinity was as high or higher than some of the immunogenic peptides. Thus, although the capacity of a peptide to bind to this class I molecule is required for immunogenicity, it does not guarantee it. This suggests that the additional factors at the level of antigen processing and the T cell repertoire may play a role in determining which HLA-A2.1 binding peptides within a viral protein are able to induce a CTL response.

25 EXA-MPLE III Peptide Specific CTL Recognize Endogenous HBenv Antigren The ability of H~sAgJ 3 3 5 3 4 3 and H~sAg 3 4 8 3 7 specific CTL-to recognize endogenously synthesized antigen was examined 30 by measuring their ability to lyse target cells that had been inetdwthto- on of recombinnt -a encode the large, middle and major envelop derived from. cloned- HBV genomes of-either s ubtypes Of HBV.

035 Recominant vaccinia viruses expri large-, Middle and-major envelope polypeptii and a-correspondinq, controllwil ye ae (Smith et al.,-Nature 302:490 (183;Cheni ccnaviruses wulcn e Polvpeptides the ayw or the adw essing the HBV des. (adw subtype) iriia were obtained

A

60:337 (1986); and Che Ing and Moss, Virol. 61:1286 (1987), each of which are incorporated herein by reference). An independent series of recombinant vaccinia viruses expressing the same three HBV envelope polypeptides of the HBV ayw subtype was derived as follows. For expression of the HBsAg, an XhoI/Sphl restriction fragment containing nucleotides 1409- 2514 of the HBV sequence was cloned into a vaccinia virus expression vector downstream from the 7.5 K early/latdpromoter. For the presi expressing vaccinia virus, a Bgl II/Sph I fragment containing nucleotides 937-2514 was used.

For cloning the preS2 coding sequence, first a short adapter oligonucleatide was synthesized which started at nucleotide 1267 six base-paris upstream from the preS2 Start codon) and spanned the Eco RI site at position 1280. After *ego 15 cloning this oligonucleotide into the vaccinia virus expression vector, the coding sequence was completed by *recloning the Eco RISh B rget(ncetds18-54 in0o i Vinrmeaes dontut eeaino eobnn vaccinia vrsswas doeaccording to standard procedures as described in Smith et al., supra. Stable transfectants that exuressed the HBV envelope Droteins (ayw subtype) were produced by transfection of B-LCL with a panel of EBV based expression vectors that contain the corresponding IIsV (ayw :subtype) coding regions, as described in Guilhot et al., J.

Virol. 66:2670 (1992), incorporated herein by reference.

An UBsAg 3 3 5 -3 4 3 specific CTL line (patient A-I) and an 0000* IBsAa 34

B

8 57 specific CTL line (patient A-4) were generated by stimulation with peptide sequences WISLLVPFV and GLSPTVWLSV, respectively. Cr-L were incubated with 5 1 labelled Y target 20 cells that had been preincubated either with media, with the inducing peptide or (in the case of, HBSAg 3 4 8 3 5 7 vith a variant peptide (GLSPTVWISA). CTL were also incubated with 2cr-labelled JY target cells that had been infected with a panel of 6 recombinant vaccinia viruses that express the HBV ajo middle (V--preS2) n lre(Vne1 envelop polyp rie dervdro avw--and aadw HBV aenoma-s- Wijld-type

K~

vaccinia viruses (V-wrtj were used as controls. .The -HBS seCiiC CTL 1line' (rgh (right..4 panel) was used at-a

I

v-li :r-I

SC

*SC

C C

C

*r 9 C

CC...

CC...

C C

-C

*C cc C

C.

E:T=40:1. The HBsAg 348 357 specific CTL line (left panel) was used at an E:T=3:1. Results shown represent lysis in a 4 hour 51 Cr release assay.

