FIELD OF THE INVENTION The present invention is related to peptides which are suitable for inducing an immune response to HBV. It further relates to the use of said peptides for the preparation of vaccines, compositions and in methods of diagnosis. It is further related to peptides predicted to bind to MHC class 1, and which stimulate cytotoxic T-lymphocytes. It further relates to a method for predicting the properties of peptides bound to MHC class I or class 11 molecules.

BACKGROUND OF THE INVENTION Cytotoxic T-cells (Tc or CD8-T lymphocytes) and helper T-cells (TH or CD4-T lymphocytes) have the capability of recognizing short, processed fragments of a protein antigen, referred to as antigenic peptides or T-cell epitopes. However, recognition does not occur by direct binding to free peptides. Specific receptor molecules on T-cells (T-cell receptors or TCRs) recognize a peptide antigen only when it is bound to another receptor known as a major histocompatibility complex (MHC) molecule. Such MHC-peptide complexes serve the role of cell markers: when the MHC contains an endogenous (self) peptide, it marks the cell as"healthy" ; when it contains a foreign peptide, the cell is marked as"infected". The MHC-mediated presentation of antigenic peptides to the repertoire of T-cells can thus be seen as the primary stimulus to elicit an immune response. Depending on the type of MHC presenting an antigen, which is correlated with the type of cell expressing it, the immune system is triggered to either destroy the antigen presenting cell or to produce antibodies directed against the infectious agent.

MHC molecules are subdivided into classes I and 11. While their general function is the same (presenting antigen), they differ in a number of aspects. MHC class I is expressed on the cell surface as a heterodimeric complex between a 46-kDa heavy chain (the a-chain) and a 12 kDa light chain (the (32-microglobulin or am chain). The a-chain consists of three domains, a,, a2 and OC3 ; the cc1 and a2 domains are responsible for binding of a peptide ligand, while the a3 domain is membrane-bound and involved in CD8 co-receptor binding. Class II MHC molecules have the same overall shape, although they are constituted of two membrane-bound chains: an oc chain of-35 kDa and a ß chain of-28 kDa. Both the a and the ß chain form two domains (a, and a2 on the one hand and pi and pz on the other). The ai and pi domain jointly form the peptide binding domain. The (3z domain is involved in CD4 co-receptor binding.

Both MHC class I and class 11 molecules show a high degree of polymorphism. They have been further subdivided into different subtypes. The existence of different MHC allotypes lies at the basis of the capacity of MHCs to bind a broad range of peptides while still preserving some specificity. Given this polymorphism, being able to predict which peptides specifically bind to which MHC subtypes, is thought to be of great value in vaccination strategies and de- immunization programs. Thanks to the recent burst of information derived from experimentally determined 3D-structures, valuable insights about the determinants of peptide binding specificity have been obtained. This, in turn, has led to the idea that a structure-based prediction of potentially antigenic peptides (or T-cell epitopes) is within reach.

Functional human leukocyte antigens (HLAs or human MHCs) are characterized by a deep binding groove to which endogenous as well as potentially antigenic peptides bind. The groove is further characterized by a well-defined shape and physico-chemical properties. HLA class I binding sites are closed, in that the peptide termini are pinned down into the ends of the groove. They are also involved in a network of hydrogen bonds with conserved HLA residues (Madden, D. R. et al., (1992) Cell 70, 1035-1048). In view of these restraints, the length of bound peptides is limited to 8-10 residues. Superposition of the structures of different HLA complexes confirmed a general mode of binding wherein peptides adopt a relatively linear, extended conformation. At the same time, a significant variability in the conformation of different peptides was observed also. This variability ranges from minor structural differences to notably different binding modes. Such variation is not unexpected in view of the fact that class I molecules can bind thousands of different peptides, varying in length (8-10 residues) and in amino acid sequence. The different class I allotypes bind peptides sharing one or two conserved amino acid residues at specific positions. These residues are referred to as anchor residues and are accommodated in complementary pockets (Falk, K. et a/., (1991) Nature 351,290-296).

Besides primary anchors, there are also secondary anchor residues occupied in more shallow pockets (Matsumura, M. et al., (1992) Science 257,927-934). In total, six allele-specific pockets termed A-F have been characterized (Saper, M. A. et al., (1991) J. Mol. Biol. 219,277-312 ; Latron, F. et al., (1992) Science 257,964-967). The constitution of these pockets varies in accordance with the polymorphism of class I molecules, giving rise to both a high degree of specificity (limited cross reactivity) while preserving a broad binding capacity.

In contrast to HLA class I binding sites, class 11 sites are open at both ends. This allows peptides to extend from the actual region of binding, thereby"hanging out"at both ends (Brown.

J. et a/., (1993) Nature 364,33-39). Class II HLAs can therefore bind peptide ligands of variable length, ranging from 9 to more than 25 amino acid residues. Similar to HLA class 1, the affinity of a class 11 ligand is determined by a"constant"and a"variable"component. The constant part again results from a network of hydrogen bonds formed between conserved residues in the HLA class 11 groove and the main-chain of a bound peptide. However, this hydrogen bond pattern is not confined to the N-and C-terminal residues of the peptide but distributed over the whole of the chain. The latter is important because it restricts the conformation of complexe peptides to a strictly linear mode of binding. This is common for all class 11 allotypes. The second component determining the binding affinity of a peptide is variable due to certain positions of polymorphism within class 11 binding sites. Different allotypes form different complementary pockets within the groove, thereby accounting for subtype-dependent selection of peptides, or specificity.

Importantly, the constraints on the amino acid residues held within class 11 pockets are in general "softer"than for class 1. There is much more cross reactivity of peptides among different HLA class 11 allotypes. Unlike for class 1, it has been impossible to identify highly conserved residue patterns in peptide ligands (so-called motifs) that correlate with the class 11 allotypes.

The different characteristics of class I and class 11 MHC molecules are responsible for specific problems associated with the prediction of potential T-cell epitopes. As discussed before, class I molecules bind short peptides that exhibit well-defined residue type patterns. This has led to various prediction methods that are based on experimentally determined statistical preferences for particular residue types at specific positions in the peptide. Although these methods work relatively well, uncertainties associated with non-conserved positions limit their accuracy. Prediction methods for MHC class li-mediated T-cell epitopes essentially follow the same strategy, but are hampered by the fact that the binding groove is open. The latter makes it difficult to locate, in a pool of peptides identified as binders, the 9-residue segment that is actually responsible for the binding. This fact, combined with the intrinsically weaker constraints of the complementary pockets in class 11 binding grooves, makes the establishment of (pseudo-) motifs very difficult (Mallios, R. R. (2001) Bioinformatics 17, 942-948). On the other hand, class 11 peptide binding motifs generally include more anchor residues than class I motifs.

Methods for MHC/peptide binding prediction can grossly be subdivided into two categories:"statistical methods"that are driven by experimentally obtained affinity data and "structure-related methods"that are based on available 3D structural information of MHC molecules.

Statistical methods have been promoted under the impulse of a growing amount of binding data. Sources of binding information are, typically, elution and pool sequencing of peptides bound naturally to MHC molecules inside cells (Falk, K. et a/., (1994) Immuno-genetics 39,230-242), phage display of peptide libraries (Hammer, J. et aL,, (1993) Cell 74, 197-203.

Fleckenstein, B. et a/., (1999) Sem. Immunol. 11,405-416), data sets compiled from reports in the literature (Brusic, V. et a/., (1998) Nucleic Acids Res. 26,368-371, Rammensee, H. G. et a/., (1999) Immunogenetics 50,213-219). A common approach is to decompose, in a statistical way, the available experimental information into MHC type-specific and peptide residue position- specific numerical values reflecting the preference for individual amino acid types at that position (Parker, K. C. et a/., (1994) J. Immunol. 152,163-175). The matrices obtained in this way may then serve as profiles from which the binding affinity of a peptide sequence of interest can be estimated. Structure-based methods generally include a first step wherein the structure of a specific MHC/peptide complex is modeled and a second step wherein the binding strength of the peptide is estimated from the modeled complex in accordance with an empirical scoring function.