Both HBsAg 33 5 3 43 and HBsAg 348 35 7 specific CTL from patients A-i and A-4 were able to lyse recombinant vaccinia virus infected target cells that synthesize all three of the HBV envelope proteins (Fig. This indicates that both of these synthetic peptides represent epitopes that are generatedby the endogenous processing of the large, middle and major HBV envelope polypeptides within infected cells.

Importantly, HBsAg 33 5 3 43 specific CTL could lyse targets that were infected by both sets of recombinant vaccinia viruses with equal efficiency, with t.e HBsAg 34 8 3 57 specific CTL lysed with ayw infected target cell panel much more efficiently than the adw targets.

The results described in the foregoing Examples illustrate that the CTL response to HBV in man appears to be quite polyvalent, presumably to afford more efficient protection against this serious viral infection. Furthermore 20 the data indicate that the peptide stimulation strategy employed herein is both efficient and effective for the identification and analysis of the polyvalent response, restricted as it is by the polymorphic HLA class I locus. As additional HLA allele specific binding motifs are identified, HBV-derived peptides containing these motifs can be used for in vitro stimulation of CTL precursors.

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.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein:for purposes of illustration, various 10 modifications may be made without deviating from the spirit S and scope of the invention. Accordingly, the invention is not limited except as by the appended .claims.

d g

B

4.

L

42 SEQ;UEKCE LISTING GENERAL INFORMATION: APPLICANT: Chisari, Francis V- (ii) TITLE OF INVENTION- PEPTIDES FOR INDUCING CYTOTOXIC T LYMPHOCYTE RESPONSES TO HEPATITIS B VIRUS (iii) NUMBER OF SEQUENCES: 27 (W-)CORRESPONDENCE ADDRESS: ADDRESSEE: Townsend and Towinsend 1B) STREET: One Market Plaza, Steuart Street Tower CITY: San Francisco STATE: CA COUNTRY: USA ZIP: 94105 (iCOMPUTER READABLE FORM: MBDIUM TYPE- Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM- PC-DOS/MS-DOS SOFTWARE* Patentir. Release Version #1_25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: SCC) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: NAME: Parmrelee, Sceven W- REGISTRATION NUMBER: 31,990 REFERENCE/DOCKET N1UMBER: 11740-4 TELECOrMMUNICATION INFORMATION: TELEPHONE: 206-467-9600 TELEFAX: 415-543-5043 INFORMATION FOR SEQ ID NO:1: (W SEQUENCE CHARACTEISTICS: LENGTH: 9 amino acids S TYPE: amino acid STRANOEDINESS: single TOPOLOGY: linear MOLECULE TYPE: peptide (xci) SEQUENCE D3ESCRIPTION: SEQ ID NOI: Phe Leu Leu Thr Arg Ile Leu'Thr Ile INFORMATION FOR SEQ ID NO.2: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids fa TYPE: amino acid STRANDEDNESS: single !Dj TOPOLOGY: linenr i) IM)LECULETYPE- petide -xpe 43 (Xi) SEQ2UENCE DESqCRIPTION: SEQ ID NO:2.: Phe le Leu Leu Lem Cys Leu le Phe Leu Leu Val LeU 1 510 INFORMATION FOR SEQ ID KO:3: Wi SEQUENCE CHARACERISTICS:7 LENGTH: 10 amino acids TYPE: amino acid STDEDNESS: single TOPOLOGY- linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:.

Phe Ile Leu Leu Leu Cys Leu le Phe Leu 1 5 0eG#*e INFORMjXJQN FOR SEQ rIN04: (i SEQUNC

CHARACERISTICS.