Examples include WO 98/59244, Altuvia, Y. et a/., (1995) J. Mol. Biol. 249,244-250 ; Doytchinova, I. A. and Flower, D. R. (2001) J. Med. Chem. 44,3572-3581). Alternatively, a molecular dynamics simulation is sometimes performed to model a peptide within an MHC binding groove (Lim, J. S. et a/. (1996) Mol. Immunol. 33,221-230). Another approach is to combine loop modeling with simulated annealing (Rognan, D. et a/., (1999) J. Med. Chem. 42, 4650-4658). Most research groups emphasize the importance of the scoring function used in the affinity prediction step. Schueler-Furman et al. (Schueler-Furman, O. et a/., (2000) Prot Sci. 9, 1838-1864) apply a statistical potential to evaluate the contacts between the peptide and the MHC receptor. Rognan et al. (1999) rely on a quantification of physicochemical effects (like H- <BR> <BR> bond formation, lipophilic contacts, desolvation, etc. ). Swain et a/. (Swain, M. T., et a/., (2001) Proceedings of the second IEEE International Symposium on Bioinformatics and Biomedical Engineering. IEEE computer Society Press, Bethesda, Maryland, pp. 81-88) also apply a heuristic scoring function based on inter-atomic contacts, electrostatic interactions and H-bond formation. Doytchinova and Flower (2001) consider essentially the same contributions but follow a quantitative structure-affinity relationship (QSAR) method to assess the binding affinity.

Logean et a/. (Logean, A., et a/., (2001) Bioinorg. & Med. Chem. Letters 11,675-679) have analyzed the performance of 7 universal scoring functions. They found that many of these scoring functions yield poor correlation with experiment, in contrast to their"Fresno"scoring function. However, it was also recognized that the Fresno function cannot be universally applied but requires recalibration for different protein-ligand systems.

There is a need to substantially improve both the structure prediction and the affinity assessment steps of methods which predict the affinity of a peptide for a major histocompatibility (MHC) class I or class 11 molecule. The main problem encountered in this field is the poor performance of prediction algorithms with respect to MHC alleles for which experimentally determined data (both binding and structural information) are scarce. It is an aim of the present invention to provide a novel method for predicting the affinity of a peptide for a major histocompatibility (MHC) class I or class 11 molecule, also in cases where experimental information is rare.

Chronic infection with hepatitis B virus (HBV) is a major cause of morbidity and mortality in all regions of the world, and is the ninth most common cause of death worldwide. Hepatitis B is transmitted primarily by blood-to-blood or sexual contact; oral transmission has also been documented. Replication of the HBV is only mildly cytopathic toward the host cell, but cellular immune responses inducing HBV-induced liver damage are a major cause of chronic hepatitis,' cirrhosis and primary liver cancer. There are an estimated 350 million carriers of HBV worldwide, and nearly one-third of these individuals are expected to develop progressive liver disease.

Consequently, the demand for reliable diagnostic methods and effective therapeutic measures is high.

First-generation hepatitis B vaccines used the hepatitis B surface antigen, the noninfectious surplus protein coat of the HBV, purified from the plasma of asymptomatic human carriers and subsequently subjected to at least two inactivation procedures.

Second-generation vaccines based on recombinant DNA hepatitis B surface antigen (HBsAg) protein produced in stable transfected yeast or Chinese hamster ovary cells have replaced plasma-derived vaccines in many countries. The latter vaccines are very effective but expensive, which limits their widespread use, especially in underdeveloped countries where HBV is endemic. Although a number of factors are known to adversely affect the antibody response to the surface antigen proteins of HBV, the mechanisms underlying non- responsiveness in humans remain largely unexplained. Vaccines incorporating significant levels of the surface components (S, pre-S1 and pre-S2) of the viral envelope proteins have demonstrated enhanced immunogenicity when compared with conventional S antigens expressed in yeast-derived vaccines. Several third-generation recombinant HBV vaccines incorporating all three S components of the viral envelope proteins are under clinical development as prophylaxis and immunotherapy for hepatitis B.

Studies of hepatitis B vaccine use demonstrate that vaccination can significantly reduce the development of chronic hepatitis B infections and decrease the risk and incidence of primary liver cancer. The U. S. FDA, the CDC and the World Health Organization (WHO) have recognized the hepatitis B vaccine as a significant tool to reduce chronic hepatitis B infections which may develop into primary liver cancer. In 1991, the CDC recommended hepatitis B immunization for all infants and in 1994 further recommended vaccination for those adolescents not previously immunized.

DNA-based immunization, represents a novel approach for the development of therapeutic and prophylactic vaccines against HBV. Genetic vaccination can stimulate both B and T cell responses. DNA vaccination has proven to decrease the production of HBsAg in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state. Data from clinical studies using DNA vaccines will determine whether DNA-based immunization can also induce virus clearance in humans.

The oral administration of antigens has traditionally been problematic, given that oral absorption is low and thus relatively large quantities of the antigen must be administered.

Certain sugars (galactose, lactose and sorbitol), however, have been shown to significantly enhance mucosal uptake of viral antigens and other mucosal immunogens. This finding has stimulated a new area of research: that of edible vaccines. ProdiGene, a Texas-based biotechnology company that develops and commercializes novel proteins from transgenic plants, is working on the development of an edible human vaccine for hepatitis B produced in transgenic plants. U. S. patent number 5,914, 123 (ProdiGene) discloses an edible hepatitis B vaccine, preferably in the form of a fruit or vegetable juice, produced in transgenic plants (e. g., tomatoes or potatoes) expressing the hepatitis B surface antigen. Scientists at the Polish Academy of Sciences obtained transgenic lupin callus expressing HBsAg, which was fed to mice. Mice fed the transgenic lupin tissue developed significant levels of HBsAg-specific antibodies. Human volunteers fed transgenic lettuce plants expressing HBsAg developed serum-specific IgG responses to the plant-produced protein.

The development of chronic hepatitis B infection results from an inadequate host immune response leading to an inability to eliminate HBV. It has been proposed that adjuvanted HBV vaccines may circumvent tolerance and produce efficacious anti-HBV responses. Thus, various adjuvants are being studied to enhance the antigen-specific immune responses when combined with hepatitis B vaccine antigen (s).

The number of chronic carriers of HBV is still around 350 million ; around 3 million of these live in Europe or the United States and many more in the developed countries in Asia; 200,000 new cases are reported in the United States every year. The current treatment for Hepatitis B consists of a two year administration of interferon alpha and lamivudine, which works in 25-35% of cases. Therefore, there still is a need to develop therapeutic vaccination.

A naked DNA/Alvac recombinant vaccinia virus approach, expressing HbsAg but not pre S1 or pre S2, was tested in a single chimpanzee chronically infected with HBV. Although right after the canarypox (Pasteur/Virogenetics) boost, viral DNA was reduced to non-detectable levels, or 2.5 logs, as assayed by quantitative PCR, and more sensitive nested PCR showed a level of about 10 copies per mL viral HbsAg, mRNA and protein levels were not affected. The decrease coincided with increased levels of interferon gamma, but not CTLs (Pancholi et al., 2001).

Although prophylactic HBV vaccination in the developed countries has brought new infections to a standstill, a large percentage of the world's population is still infected with HBV.

People that are chronically infected have a 10% chance of developing liver cancer, and a 10- 25% chance of developing liver cancer and cirrhosis if they were infected as a child. An average 8% of the people in Africa, South-East Asia (including Korea, The Philippines and Indonesia) are chronically infected; in eastern and southern Europe, Japan, central Asia and Russia the average varies between 2-7%, while in western Europe and the US the rate stands at 2%.

Current therapeutic practice involves the application of various forms of interferon alpha, lamivudin, a combination of these two or thymosin-alpha, with success rates between 40-70%.

Presently there are no therapeutic vaccines for HBV that are effective, the reason being that immunogenic epitopes of HBV are not known. Thus, there is a need for therapeutic vaccines which aim at inducing a strong T-cell response and at the same time prevent destruction of liver tissue. It would also be desirable to elicit more effective immunity, such as by increasing or diversifying the immunogenicity of the vaccines.

The immune response to HBV is believed to play an important role in controlling hepatitis B infection. A variety of humoral and cellular responses to different regions of HBV including the nucleocapsid core, polymerase and surface antigens have been identified. T-cell mediated immunity, particularly involving class I human leukocyte antigen-restricted cytotoxic T lymphocytes (CTL), is believed to be crucial in combatting established HBV infection.

Class I human leukocyte antigen (HLA) molecules are expressed on the surface of almost all nucleated cells. CTL recognize peptide fragments, derived from intracellular processing of various antigens, in the form of a complex with class I HLA molecules. This recognition event then results in the destruction of the cell bearing the HLA-peptide complex <BR> <BR> directly or the activation of non-destructive mechanisms e. g. , the production of interferon, that inhibit viral replication.

In view of the heterogeneous immune response observed with HBV infection, induction of a multi-specific cellular immune response directed simultaneously against multiple epitopes appears to be important for the development of an efficacious vaccine against HBV. There is a need to establish vaccine embodiments that elicit immune responses that correspond to responses seen in patients that clear HBV infection. Epitope-based vaccines appear useful.

AIMS OF THE INVENTION It is an aim of the invention to provide immunogenic peptides which may be used for the preparation of therapeutic and prophylactic vaccines for the treatment and prevention of HBV infections. It is a further aim of the invention to provide compositions comprising said peptides as well as methods for their preparation.