IA) LENGTH. 9 &Mino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear *.So(ii) MULECUrE TYPE: peptide Go [xi) SEQUENCE DESCRIPTION: SEQ ID NO :4: Ile Leu Leu LuCys L le Phe Leu 6 13 2) INFORMATION FOR SEQ ID Wi SEQUENCE

CHARACTISCS

LENGTH: 10 aioacids TYPE: amino acid STRANDEDNESS: single *@0*see TOPOLOGY: linear (ii) MOLECUjLE TYPE: peptide (xi) SEQUENCE DESCRIPTION: E ID le Lau Leu Leu Cys Leu le Phe Leu Leu 1 M2, INFORMATION FOR SEQ ID mO_:6: SEQUENCE

CHARACTERISTICS:-

LENGT 9 amino acids ()TYPE: amino acidI STRANDEDNESS: single TOPOLOGY. linear (il) M4DLECULE TYPE: pept ide 44 (Xi) SEQUENCE DESCRIPTION: SEQ ID) NO.6: Len Leu Leu Cys Leu le Phe Len Leu (23 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH~: 9 amino acids TYPE: amino acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID) NO:7: Leu Leu Cys Leu le Phe Len Leu Val 1 INFORMATION FOR SEQ TO NO:8: ED) SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid *:94 STMANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Leu CYS Leu Ile Phe Leu Len Val Leu 1 5 INFORMATION FOR SEQ ID N0:9: W SEQUENCE CHARACERISTIc-s: LENGTH: 10 amino acids TYPE:- amino acid SRANDEDNESS: single4 TOPOLOGY: linear S(ii) MOEUETYPE: peptide- I SEQUENCE DESCRIPTION-: SEQ ID NO:9: LuLeu Asp Tyr Gla Gly Met Leu ProVa 1 510 INFORMATION-FOR SEQ ID 110:10: till SEQUENCE CHARACTERISTICS: LENGTH.. 9 amino acids TYPE: amino acid STRI4NDEDNrESS:. single E D) TOPOLOGY: linear (ii) MOLECULE TYPE-. peptide (xi) SEQUENCE DESCRIPTION: SEQ ID 140:10: Trp LeL Ser Leu Leu Val Pro Phe Val

I

INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid XC) STRIJ4DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TfPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: Ser Ile Leu 5cr Lys Thr Gly Asp Pro Val 1 510 3:44.9(2) INFORMATION FOR SEQ ID 140:12: (ii SEQUENCE CHARACTERISTICS: ,Ago* LENGTH: 9 amino acids TYPE: amino ai STRANDEDNESS: single TOPOLOGY: linear (ii) MLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ in 140-12: Val Len Gln Ala Gly Phe Phe Leu Leu INFORMTION FOR SEQ ID NO:13: li) SEQUENCE CHARACTERSTICS:- LENGTH: 9 amino acids TYPE: amino acid 3 C(C) STRANIJEDNESS: sinale TOPOLOGY: linear (ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID 140:13: Phe Leu Gly Gly Thr Pro Val Cys Leu 1 5 INFORMATION FOR SEQ ID NO:14- SEQUENCE CHAkRACTERISTI1CS: LE2GTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear [ii) MOLECULE'TYPE: peptide 46 (xi) SEQUENCE DESCRIPTION: SE~f ID NO:14: Ser Ile Val Ser Pro Pbe Il1a Pro Lau Lau 1 5 INFORMATION FOR SEQ ID W1 SEQUENCE CHARACTERISTICS: LENGIX: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLO3GY: linear (ii) )M3LECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: Ser Trp Leu Ser Leu Leu Val Pro Phe Val.

1 5 INFORMATION FOR SEQ ID NO;16; 4 Wi SEQUENCE CMMRCTERISTLICS:t LEN=~: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECU-E TYPE; peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Trp Leu Ser Leu Leu Val Pro Phe Val Gin I S4 INFORMATION FOR SEQ ID MO:17: SEQUENCE CMARACTrERISTICS: :ENGTH: 2 0 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear iiJ) MOLECULE TYPE: pepcride SEQUENCEM DESCRIPTION: SEQ ID KO:17: Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser P.he Lau Pro !NFORMATION FOR SEQ ID NO:18: Li) SEQUENCE CHARACTERISTICS:.

LEN=G1: 20 amino acids TMPE: amino acid STRMIDEDNESS- single.- TOPOLOGY: linear -MOLECUJLE TYPE: peptide 2

Z'

I

I

46 Xi), SEQUENCE DESCRIPTION: SEQ ID NO:14: Ser Ile Val Ser Pro Phe Ile Pro LeU Leu INFORMA.TION FOR SEQ ID Wi SEQUENCE CHARACT1ERISTICS: LENGTH: 10 amino acids TYPE. amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 9; e a.

C S to .r *4 a.

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S

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4 04 *L S. a 1' 14 txi) SEQUENCE DESCRIPTION: SEQ ID Ser 'rrp Lau Ser Leu Leu Val Pro Phe Val 1 5 INFORMATION FOR SEQ IDINO:16: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid (Cs STRANDEDNESS- single TOPOLOGY; linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No:16: Trp Leu Ser Leu Leu Val Pro Phe Val Gin 15 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH:. 20 amino acids IS) TYPE: amino acid STR.ANDEDNESS single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:.17: Met Asp Ile Asp' Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Len Leu 1 5 V10 Ser Phe Leu Pro- INFORMATION FOR SEQ ID NO:18: SEQUENCE V CHARACTERISTICS: LENGTH:. 20 amino acids TYPE: amino acid STRANDEDNESS:- single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide.

F'

U

9 4 0 i 4* 4 4493 3909 *9 @8 .9 9 .9 U 869 47 (xi) SEQUENCE DESCRIPTION: SEQ ID NO-18: Pro His His Tyr Ala LeU Arg Gin Ala Ile LeU Cys9 Tip Gly Glu Leu 1 -S10 is Met Tyr Leu Ala INFORMATION FOR SEQ ID No:l9: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:L9: Leu Leu Tip Phe His le Ser Cys Lau Thr Phe Gly Arg Glii Thr Val 1 5 10 Ile Glu Tyr Leu INFORMATION FOR SEQ ID (i),SEQUENCE CHARACTERISTICS: LENGTH: 1S amino acids TYPE: amino acid STRANDEDNESS: single (D).TOPOLOGY:Ilinear- (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:- SEQ ID Glu TYr Leu Val Ser Phe Gly Val Tip Ile Arg Thr Pro Pro Ala 1 -5 10 1 INFORMATION FOR SEQ, ID NO:21: SEQUENCE CHARACTERISTICS. LENGTH: 20 -amino acids TYPE: amino acid STRANflEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide -SEQUENCE DESCRIPTION: SEQ ID NO:.21: Val Ser Phe Glyr Val T p Ile Arg Thr Pro Pro Ala .Tyr Arg Pro Pro Asn Ala Pro Ilie NFOMATON-FOR SEQ ID NO.22: 39 90 9 ~9 9: ~0 '9 9 *994~.~ S 0

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800844 8 4 99 04 00 3 a

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~a 48 ti) SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 0 *0 00000 0* 0.

@0.00 0 -0 0*00.0

L

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ala Ser Ala Arg Pile Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gin 1 5 10 Trp Phe Val Gly INFORMATIONJ FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE- amino acid STRMANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide, (xi) SEQUENCE DESCRIPTION. SEQ ID N0:23: Phe Leu Pro Ser Asp Pile Pile Pro-Ser Val 1 5 10 INFORMATION FOR SEQ ILI NO:24: SEQUENCE CHARACTERISTICS: (A LENGTH: 10 amino acids (BI TYPE: amino acid STR-ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi),SEQUENCE DESCRIPTION: SEQ ID NO:24: Ser Lau Asn Pile Leti Gly Mly Thr Thr Val 1 5 -10 INFORMATION FOR SEQ ID Ci) SEQUENCE.CHARACTERISTICS: LENGTH: 10 amino acids TYPE: <amino acid C(C) STRANDEDNESS: single CD) TOPOLOGY. linear fii) MOLECULE ITYPE. peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Leu Val Pro- Pile Val Gln Trp PYle.Val .1 -~-s10 '2 2 Iv 49 INFORMATION FOR SEQ ID) N0:26: iSEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Gly Leu Ser Pro Thr Val Trp Leu Ser Val 1 5 INFORMATION- FOR SEQ ID NO.27: Wi SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRAN2DEDNESS: single D) TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO:27:.