SUMMARY OF THE INVENTION The present invention is related to peptides that are predicted to bind with a high affinity to HLA-A2, HLA-A24, HLA-CW4, HLA-A3, HLA-A1. The peptides of the invention are suitable for inducing an immune response to HBV. Said peptides are predicted to interact with MHC class I according to a method of the invention, and suited to provoke an immune response by stimulation of cytotoxic T-cells. Furthermore, the peptides of the invention are predicted to react with multiple HLAs and therefore are capable of stimulating a strong CTL response.

Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. There is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations. The epitopes for inclusion in an epitope-based vaccine are selected from conserved regions of viral or tumor-associated antigens, which thereby reduces the likelihood of escape mutants. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.

An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes, and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.

Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.

An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen. Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from that pathogen in a vaccine composition. A"pathogen"may be an infectious agent or a tumor associated molecule.

One of the most formidable obstacles to the development of broadly efficacious epitope- based immunotherapeutics, however, has been the extreme polymorphisin of HLA molecules.

To date, effective non-genetically biased coverage of a population has been a task of considerable complexity ; such coverage has required that epitopes be used specific for HLA molecules corresponding to each individual HLA allele, therefore, impractically large numbers of epitopes would have to be used in order to cover ethnically diverse populations. Thus, there has existed a need to develop peptide epitopes that are bound by multiple HLA antigen molecules for use in epitope-based vaccines.

The greater the number of HLA antigen molecules bound, the greater the breadth of population coverage by the vaccine. Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to multiple HLA alleles. The peptides of this invention have the additional advantage that each peptide itself binds to multiple HLA alleles.

Furthermore, as described herein in greater detail, a need may exist to modulate peptide binding properties, for example, so that peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response.

Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor to prevent or clear an infection in a diverse segment of the population. Such a response can also target a broad array of epitopes. The technology disclosed herein provides for such favored immune responses.

In a preferred embodiment, epitopes for inclusion in vaccine compositions are evaluated for the affinity for a major histocompatibility (MHC) class I or class 11 molecule. Said peptides are then tested for the ability to bind to the MHC HLA molecule. Those peptides predicted and

tested with an intermediate or high affinity are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptides are selected for inclusion in vaccine compositions.

High affinity peptides may additionally be tested for the ability to bind to multiple alleles within the HLA supertype family. Moreover, peptide epitopes may be analogue to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype.

An alternative modality for defining the peptides in accordance with the invention is to recite the physical properties, such as length ; primary, potentially secondary and/or tertiary structure; or charge, which are correlated with binding to a particular allelespecific HLA molecule or group of allele-specific HLA molecules. A further modality for defining peptides is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e. g. pocket configuration and charge distribution) and reciting that the peptide fits and binds to said pocket or pockets.

The present invention relates to an isolated or purified peptide comprising an MHC class I restricted T-cell stimulating epitope of the hepatitis B virus (HBV) surface polypeptide selected from the group of peptides represented by SEQ ID NOs 540,546, 552,414, 347,348, 402,437, 346,443, 280,542, 342,224, 350,539, 183,547, 227,544, 551,541, 182,343, 228,300, 340, 410,299, 245,538, 246,265, 298,238, 220,318, 429,248, 223,413, 296,185, 314,339, 250, 409,351, 335,268, 247,281, 235,545, 325,548, 218,341, 221,317, 372,373, 222,344, 338, 177,175, 311,241, 244,332, 415,179, 292,178, 461,412, 217,406, 368,401, 375,442, 459, 439,396 and 371 characterized in that said peptide binds at least two, preferably at least three different MHC class I HLA types selected from the group comprising HLA-A2, HLA-A24 and HLA-CW4. Most preferably said isolated or purified peptide binds to at least two, three, four or all of the following five MHC class I HLA types: HLA-A2, HLA-A24, HLA-CW4, HLA-A3 and HLA-A1 and is selected from the group of peptides represented by SEQ ID NOs 540,348, 347,414, 416, 542,402, 443,346 and 552.

The present invention further relates to an isolated or purified peptide comprising an MHC class I restricted T-cell stimulating epitope of the hepatitis B virus (HBV) core polypeptide selected from the group of peptides represented by SEQ ID NOs 5,68, 85,130, 64,47, 8,60, 138,108, 65,58, 57,132, 83,66, 129,139, 84,136, 7,61, 134,135, 10,25, 163,42, 79,9, 70, 80,43, 62,152, 161,148, 63,3, 46,71, 59,4, 6,82, 55,154, 67,81, 56,153, 48,159, 156,171, 167 and 69 characterized in that said peptide binds at least two, preferably at least three different MHC class I HLA types selected from the group comprising HLA-A2, HLA-A24 and HLA-CW4. Most preferably said isolated or purified peptide is represented by SEQ ID NO 5 and binds to the following five MHC class I HLA types: HLA-A2, HLA-A24, HLA-CW4 and HLA-A3.

The present invention also relates to an isolated or purified peptide comprising an MHC class I restricted T-cell stimulating epitope of the hepatitis B virus (HBV) polymerase polypeptide selected from the group of peptides represented by SEQ ID NOs 1120,1042, 1229,1039, 1046, 662,1353, 668,660, 1049,1038, 1060,987, 1069,1145, 1233,667, 661,1347, 1144,1388, 1041,1148, 986,556, 684,1062, 1043,663, 873,1150, 1070,1044, 692,1272, 1066,1020, 1141,682, 1074,670, 1037,621, 982,555, 984,1227, 1071,677, 1361,992, 989,952, 1122, 1143,1045, 1118,1214, 1234,627, 665,687, 1239,1268, 1230,1270, 1354,749, 1048,999, 985,954, 696,1151, 1385,991, 890,1199, 1040,1231, 1232,601, 988,1164, 1377,1260, 891, 664,799, 955,1349, 1209,1210, 1271,755, 1350,681, 757,678, 802,1198, 1050,1092, 1138, 1310,689, 646,1061, 1146,1073, 1098,1235, 1224,1382, 1052,871, 644,1124, 674,1173, 1343,1064, 669,983, 1047,1317, 1237,1072, 1152,800, 679,607, 1188,1267, 1035,750, 675,878, 1156,838, 1142,812, 1108,1383, 737,1115, 1157,1082, 1149,630, 705,804, 1200, 996,1236, 709,1112, 1348,1093, 625,1185, 1269,763, 1290,1311, 1256,1255, 722,995, 914,836, 1013,1345, 1283,797, 1128,1063, 1019,1312, 554,1106, 1371,849, 856,866, 933, 835,854, 998,683, 876,648, 925,1297, 619,1315, 1346,1355, 617,1169, 1000,1116, 981, 566,1107, 1168,1298, 828,1254, 1246,570, 1273,1121, 1257,629, 833,888, 1184,872, 837, 618,1362, 752,609, 680,1097, 935,718, 792,645, 1123,721, 1003,658, 671,972, 821,898, 756,1001, 892,1352, 912 and 1068 characterized in that said peptide binds at least two, preferably at least three different MHC class I HLA types selected from the group comprising HLA-A2, HLA-A24 and HLA-CW4. Most preferably said isolated or purified peptide binds to at least two, three, four or all of the following five MHC class I HLA types: HLA-A2, HLA-A24, HLA- CW4, HLA-A3 and HLA-A1 and is selected from the group of peptides represented by SEQ ID NO 1120,1042, 1229,662, 1038,1039, 1046,1060, 1069,1144, 1145,1353, 660 and 873.

The present invention also relates to an isolated or purified peptide comprising an MHC class I restricted T-cell stimulating epitope of hepatitis B virus (HBV) selected from the group of sequences represented in Tables 1,2, 3,4, 5,6 and 7.

The present invention also relates to a peptide consisting of multiple repeats, combinations, or mimotopes of any of the peptides as mentioned above, with said combinations comprising at least one peptide according to any of the preceding claims possibly joined with any other peptide into a single structure and with said mimotopes having one or more amino acid variations compared to said peptides as long as said mimotope peptides are capable of providing for immunological stimulation after which the T-cells are reactive with HBV.

The present invention further relates to the use of an HBV peptide as mentioned above for the preparation of an HBV immunogenic composition.

The present invention also relates to the use of a recombinant expression vector comprising a nucleic acid insert encoding a peptide as mentioned above for the preparation of an HBV immunogenic composition.

The present invention further relates to said use, wherein said composition induces a cytotoxic-T lymphocyte (CTL) response.

Further, the present invention relates to said use, wherein said composition is a prophylactic vaccine composition.

The present invention also relates to said use, wherein said composition is a therapeutic vaccine composition.

The present invention further relates to said use, wherein said composition is a diagnostic composition.

Further, the present invention relates to a composition comprising at least one of the peptides as mentioned above, mixed with a pharmaceutically acceptable excipient.