Leu Leo Pro Ile Phe Pbe Cys Leu Trp Val 1 5 1 y

Claims (7)

1. A CTL inducing peptide comprising the following sequence-. HBenvla3-191 (Seq. ID No. 1) PeLuLuTrAgIeTuTrie

2. The CTL inducing peptide of claim 1, wherein the peptide also contains a T helper epitope.

3. The peptide of claim 1, conjugated to a -immunogenic lipid carrier. J

4. A CTt. inducing peptide comprising from eight to thirteen amino acids, wherein at least a majority of the amino is1 acids are homologous to a corresponding portion of HBenv having the sequence: H~env248-260 (Seq. ID N.2) SS. The CTt. inducing peptide Of claim 4, which comprises from nine to eleven amino acids. The CTL inducing peptide of claim 4, which

5.03 25 comprises: i{Berlv246-257 (Seq. ID No. 3) *HBenv24g-9 (Seq. ID No. 4) Ile-Lu-LeuLeus-Leuys.ile-.PheuLe;W 35 Benv250-2 58 (Seq. ID No. L eu e u e -C s -L u 7 I -Ph -L e u L e u H~env251-259 ID N.7 LeU-LeU-cys-LeuIe-Pheu..r.uL.Val; or I{env25l-260 (Seq. ID No-. 8) A CTL inducing peptide comprising from nine to eleven amino acids, wherein at least a majority of the amino acids are homologous to a corresponding pdti-tion of Hflenv having the sequence: HBenv334-344 (Seq. !D No.) Trp-Leu-Ser-Leu-Lu-Val-Pro-Phe-.Vai. T, S The CTL inducing peptida of claim 7, which comprises: H~env335-343 -(Seq. ID No. HBenv334-343 (Seq. ID. No. Se- pLu-e-LuLeme Po-h-Vl or -HBenv335-344 (Seq. ID. No. 16)

9. The peptide of claim 8, conjugated to a 25 immunogenic lipoprotein carrier. 50*55510. The peptide of claim 9, suspended in pharmaceutically acceptable carrier which comprises a liposome. mm

511. A. method for treating or preventing hepatitis B infection, which comprises administering an effective amount4 of a peptide to a host having an HIL-A2 haplotype, wherein the peptide. is a CTL inducing peptide comprising the following -sequence: HBenvlS3-191 (Seq. ID No. 1) Phe-eu-L u-Th -Ar -TL1 -Leu Thr4l-i 52 12. A methlod for treating or preventing hepatitis infection, which comprises administering an effective anouit of a Peptide to a~ host having an HLA-a2 halplotype, wherein the peptide is 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 IIBeHV having thle sequence: HBenv24 8 2 6 0 (Seq. ID No. 2) Phe-Ile-LeuLu-j or ucsLuIePeLuLuVlLu J7 a a S S S 0. S at S S C 0 HB~fV335- 3 4 3 (Seq. ID Ito. 13. The method of claim 12,weinteCLndcg p e p t id e i s w e e n t e C L i d c n HBenv248. 257 (Seq. ID No. 3) HBenv249- 25 7 (Se. ID No. 4) leLeu-reLeuCys-LuIePeLu HBenv249-. 2 5 8 (Seq. ID No. IleLe-Lu-Lu-yILeuheI1eu.Ph VLeuoru HBenV25.. 2 58 (Seq. ID go. 8) 'leu-LeU-Lucys -LUIeih-eULu Leu L u ys eu l e h Leu-L u V aj..q or 14.TheMe~~d'Ofclaijm 13, wherein the host has chronic hepatitis B infection Or: is a hepatitis B carrier. k I 4; 53 The method of claim 12, wherein the CTL inducing peptide is: HBenV335-3 43 (Seg. ID No. TrP-Leu-Ser-Lu..u-ValPro-Pile-Val HBenv334-343 (Seq. ID. No. Ser-Trp-Leu-Ser-Lu-Leu-Val-Pro-Pile-Val; or IIBenv335-344 (Seq. ID. No. 16) Trp-euSer-L-e u-u. VaPro-Phe..Val-Gl 16- Tile method of claim 12, wherein the host has #:Goes acute--hepatitis B infection. 00.. 17- The method of claim 12, wherein the CTL inducing peptide is administered prophylactically. 1S. The method of claim 13, wherein the host has chronic hepatitis B infection or is a hepatitis B carrier 19. The method of claim 12, wherein the CTrL inducing peptide is administered to the host with a second peptidie which elicits a Tr helper response to HEy. 5* A The method of claim 19, wherein the CTL idcn peptide and the T helper inducing peptide are linked. v 1. A CTL including peptide comprising from eight to thirteen amino acids, wherein at least a majority of the amino acids are homologous to a corresponding portion of HIBenv having the sequence: HBenvlS3-191 (Seq ID No.. 1). 2. The peptide of claim 1, which comprises nine or ten amino acids. The peptide of claim 1, which comprises from ten to twelve amino acids. 4. 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 IlBenv having the sequence: HBenv248-26O (Seq. ID No. 2) Ph-t......-.ysLu-l-Pc-e-euVl- The CTL inducing peptide of claim 4, which comprises: HBenv249-257 (Seq. lID No. 3) Ph-eLuU-nCy-e-ePeLu HBenv249-257 (Seq. IID No. 4) ***lieLeuU1u-uCys-Leu-Ile-Phe.-Leu, H7Beny249-258 (Seq. ED No. -Iie-Leu-Leu-Leu-Cys-Leu-IlePhe-Leu 4 eu; llBenv25O-258 (Seq. JID No. 6) Leu-Leu-Leu-Cys-LeI-le-Phe-Leu-Leu, HBenv25l-259 (Seq. ID No. 7) 4.4 Leu-Leu-Cys-LeI1Ile-PheUu-Leu-Val; or HBenv251-260 (Seq. lID No. 8) Leu-Leu-Cys-Leu-Ile-Phe4.CLeuLuVal-Leu. 6. A CTL inducing peptide comprising from nine to eleven amino acids, wherein 4 at least a majority of the amino acids are homologous to a corresponding portion of llBenv having the sequence: HBenv335-343 (Seq. ID No. .4.44:Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val. 30 7 The CTL inducing peptide of claim 6, which comprises: HBetiv335-343 (Seq. ID) No. Tr--uSrU-euVlPoPeVl HBenv334-343 (Seq. ID. Se-r-e-e-e-LuVlPoPeVl or as HBenv335-344 (Seq. ID. No. 16) Trp-Leu-Ser-Let-Leu-VaI-Pro-Phe-ValIGln. 8. Thetpeptide of claim 7, conjugated to a immunogenic lipoproteini carrier. 9.The peptide of claim. 