The present invention also relates to a composition as mentioned above wherein the peptide is admixed or linked to a second molecule.

Further, the present invention relates to said composition wherein said second molecule is a helper T lymphocyte epitope.

Further, the present invention relates to the above-mentioned composition further comprising a liposom.

The present invention also relates to this composition, wherein the peptide is complexe with an MHC class I molecule that is present on an antigen presenting cell.

The present invention further relates to the composition as mentioned above which is an immunogenic composition.

Further, the present invention relates to said composition which is a vaccine composition.

The present invention also relates to a composition as mentioned above which is a prophylactic vaccine composition.

The present invention further relates to a composition as mentioned above which is a therapeutic vaccine composition.

The present invention also relates to this composition, comprising an additional peptide containing at least one B-cell stimulating epitope of HBV, and/or a structural HBV polypeptide, and/or a non-structural HBV polypeptide.

The present invention also relates to said composition, wherein said additional peptide is mixed with HBsAg or HBsAg particles, HBV immunogens, HCV immunogens, HIV immunogens and/or HTLV immunogens.

The present invention further relates to a composition as mentioned above, capable of a cytotoxic-T lymphocyte (CTL) response.

Further, the present invention relates to an HBV immunogenic composition comprising a recombinant expression vector comprising a nucleic acid insert encoding a peptide as mentioned above, for the preparation of the HBV immunogenic composition for DNA-based immunisation.

The present invention further relates to an in vitro method of detecting in lymphocytes of a mammal, CTLs that respond to a MHC class I restricted T-cell stimulating peptide of HBV, comprising the steps of : (a) contacting target cells with a polypeptide comprising at least one of the peptides as mentioned above, wherein said target cells are of the same HLA class as the lymphocytes to be tested for said CTL, (b) contacting said lymphocytes to be tested for said CTL with a peptide comprising at least one of the peptides as mentioned above, and (c) determining whether said lymphocytes exert a cytotoxic effect on said target cells.

The present invention also relates to in vitro use of a peptide as mentioned above for the preparation of an immune response provoking vaccine in the event of HBV infection, said vaccine being prepared by contacting said peptide in an immune response-provoking amount of specific CTL.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, a"peptide"refers to at least two covalently attached amino acids which includes polypeptides and oligopeptides. The peptide may be made up of naturally occurring amino acids and peptide bonds, or non-naturally-occurring amino acids or synthetic <BR> <BR> peptidomimetic structures, i. e. , "analogs"such as peptoids [see Simon, R. J. et a/., (1992) Proc.

Natl. Acad. Sci. U. S. A. 89 (20), 9367-9371], generally depending on the method of synthesis.

"Amino acid", or"residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline, and noreleucine are considered amino acids for the purposes of the invention."Amino acid"also includes imino acid residues such as proline and hydroxyproline. In addition, any amino acid representing a component of the variant proteins of the present invention can be replaced by the same amino acid but of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D-amino acid but which can additionally be referred to as the R-or the S-, depending upon its composition and chemical configuration. Such derivatives have the property of greatly increased stability, and therefore are advantageous in the formulation of compounds which may have longer in vivo half lives, when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non- naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly ; see van Hest et al., FEBS Lett 428: ( 1-2) 68-70 May 221998 and Tang et a/., Abstr. Pap Am. Chem. S218: U138-U138 Part 2 August 22,1999, both of which are expressly incorporated by reference herein.

Aromatic amino acids may be replaced with D-or L-naphylalanine, DM or L- Phenylglycine, D-or L-2-thieneylalanine, D-or L-1-, 2-, 3-or 4-pyreneylalanine, D-or L-3- thieneylalanine, D-or L- (2-pyridinyl)- alanine, D-or L- (3-pyridinyl)-alanine, D-or L- (2-pyrazinyl)- alanine, D-or L- (4-isopropyl)- phenylglycine, D- (trifluoromethyl)-phenylglycine, D- (trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D-or L-p-biphenylphenylalanine, D-or L-p-methoxybiphenylphenylalanine, D-or L-2-indole (alkyl) alanines, and D-or L-alkylainines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono) alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfate (e. g.,-SO3H) threonine, serine, or tyrosine.

Other substitutions may include unnatural hyroxylated amino acids may made by combining"alkyl"with any natural amino acid. The term"alkyl"as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur.

Preferred alkyl groups herein contain 1 to 12 carbon atoms. Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, <BR> <BR> arginine, ornithine, citrulline, or (guanidino) -acetic acid, or other (guanidino) alkyl-acetic acids,<BR> where"alkyl"is define as above. Nitrile derivatives (e. g. , containing the CN-moiety in place of COOH) may also be substituted for asparagine or glutamin, and methionine sulfoxide may be substituted for methionine. Methods of preparation of such peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage in any of the variant polypeptides can be replaced by a ketomethylene moiety. Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

Additional amino acid modifications of amino acids of variant polypeptides of to the present invention may include the following : Cysteinyl residues may be reacted with alpha- haloacetates (and corresponding amine), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta- (5- imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, P-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7- nitrobenzo-2-oxa-1, 3-diazole.

Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e. g. , at pH 5.5 to 7.0 because this agent is relatively specific for the histidyl<BR> side chain, and para-bromophenacyl bromide may also be used, e. g. , where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues.

Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters e. g. , as methyl picolinimidate ; pyridoxal phosphate; pyridoxal ; chloroborohydride ; trinitrobenzenesulfonic acid; O-methylisourea ; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1, 2- cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosyl residues per se is well-known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.

N-acetylimidizol and tetranitromethane may be used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as 1-cyclohexyl-3- (2-morpholinyl- (4- ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4, 4-dimethylpentyl) carbodiimide. Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.

As used herein"side-chain placement algorithm"refers to methods for optimizing the side-chain conformations of residues. Non-limiting examples of such methods include International Patent Application No. WO 01/33438, De Maeyer et a/ (De Maeyer et a/., (2000) Methods in Molecular Biology, vol. 143 : Protein Structure Prediction : Methods and Protocols. <BR> <BR> <P>Webster, D. (Ed. ) Humana Press Inc., Totowa, NJ, pp. 265-304), Koehl, P. and Delarue, M. (J.

Mol. Biol. (1994) 239,249-275), Shenkin, P. S. et al., (Shenkin, P. S. et al., (1996) Proteins 26, 323-352), Tuffery et al. (Tuffery, P. et al., (1997) Protein Eng. 10,361-372), Holm and Sander (Proteins (1992) 14,213-223 1992). Further included are methods which explicitly account for pair-wise side-chain/side-chain interactions.

As used herein,"dead-end-elimination"or"DEE"refers to methods for testing which side-chain conformations are energetically incompatible with the globally optimal side-chain <BR> <BR> arrangement onto a protein backbone (or template) structure (e. g. Desmet, J. et a/. , (1992) Nature 356,539-542). In a protein system to be tested, each amino acid residue is first represented by a limited set of discrete side-chain conformations obtained from a library of theoretically possible conformations, also known as a rotamer library. To arrive at a globally optimal conformation for the protein system, rotamers are screened in accordance to one or more mathematical expressions, called DEE criteria. Different valid elimination criteria have <BR> <BR> been identified in the past (De Maeyer, M. , Desmet, J. and Lasters, I. (2000) The dead-end elimination theorem: mathematical aspects, implementation, optimizations, evaluation and <BR> <BR> performance. in: Methods in Molecular Biology, vol. 143 : De Maeyer, M. , Desmet, J. and Lasters, I. (2000) and references therein). Upon convergence, all but one rotamers have been eliminated for each modeled side-chain so that the final, unique assignment of rotamers corresponds to the global optimum. If convergence cannot be reached by merely applying DEE criteria, some additional end-stage routines are required (Desmet et a/., 1997).

As used herein"fast and accurate side-chain topology and energy refinement"or "FASTER"refers to methods of International Patent Application No. WO 01/33438 which is incorporated herein by reference.

With regard to a particular amino acid sequence, an"epitope"is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor (TCR) proteins and/or Major Histocompatibility Complex (MHC receptors). In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, TCR or HLA molecule. Throughout this disclosure epitope and peptide are often used interchangeably.

It is to be appreciated that protein or peptide molecules that comprise an epitope of the invention as well as additional amino acid (s) are still within the bounds of the invention.

"Nested epitopes"occur where at least two epitopes overlap in a given polypeptide.

A"minigene construct"encodes a peptide comprising one or multiple epitopes.

"Human Leukocyte Antigen"or"HLA"is a human class I or class li Major Histocompatibility Complex (MHC) protein.

"Major Histocompatibility Complex"or"MHC"is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses.

In humans, the MHC complex is also known as the HLA complex. For a detailed description of <BR> <BR> the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RDED. , Raven Press, New York, 1993.