8, suspended in Pharmaceutically acceptable carrier which comprises a liposome. I 2. I I.> I I- r 10- A method for treating or preventing hepatitis B infection, which complises; administering an effective amotint of a Up-ptide to a host having an HLA-A2 haplotype, wherein the peptide is a CTL inducing peptide comprising the following sequence: BBenv183-191 (Seq. ID No. 1) Phe-Leu-Leu-Thr-Arg-lle-Leu-ThIle. 11. A method for treating or preventing hepatitis B infection, which comprises administering an effective amount of a peptide to a host having an HLA-A2 haplotype, wherein the peptide is 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 io portion of HBenv having the sequence: llBenv248-26O (Seq. IID No. 2)- LlBenv335-343 (Seq. ID No. Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val. 12. The method of claim 11, wherein the CTL inducing peptide is: HBenv248-257 (Seq. 11) No. 4) :HBenv249-258 (Seq. ID No. le-eu-Leu-Leu-Cys-Leu-Ile-Phe-eu-eu; HBenlv250-258 (Seq. ID No. 6) Leu-Leu-Leu-Cys-Leu-lle-Phe-Leu-eu; Bew.v251-259 (Seq. ID No. 7) V. Leu-eu-Cys-LeuIle-Phe-Leu-Leu-Val; or HBeinv251-26O (Seq. ID No. 8) 2 5 Leu-Uu-uCys-LU-Ile-Phe-leu-L-eu-Val 4 .eu. *13. The method of claim 12, wherein the host has chronic hepatitis B infection or is a hepatitis B carrier. 14.- The method of claim 11, wherein the CTL inducing peptide is: HBenv335-343 (Seq. ID No. Trp-eu-Ser-LeI-Leu-Val-PrQ-Phe-Val; IIBenv334-343 (Seq. ID. No. Ser-Trp-euSerLu-LUn-ValbPrO-Phe-Val; or' HBenv335-344 (Seq. IID._ No. 16) Tr u-SerLeu-cu-a-Pro-Phe-Va1-Glfl. 15', The method of claim 11, wherein the host has acute hepatitis B infection. 16., The method of claim Ii, wherein the CTL inducing pptide isadm-infistered prophylactically.. 17. The method of claim 12, wherein the -host has chronic hepatitis B infection or is a hepatitis B carrier. r,7; I 56 18. The method of claim 11, wherein the CTL inducing peptide is administered to the host with a second peptide which elicits a T helper response to HIBV. 19. The method of claim 18, wherein the CTL inducing peptide and the T helper inducing peptide are linked. 20. An immunogenic composition comprising the peptide of any one of claim I- 9, and a second immunogenic peptide. 21. The immunogenic composition of claim 20, wherein said peptide of any one of claims 1 -11 and said second immunogenic peptide are conjugated to form a polymer. 22. The immunogenic composition of claim 20 or 21 wherein said second immunogenic peptide contains a T helper epitope. 23 A CTL inducing peptide, containing between six and-twenty~five residues in length, substantially as hereinbefore described with reference to any one of the Examples, excluding the comparative examples. 24. A method for treating of preventing hepatitis B infection, substantially as hereinbefore described with reference to any one of the Examples. A CTL inducing peptide comprising the following sequence: ll0envl83-191 (Seq. ID No. 1) 4 Phe-Leu-Leu-Thr-Arg-lle-Leu-Thr-Lle; when used in the treatment and/or prevention of hepatitis B infection, in a host having an HLA-A2 haplotype. 26- A CTh 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 HBenv having the sequence: H-Benv,248-260 (Seq. ID No. 2) Phe-Ite-Leu-Leu-Leu-Cys-Leu-lle-Phe-Leu-Leu-Val-L--u, or 1IBenv335-343 (Seq. ID No. A Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val;. when used in the treatment andior prevention of hepatitis B infection, in a host having an ILA-A2 haplotyp. 27. The CTL inducing peptide of claim 26, wherein the CTL inducing peptide is: Hlienv248-257 (Seq. ID No. 4) lle-Leu -U-1 -e -Cys-LU-1 -Phe-Leu; HBenv249-258 (Seq._ ID No. lle-Icu-Lecu-Leu-Gys-Leu-Ile-Phe-Leu-Leu- -HBenv25O-258 (Seq-. ED No. 6) Leu-Leu-Leu-Cys-Lezu-lle-Plie-Leu-Leu; HMenv25I-259 (Seq. 11) No., 7) Benv251-6 (Seq. 11D No. 8 Leu-Leu.Cys-Leu-lle-Phe-Leu-LUt-Val-Le. 57 28. The CTh inducing peptide of claim 27, wherein the host has chronic hepatitis B infection oris a hepatitis B carrier.-o 2.The CTL inducing peptide of claim 26, wherein the CTL inducing peptide is: IIBenv335-344 (Seq. ID. No. 1) Trp -Leu-Ser-Leu-Leu-V~al-Pro-Phe-Val;Gn 30. The CTL inducing peptide of claim 26, wherein the host has acute hepatitis B infection- 31. The CTh inducing peptide of claim 26, wherein the CTL inducing peptide is administered prophylactically. 32. The CTL inducing peptide of claim 27, wherein the host has chronic hepatitis B infection or is a hepatitis B carrier. 33. The CTL inducing peptide of claim 26, wherein the CTL inducing peptide is administered to the host with a second peptide which elicits a T helper response to HBV. 34. The GTh inducing peptide of claim 33, wherein the CTh inducing peptide and the T helper inducing peptide are linked. 35. Use of a CTL inducing peptide comprising the following sequence: Llenvl83-191 (Seq. ID No. 1) '*Phe-Leu-Leu-Thr-Arg-Ile-Leu-Thr-Ile; in the preparation of a medicament for use in the treatment andlor prevention of hepatitis B infection, in a host having an IILA-A2 haplotype. 215 36. A CTL inducing peptide when used in the treatment and/or prevention of hepatitis B infection, substantially as hereinbefore described with reference to any one of the Examples. 37. Use of a CTh inducing peptide comprising from eight to thirteen amino acids, S wherein at least a majority of the amino acids are homologous to a corresponding portion of RBenv having the sequence: HBenv248-260 (Seq. ID) No. 2) Phe-lle-Leu-Leu-Leu-Cys.-Leu.-fle-Phe-Leu-Leu-Val-L-au, or HBenv335-343 (Seq. ID) No. Trp-Leu-Ser-Leu-Leu-Vlal-Pro-Phe-Val;- -in the preparation of a muedicament for use in the treatment andlor prevention of hepatitis B infection, in a host having an HIA-A2 haplotype. 38. The use of claim 37, wherein the CTh inducing peptide is: IlBen~v248 -257 (Seq. ID No. 4) lle-Lcu-Leu-Leu-Cys-Leu-lle-Phe-Leu; llev4-258 (Seq. ID No. -Ifflnv24 Hienv25O-2 58 (Seq. ID No. 6) Leu-LUL-CLeYuePhe-uLu; 1-259 (Seq. ID No. 7) uLuCs-e-l-h-lr-uVl or 1-260 (Seq. ID No- 8) UUL -y-e-l-heu-e-a-Lu 39. The use of claim 38, Wherein fth host has chronic hepatitis B infection or is a hepatitis B earnier. 40. The use of claim 37, wherein the CTL inducing peptide is-- HjBenv335- 343 (Seq. lIDNo. lOV Tr-e-e-uLuVlPoPeVl HBenv33 4 343 (Seq. ID). No. se-r-usrLuL-1VtPOPeVl or is ILBenv335- 344 (Seq. ID. No. 16) Gas Trp-Leu-er--eu- ofU41. The use of claim 37, wherein the host has acute hepafitis B3 infection. 42. The use of claim 37, wherein the CTL inducing peptide is administered prophylactically. *O Va2 43. The use of claim 38, wherein the host ha/hoieaii Bifcino saf hepatitis B carrier. The use of claim 37, wherein the CTL inducing peptide is administered to the host with a second peptide which elicits a T helper response to IJBY. me a 45. The use of claim 44, wherein the CTL inducing peptide andi the T helper inducing peptide are linked. 46. Use of a CTL inducing peptide when used in the preparation of a medicamfent for use in the treatment and/or prevention of hepatitis B infectosbanily s he hreinbefore described with reference to anY One of the Examples. B Dated 13 November, 1998 The Scripps Research InstitUte patent Attorneys for -the Appfizornt/Nornifated Person SPRUSON FERGUSON