"IC50"is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Binding is expressed relative to a reference peptide. The assessment of whether a peptide is a good, intermediate, weak or negative binder is based on its IC5o relative to the IC50 of a standard peptide."High affinity"with respect to HLA class I <BR> <BR> molecules is defined as binding with an ICgo of 50 nM or less ; "intermediate affinity"is binding with an IC50 value of between about 50 nM and about 500 nM.

"Binding"of the peptide to the HLA-antigen forms an HLA-peptide complex, or a peptide- receptor complex.

The terms"identical"or percent"identity, "in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithms or by manual alignment and visual inspection.

An"immunogenic peptide"or"peptide epitope"is a peptide that will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.

The term"peptide"is used interchangeably with"oligopeptide"and"polypeptide"in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. In some embodiments, the preferred CTL-inducing polypeptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues.

"Pharmaceutically acceptable"refers to a generally non-toxic, inert, and physiologically compatible composition.

A"protective immune response"or"therapeutic immune response"refers to a CTL and/or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.

"Promiscuous recognition"is where a distinct peptide is recognized by the same T cell clone in the context of multiple HLA molecules. Promiscuous binding is synonymous with cross- reactive binding.

As used herein, a"vaccine"is a composition that contains one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more epitopes of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e. g. , a minigene that encodes a polyepitopic peptide. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class 1-binding peptides of the invention can be admixed with, or linked to, HLA class 11-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can also comprise peptide-pulsed antigen presenting cells, e. g. , dendritic cells.

Method for structure-based prediction of the affinity of potentially antiqenic peptides for maior histocompatibility (MHC) receptors.

The present invention relates to the use of a method for structure-based prediction of the affinity of potentially antigenic peptides for major histocompatibility (MHC) receptors. More specifically, a method to provide a quantitative assessment of the affinity of a selected peptide sequence for a selected MHC allotype through (i) analysis of the three-dimensional structure of an MHC peptide binding domain, (ii) by generating multiple conformations for the backbone of the selected peptide, (iii) by optimizing the side-chain conformation for each MHC/peptide main- chain structure, and (iv) by computing the expected binding affinity of the MHC/peptide complex, thereby including a conformational entropy component derived from the set of generated conformations. The application of this method to multiple peptides and/or multiple MHC receptor types may be helpful to identify the most antigenic peptides originating from a common source, for example from a specific viral or bacterial species or a therapeutic protein molecule. This, in turn, may be useful in vaccination or de-immunization applications.

A first step comprises receiving an experimentally determined three-dimensional (3D) structure for a selected MHC class I or class 11 allotype is retrieved. If a suitable 3D structure is not available, it is modeled by homology to a known structure which preferably has a maximal amino acid sequence identity with the selected MHC allotype. The retrieved or modeled structure consists, at least, of those amino acid residues forming the peptide binding site.

In a second step, multiple conformations for the main-chain of the selected peptide are generated, either by retrieval from an MHC/peptide main-chain library or by a suitable computer modeling algorithm, preferably a docking algorithm. The said library may be a compilation of experimentally determined structures or structures generated in advance by a suitable computer modeling algorithm, preferably a docking algorithm.

In a third step, for each peptide main-chain conformation generated in the second step, the conformation of side-chains of the selected peptide are modeled by applying a suitable side- chain placement algorithm, preferably a FASTER or a DEE method, in conjunction with a first energy-based scoring function, preferably a potential or free energy function. The co-modeling of the MHC receptor structure with that of the peptide is a preferred option. The result of this third step is a set of full complex structures at atomic level of detail.

In a fourth step, the ensemble of modeled structures obtained in the third step is evaluated in accordance with a second scoring function hereinafter called the"affinity scoring function". The latter is suited especially to evaluate the binding affinity of a peptide ligand to a receptor. The affinity scoring function preferably includes components related to the conformational energy, the effect of solvent, and parametrized amino acid type-based terms. An essential component of the affinity function is the incorporation of an entropical contribution, preferably derived in accordance with statistical mechanical laws and applied to the complete ensemble of modeled structures, as generated in the third step. The explicit generation of structural ensembles is intended to account for, essentially, the conformational freedom (or flexibility, micro-states, entropy etc. ) of the complex.

A method as used in the present invention concerns the quantitative prediction of the binding affinity of a given peptide for a given MHC allotype. A method might be applied to multiple peptides and/or multiple receptors by repeated application of the basic method for a single peptide/receptor system. The considered MHC molecules are of any class, preferably of class I and class il.

There are no limitations to the amino acid composition or the length of the simulated peptide. The length of simulated class l-binding peptides may be less than 30 residues, preferably less than 20 and more preferably between 8 to 10 residues. The length of class 11 simulated peptides may be less than 30 residues, preferably less than 20 and more preferably restricted to nonapeptides (9-residue peptides) in view of the experimental evidence that fragments of this length form the region of contact with the receptor binding groove.

A method as used in the present invention relates to the quantitative prediction of affinity values. Properties that are directly related with binding affinity comprise binding free energy, association/dissociation constants and IC50 values. The prediction of these values forms part of the invention. Properties that are indirectly related with binding affinity comprise, for example, association/dissociation rates (on/off rates), immunogenicity and conformational flexibility. An aspect of the present invention may be the use of a method for prediction of kinetic and immunogenic properties. Another aspect of the present invention may relate to the use of a method for simulation and quantification of conformational flexibility.

The method as used in the present invention provides a novel approach to structure- based prediction of MHC/peptide affinities, comprising a quantitative assessment of the affinity of a selected peptide sequence for a selected MHC allotype through four computational steps.

The first three steps relate to the prediction of multiple 3D structures for the selected MHC/peptide complex by gradually adding levels of detail in the consecutive modeling steps.

The fourth step analyzes structural information and applies a specific scoring function in order to translate the structural information into a predicted peptide binding affinity. A method of the present invention comprises steps 1 to 4, summarized as follows (see also FIGURE 1).

1. MHC template construction. A suitable 3D model for the selected MHC allotype is generated, either by retrieval from the Protein Databank (PDB) or by a standard homology modeling method. This model serves as an input template structure for the next steps. The model is devoid of any peptide structure, i. e. the binding groove is"emptied". For the purpose of this section only, the model is referred to as"MHC".

2. MHClpeptide main-chain construction. The MHC template structure from step 1 is complemented with an ensemble of peptide backbone (i. e. main-chain) conformations. This leads to an ensemble of 3D structures consisting of a structurally constant part, MHC, and a variety of peptide main-chain structures. For the purpose of this section only, the said ensemble is named"{Pmc}"-The union of MHC and the multiple representations of peptide backbones is denoted as''{MHC/pmc} Xw in this description. The latter set of structures may be generated, for example, by a suitable computer modeling algorithm that yields multiple energetically feasible peptide backbone configurations in relation to MHC, called, for the purpose of this description, a "docking approach". In another example, the set of structures may be generated by a method which retrieves pre-oriented peptide structures from a library, said method called the"database approach"for the purpose of this description. Both approaches are discussed in detail below.

3. MHClfull peptide construction. A third step concerns the addition and modeling of side- chains. In accordance with the amino acid sequence of the selected peptide, each residue position of pmc in each structure of the set {MHc/pmc} is provided with the correct side-chain. In the event that the correct side-chains are already present (for example, if step 2 was performed by docking of the same peptide), the mutation step may be skipped. More important is the modeling of each MHC/pmc. In one embodiment of the present invention, this is accomplished by a suitable side-chain placement algorithm such as a FASTER or a DEE method. The modeling of side-chains may not necessarily be limited to those of the peptide; one aspect of the invention is to include in this step a number MHC side-chains as well. Even if step 2 was performed by a docking method, the invention allows for the re-modeling of at least all receptor side-chains in contact with the peptide, in addition to the side-chains of the peptide itself. Thus, step 3 of a method of the present invention delivers an ensemble of full complex structures at atomic detail, denoted as {MHC/pf,, for the purposes of this description, wherein the side-chain conformations are optimally adapted to each pmo structure in relation to MHC.

4. MHClpeptide affinity assessment. One aim of step 4 is to compute a single scoring value reflecting the binding affinity of the selected peptide for the selected MHC allotype. A source of input data is the structural information obtained in step 3. The final score of the considered system is obtained by applying a function called the affinity scoring function, F, for the purpose of the present description, which has been optimized so as to correlate with the true thermodynamic free energy of binding. As explained further below, this function comprises preferably components related to the conformational energy, the effect of the solvent, and specific amino acid type-based terms that have been parametrized. These types of contributions are not ensemble properties, i. e. they are computed for each individual structure of the set MHClpf"n}. Yet, working with multiple structures, or ensembles, enables certain structure- derived contributions to be averaged, thereby reducing the noise level. Processing these contributions leads to a first component of the predicted affinity under the form of an average energy component for the whole ensemble, termed <E> for the purpose of the present description. Another essential component of F is the entropical contribution (termed S for the purpose of the present invention), derived in accordance with statistical mechanical rules and accounted for by an equation: F = <E>-c S In equation [1], c is a parametrized constant which theoretically corresponds with the absolute temperature (in degrees Kelvin) at which the MHC/peptide system is simulated. The entropy contribution S is preferably taken to be the logarithm of the number of energetically acceptable structures within the set {MHC/pfull}. Clearly, S is an ensemble property reflecting the overall conformational flexibility of the selected peptide in the complex. It is also noteworthy that the more negative <E> and the more positive S, the lower will be F, thus the higher will be the predicted affinity, in agreement with thermodynamic principles.

In step 2 of the method of the present invention-obtaining an ensemble of multiple conformations for the main-chain of the peptide located in the target-MHC binding site-two means for generating said ensembles are suggested as examples: (A) A basic method, also referred to as the"docking approach", wherein peptide main- chain conformations or"binding modes"are generated via molecular modeling, preferably peptide docking.

(B) An advanced method, also referred to as the"database method", wherein peptide main-chain conformations are retrieved from a database of structures.

An underlying hypothesis of the database method might be explained by the following : peptides can assume only a limited number of binding modes, irrespective of their amino acid sequence. Assuming the validity of this hypothesis, this means that different independently performed docking experiments of peptides varying in sequence (but not in length) are likely to show some partial overlap between the generated ensembles. In a more formal notation this corresponds to the situation wherein- {MHC/pmc} {MHC/p'mc} W [2] The merging of a sufficient number of ensembles resulting from independent docking experiments with different peptide sequences may therefore lead to the establishing of a generalized ensemble of possible MHClpmc structures, hereby denoted as {MHClPmc}. The exact amino acid sequence of each peptide in this ensemble then becomes irrelevant (in view of the structural overlap between the constituting populations). In other words, the set {MHC/Pmc} might be seen as the structure MHC provided with a variety of pure peptide backbone conformations, or"poly-alanine"peptide conformations.

An aspect of the present invention in which peptide main-chain conformations are retrieved from a library has advantages over other methods. One advantage is of course a drastic reduction of the computational time per peptide. Docking simulations are often extremely demanding in computing time because of the huge search space. (The latter consists of three translational, three rotational and a large number of conformational degrees of freedom, making <BR> <BR> up a total space with very high dimension. ) An indirect advantage is the fact that the prediction accuracy can be improved because more attention can be paid to the important side-chain placement and affinity prediction steps. Finally, for various technical reasons some peptide binding modes may be missed in a docking experiment, whereas they are de facto represented in the generalized ensemble, on condition that the latter covers the full accessible space.

An ensemble {MHC/PmC} only depends on two variables: MHC allotype and peptide length. Any sequence information may be suppressed in view of the scope of any such ensemble: representing peptide main-chain binding modes. In one embodiment of the present invention, MHC/Pmc structures are preferably stored in a format wherein the peptides are converted into poly-alanine fragments. In another embodiment, a generic database may be compiled from different MHC allotype-specific and peptide length-specific structural libraries.

Such a database may be used, for example, to predict affinities for peptides of different length or to predict the affinity of a given peptide for different MHC types.

Detailed steps of a method as used in the present invention comprise the following : 1. Construction of an MHC template A method as used in the present invention requires two basic elements of input data, besides a number of execution parameters (see FIGURE 2 for a schematic overview of the complete method). The first element is the selection of an MHC allotype of interest, the second one is the sequence of a peptide as present in a protein source of interest, for example a viral protein. Selecting an MHC allotype is equivalent to selecting the amino acid sequence representing the MHC allele. With this sequence (or a reference to it) it is possible to search the protein data bank (PDB) for the presence of 3D structures sharing the same amino acid sequence. lf such structure exists, it can be retrieved from the PDB (Berman, H. M. et al., (2000) Nucleic Acids Res. 28,235-242) and used as a three-dimensional MHC template structure in the further prediction steps. In the event that more than one candidate structure is available, the user has to decide which one is the most preferred starting structure. Useful criteria for this purpose are the crystallographic resolution and refinement, the absence of missing atoms, and/or the criteria applied by structure validation tools such as the Biotech Validation Suite (www. embl-heidelberg. de, and follow links therein for the Biotech Validation Suite).

In the case that neither the PDB database nor available publications describe the structural co-ordinates of a sequence identical to that of the selected MHC allotype, a template structure may be constructed by homology modeling. Various methods for homology modeling include, for example Swiss-Model (Guex, N. and Peitsch, M. C. (1997) Electrophoresis 18,2714- 2723,1997) or SCWRL (Bower, M. et a/., (1997) J. Mol. Biol. 267,1268-1282). Because the modeling of MHC binding grooves involves no insertions or deletions, a pure side-chain placement algorithm can be applied. A preferred method to accomplish this is a DEE method (De Maeyer et al., 2000) or the FASTER method as described by Desmet et al. (Desmet, J. et al., (2002) Proteins 48,31-43). Once a template structure has been retrieved or modeled, it is within the scope of the present invention to refine it by performing 100-200 steps of steepest descent energy minimization, or by any equivalent energy minimization procedure. Such energy minimization action is a standard procedure in protein modeling and serves to solve potential atomic conflicts or suboptimal positioning.

In one embodiment of the invention, a method which is followed by a user in advanced execution mode i. e. the database approach, merely involves the selection of the appropriate {MHC/PmC} ensemble from the database, said ensemble corresponding with the MHC allotype of interest. In this case the MHC template construction step may not be explicitly executed but is implicitly present in the structure retrieved from the database.

2. MHC/peptide main-chain construction One step of the present method is the construction of an ensemble of peptide main-chain configurations {Pmc} in relation to the MHC template, or {MHc/pmc} The selected peptide p is characterized by a well-defined amino acid sequence. It is logical to assume that the sequence of p has at least some influence on the ensemble of binding modes or, in other words, that {MHC/pmc} is sequence-specific. On the other hand, the very nature of MHC class I and class 11 binding grooves also suggests that the number of distinct binding modes is limited. Therefore, the construction of peptide backbones might be performed in more than one way. For example a sequence-specific {MHc/pmc} ensemble is created for each new peptide. Or in another example a generalized ensemble {MHc/pmc} might be made available, representing at least the conformational space of the selected peptide p. An over-representation of the space is not so much of a problem because the generalized ensemble {MHClPm} may be reduced to the peptide-specific ensemble {MHc/pmc} in step 3 of a method wherein MHC-incompatible binding modes are identified after side-chain placement. Furthermore, the establishing of a generalized ensemble can be accomplished in a straightforward manner by unifying different peptide-specific ensembles until a sufficient overlap between the populations is observed. Consequently, step 2 of a method of the present invention reduces to the problem of generating peptide-specific {MHClpm ensembles.

An example of a method of constructing the peptide backbone is found in Desmet et al.

(1997,2000). This docking method is a combinatorial algorithm for flexible docking of peptides to the binding site on a protein receptor molecule in which the peptide is constructed from scratch in relation to the chosen receptor structure, thereby avoiding any potential bias from a starting structure of the receptor/peptide complex. It yields a collection of different, energetically favorable complex structures wherein the peptide assumes, typically, between 0 and 500 distinct binding states. This de novo peptide building method is therefore the most preferred approach to generate the contemplated {MHc/pmc} ensembles. The method of Desmet et a/. (1997,2000) is herein explicitly incorporated by reference. its essential execution steps and characteristics are outlined in the following.

The docking method referred to above consists of a combinatorial buildup algorithm that "grows"the peptide by gradual addition of a single residue adopting a specific main-chain conformation. For each residue type there are 47 low energy main-chain rotamers and for each main-chain rotamer there are a variable number of backbone-compatible side-chain rotamers.

Glycine, proline and N-or C-terminal residues form an exception and have 125,35 and 12 main- chain rotamers, respectively. The rotamer library thus represents the entire conformational space for each residue type.

The docking algorithm starts from a peptide fragment of length one, ie. a user-selected root residue. (This can be any residue of the peptide. ) The accessible space for the root residue is searched by a combined translational, rotational and conformational exploration. Translations and rotations are performed in a discretized fashion in accordance with a grid approach. The conformational sampling is done separately for the main-chain and side-chain parts of the system. The main-chain conformation is only varied for the peptide, whereas that of the receptor is strictly kept fixed. Possible main-chain conformations for the peptide, in this case the root residue, are selected from the main-chain rotamer library (containing mostly 47 rotamers per residue type). Possible side-chain conformations are retrieved from a backbone-dependent side- chain rotamer library. Besides the side-chain of the peptide's root residue, up to about 40 side- chains from the receptor can be modeled simultaneously. The side-chain placement step is fully repeated for every translational-rotational-(backbone)-rotameric combination of the root residue, one such step called a single docking step. The side-chain placement itself is performed by a standard DEE method (Desmet et a/., 1992). The net result of each docking step is an energetical value, Ebind, reflecting the"quality of fit"of the peptide's root residue in the considered binding mode. Ebi, d is computed by a rich function, including the interaction energy between the peptide (root) fragment and the receptor, the total fragment self-energy and the augmentation of the receptor self-energy due to conformational changes induced by the presence of the fragment. This value serves as a discriminator between energetically acceptable and prohibited binding modes (applying a user-defined threshold value). All energetically acceptable single-residue fragments are added to a peptide fragment repository.

The buildup of the peptide continues by combining each previously accepted fragment in the repository with the available main-chain rotamers of an adjacent residue. Each new combination is again processed individually by the DEE-based side-chain placement algorithm.

All energetically favorable fragments are added to the peptide fragment repository. This buildup process continues until all residues of the peptide have been extended to their full length. Thus, in the end the peptide fragment repository contains only energetically acceptable full-length peptides.

One aspect of a fragment repository is that it may hold only information related to the binding mode of the peptide's main-chain; reference to a specific conformation for the side- chains may not be stored.

One embodiment of the present invention is the storage of modes identified by the docking method into a general database of {MHc/pmc} ensembles. In view of the usage of such database in providing a generic source of binding modes (i. e. when applying the advanced database-related operation mode of a method of the present invention), the peptide conformations are preferably stored as poly-alanine or poly-glycine constructs. The only form of specificity in the database concerns the MHC allotype and length of the generic peptide fragments.

3. MHC/full peptide construction Step 3 of a method of the present invention involves the reconstruction of peptide and optionally the receptor side-chain conformations in order to build full complex structures. This structural information forms the main source of input information for the next step 4 of the present method.

In view of the fact that the present invention is almost exclusively based on properties derived from predicted structures, the accuracy of this step is directly related to the prediction accuracy of the peptide binding affinity, i. e. an important aim of the present invention.

The accuracy of any side-chain placement method may be determined by three aspects: (i) the search method that is used to determine the optimal global side-chain arrangement, (ii) the rotamer library from where potential side-chain conformations are retrieved, and (iii) the quality of the scoring function used during conformational search. A fourth determinant of accuracy, i. e. the coupling between main-chain and side-chain conformational changes, is also considered. It may be implicitly calculated from the above because side-chain conformations are generated for a broad ensemble of peptide main-chain structures. The first three determinants of prediction accuracy are discussed in more detail.

1. Preferred side-chain confonnational search method The present inventors have recently developed a novel method for fast and accurate side-chain modeling called the"fast and accurate side-chain topology and energy refinement method"or FASTER method (Desmet et a/., 2002). In view of its characteristics, the FASTER method is highly preferred to perform step 3 of the present method. The main reason for this is that FASTER allows a rapid yet accurate search for the globally optimal side-chain arrangement, which is one of the key-aspects of the present invention. More specifically, for each MHC/PmC structure of the ensemble generated in step 2, all side-chains of the peptide and a significant number of side-chains from the MHC receptor (typically 10-30) are modeled simultaneously in order to find the globally best packing arrangement. In doing so, all possible pair-wise interactions between two flexibly treated side-chains are taken into account during the modeling.

This is in contrast to other methods (e. g. Swain et al., 2001) which only score the side-chain conformations of the peptide and which independently do this for each side-chain.

Apart from the FASTER method, other side-chain placement methods are suitable for performing step 3 of the present invention, such as DEE (De Maeyer et al., 2000), self- consistent mean field optimization (Koehl, P. and Delarue, M. (1994), J. Mol. Biol. 239,249-275), simulated annealing (Shenkin, P. S. et a/., (1996) Proteins 26,323-352), a genetic algorithm (Tuffery, P. et al., (1997) Protein Eng. 10,361-372) or Monte Carlo simulation (Holm, L. and Sander, C. (1992) Proteins 14, 213-223). In general, methods which explicitly account for pair- wise side-chain/side-chain interactions are preferred. Such methods may follow either a rotameric or a non-rotameric strategy.

2. Rotamer library When performing step 3 on basis of the FASTER or a DEE method, the algorithm requires access to a library of discrete, preferential side-chain conformations or rotamers. Such library may be called a rotamer library. Non-limiting examples include Ponder and Richards (Ponder, J. W. and Richards, F. M. (1987) J. Mol. Biol. 193,775-791), Tuffery et al. (Tuffery, P. et al., (1991). J. BiomoL Struct. Dynam. 8,1267-1289), Holm and Sander, (1992); Schrauber et a/., (Schrauber, H. et aL, (1993) J. MoL Biol. 230,592-612), Dunbrack and Karplus, (Dunbrack, R. L. Jr. and Karplus, M. (1993) J. Mol. Biol. 230,543-574), De Maeyer et a/., 1997, Mendes et a/.

(Mendes, J. et a/. (1999) Proteins 37,530-543), Xiang and Honig, (Xiang, Z. and Honig, B.

(2001) J. Mol. Biol. 311,421-430). One way to define rotamers is to store them as a list of torsional angle values for all rotatable bonds within a particular side-chain type and for the chemical bond that connects it to the backbone. Alternatively, rotamers in the library may be stored as sets of atomic co-ordinates in a given reference frame. Whatever rotameric representation is chosen, it is preferred that the rotamer library provide the necessary and sufficient information to reconstruct side-chain conformations in an unambiguous way onto a polypeptide backbone. One example of a preferred rotamer library is the one devised by Mendes et al. (1999), comprising so-called"flexible rotamers". Herein, a flexible rotamer is essentially defined as an ensemble of sub-rotamers deviating slightly in structure from a classic rigid rotamer. The latter type of rotamers is especially suited for the present method since it enables quantification of side-chain entropical effects, both for peptide and receptor side-chains, in a similar fashion as for the peptide main-chain. Also preferred are highly detailed libraries of classic rigid rotamers, whether backbone-dependent (Dunbrack & Karplus, 1993; Bower et a/., 1997, Desmet et a/., 1997) or backbone-independent (De Maeyer et a/., 1997; Xiang & Honig, 2001). A less preferred method for assigning side-chain conformations is by applying a non- rotameric approach such as a molecular mechanics or dynamics method, or a combination protocol (Rognan et a/., 1999). Non-rotameric methods are preferred less because they are slower and less efficient in conformational sampling (Mendes et al., 1999), though they fall within the scope of the present invention.

3. Scoring function for side-chain placement A method as used in the present invention distinguishes between two separate scoring functions, the first being applied to structure prediction of side-chains (and also peptide main- chains, if step 2 of the present method is performed by way of docking), and the second scoring function being applied in the affinity prediction step (see step 4. MHC/Peptide Affinity Assessment). As it is intended for usage in conjunction with a method for searching (sampling) huge conformational hyperspaces, the first scoring function is preferably intrinsically rapid to evaluate and, also, it does not have to include as many energetical components as an affinity scoring function. One purpose of the said scoring function is to allow the determination of the correct conformation of a specific MHC/peptide complex. For this reason, a standard potential or free energy function might be applied that accounts for the intramolecular interactions. Such a function is usually called a force field function. Non-limiting examples of widely used force fields include the CHARMM force field (Brooks, B. R. et al., (1983) J. Comput. Chem. 4,187-217), the AMBER force field of Kollman and co-workers at UCSF (Weiner, S. J. et al., (1984) J. Am. Chem.

Soc. 106,765-784) and the DREIDING field (Mayo, S. L. et al., (1990) J. Phys. Chem. 94,8897- 8909). The applied energy function may include as many relevant energetic contributions as possible, non-limiting examples of which include van der Waals interactions, H-bond formation, electrostatic interactions and contributions related to chemical bonds (bond stretching, angle bending, torsions, planarity deviations). The present inventors have shown that these energy terms suffice to reach the currently highest possible accuracy in side-chain prediction while allowing very rapid modeling (Desmet et al., 2002). The scope of the present invention allows for force fields which satisfy any of the above. In one embodiment of the present invention, the preferred force field is CHARMM (Brooks et a/., 1983).

4. MHC/peptide affinity assessment The ligand binding affinity (Kb) is related to the binding free energy (AG) by the following equation.

AG =-RT In (Kb) [3] where R is the ideal gas constant (8.31 J mol~1 K-1) and T the absolute temperature in degrees Kelvin. Further, Kb is the inverse of the dissociation constant (Kd) which is approximately equal to the often mentioned IC50 value.

AG = RT In (Kd) RT in (iCso) [4] The binding free energy, AG, is the difference in Gibbs free energy between the free receptor molecule plus the free peptide ligand on the one hand and the receptor/ligand complex on the other hand. Strongly negative AG values indicate strong binding. Differences in AG for different peptides and/or different MHC subtypes may be due to a variety of reasons, including enthalpic and entropic effects related to any of the free or bound states. Since many of these effects can by no means be deduced from theoretical simulations, affinity scoring functions might include more than one parametrized components. A basic approach of the present invention is then to incorporate into the predicted binding free energy, AGpred, as much relevant structural information as possible, and to cover all other effects by empirical components. Assuming that the different contributions are independent and additive, the following is an example of a general expression which reflects the predicted binding free energy: Ns Np AGpred = EsjS ; +pjP ; [5] i=l i=l In equation [5], Si and Pi are structure-derived and non-structure derived contributions, respectively. Ns and Np are the number of considered contributions of both types while si and pi are their respective weight coefficients. It should be noted, however, that most methods consider either structure-based or non-structure based terms but seldomly both. The coefficients si and the number of structural components Ns are in fact parameters as well since they need to be calibrated. The coefficients pi are in many methods set equal to unity.

With respect to the structure-related terms in Eq. [5], one approach is to sum over all contributions provided by a force field function (e. g. electrostatic, van der Waals, H-bonding <BR> <BR> terms, etc. ). However, pure standard force field terms generally do not yield an optimal correlation with experimental data. Including additional effects, non-limiting examples of which include desolvation, freezing of rotatable bonds, special hydrophobicity terms, may significantly enhance correlation. The"Fresno"method (Rognan et al., 1999) considers five individual contributions: H-bonding, lipophilic contacts, rotatable bond freezing, burial of polar atoms and desolvation. This scoring function requires re-calibration of the weight coefficients for different MHC subtypes. The method of Schueler-Furman et al. (2000) only considers MHC side- chain/peptide side-chain contacts (with a special treatment of MHC side-chains in contact with the peptide backbone) in conjunction with a statistical pairwise potential.

Scoring functions based on experimental data often rely on the frequency of amino acid types observed at each position in a population of peptides (e. g. self peptides) that are known to bind to a specific MHC allele (Rammensee et a/., 1999). Alternatively, the contribution of individual amino acid types at each position in a peptide sequence to the peptide's total binding affinity may be estimated by a number of statistical analyses. This can be done for a set of known binding peptides (Parker et al., 1994) or experimentally constructed peptides (Hammer et al., 1993; Fleckenstein et al., 1999).

A method as used in the present invention is predominantly based on 3D structural contributions. Structural contributions preferably comprise: (i) all terms that can be computed, using a force field e. g. CHARMM (Brooks et al., 1983), for a MHC/Pfulß complex resulting from step 3 of a method; (ii) contributions computed in the same way for separately modeled reference states of the free peptide and receptor; (iii) contributions accounting for desolvation of both the receptor and the peptide upon complex formation, and (iv) importantly, entropical contributions derived in accordance with a statistical mechanical analysis of the ensemble of structures obtained in step 3,/. e. {MC/Pfu.

When following the standard docking approach to generate the latter ensemble, one generally obtains a limited set of complex structures that are all energetically relaxed. In one embodiment of a method of the present invention, the contributions (i) to (iii) are added up for each structure of the ensemble and each sum is given the weight coefficient sj = 1/(Nsol), where Nso, is the number of solutions in the ensemble. This yields the energetical term <E> in Eq. [1].

The structure-related component (iv), corresponding to the entropical contribution S in Eq. [1], may be set equal to In (NsO), or kB In (sot) where kB is Boltzmann's constant. The latter constant <BR> <BR> may be included in the weight coefficient (c in Eq. [1], corresponding to sen, rOpy in Eq. [5] ). This coefficient is subject of global parameter optimization, which is to be executed by a suitable parameter optimization method. A non-limiting example illustrating the importance of including an entropical component is provided in EXAMPLE 4.

When a method of the present invention is performed in accordance with the advanced database-related execution mode, a more sophisticated method may be needed to determine the appropriate weight coefficients of aforementioned contributions (i) to (iv), preferably on the basis of statistical mechanical relationships.

Besides structure-related contributions (Si in Eq. [5] ), it is within the scope of the present method to consider a number of non-structural terms (Pj in Eq. [5]). A first possibility is a combination method formed by fusing a structure-based and an experimental method. This is accomplished by determining the globally optimal set of weight coefficients {si, pi), applying a suitable parameter optimization method.

A preferred possibility is to include topology contributions, for example the"Type and Topology Specific" (TTS) contributions of Desmet et a/. (International Patent Application No. WO 02/05146) which has been invented in the context of protein design. This method considers a limited number of topology classes (typically 2 or 3), depending on a residue's degree of burial in a complex. The notion topology may also be extended so as to reflect, besides shielding from solvent, the chemical nature of a residue's environment, for example a measure of polarity.

Furthermore, it is within the scope of the present invention to consider an alternative to the residue type dimension in the concept of TTS parameters, namely distinguishing chemical groups instead of residue types. A preferred classification of chemical groups is the following : 1, CHX aliphatic ; 2, CHx aromatic; 3, NHx aromatic; 4, OH; 5, S+SH; 6, NH3+ ; 7, COO- ; 8, CONH2 ; 9, NHC (NH2) 2+. This way, the type-dimension in the set of TTS parameters can be restricted to 9 groups (instead of 20 residue types). The option to work with chemical groups is fully compatible with the broader definition of topology. This creates a landscape of possibilities that can be explored by applying a suitable data mining and parameter optimization strategy, which is within the scope of the present invention. It is further within the scope of the invention to identify and quantify the most relevant contributions in the attempt to enhance the correlation between predicted and experimental AG values. The incorporation of type and topology-specific contributions again leads to a fully structure-based method.

Hepatitis B virus (HBV) MHC Class I restricted T cell epitopes The present invention particularly provides peptides derived from HBV proteins for use in compositions and methods for the treatment, prevention and diagnosis of HBV infection. The peptides stimulate MHC HLA-class I restricted cytotoxic T lymphocyte responses to HBV infected cells.

The large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to vaccine development. To address this factor, epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele specific HLA molecules.

The correlation of binding affinity with immunogenicity is an important factor to be considered when evaluating candidate peptides. Thus, by using the method of the present invention and HLA-peptide binding assays, candidates for epitope-based vaccines have been identified. After determining their binding affinity, additional confirmatory work can be routinely performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of antigenicity and immunogenicity. The following describes the peptide epitopes and corresponding nucleic acids of the invention.

CTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC50 or binding affinity value for class I HLA molecules of 500 nM or less. HTL-inducing peptides preferably include those that have an IC50 or binding affinity value for class 11 HLA molecules of 1 000 nM or less. For example, peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross- reactive binding are then used in vaccines or in cellular screening analyses.

As disclosed herein, high HLA binding affinity is correlated with greater immunogenicity.

Greater immunogenicity can be manifested in several different ways. immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response. Higher binding affinity peptides leads to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high binding epitopes are particularly desired.

The peptides of this invention have been identified according to a modified version of the method as disclosed herein.

Peptides derived from HBV proteins which are predicted according to the present invention to interact with MHC HLA-class I and stimulate CTL responses to HBV infected cells may be further evaluated by measuring their interactions with MHC class I molecules. Ways of so evaluating the peptides are known by persons skilled in the art, using known methods and include, but are not limited to, assays to measure IC50 values (S. H. van der Burg et al. (1995), Humari Immunology 44,189-198), inhibition of antigen presentation (Sette et al, (1991) J.

Immunol. 141, p 3893), in vitro assembly assays (Townsend et al (1991), Cell, 62, p285), measure of dissociation rates (S. H. van der Burg et al. (1996), The Journal of Immunology 156, 3308-3314). Assays to evaluate the CTL-response to the peptides of the invention as also known by persons skilled in the art. Such methods include, but are not limited to those described in S. H. van der Burg et al. (1995), AIDS 9,121. In respect of methods for evaluating the binding to HLA and the CTL-response of the peptides of the present invention, PCT application number WO 02/19986 A1 is incorporated herein by reference.

Various strategies can be utilized to evaluate immunogenicity.

1. Evaluation of primary T cell cultures from normal individuals (Wentworth, P.

A. et al., Mol. Immunol. 3 2: 603,1995 ; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91: 2105,1994 ; Tsai, V. et al., J Inimunol. 15 8: 1796,1997 ; Kawashinia, l. et al., Human IMMunol. 59: 1,1998) ; This procedure involves the stimulation of PBL from nonnal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using a 5 1 Cr-release assay involving peptide sensitized target cells.

2. Immunization of HLA transgenic mice (Wentworth, P. A. et al., J Immunol.

26.97, 1996; Wentworth, P. A. et al., Int. Iminunol. 8: 651,1996 ; Alexander, J. et al., J Iminunol.

159: 4753,1997) ; In this method, peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week.

Peptide-specific T cells are detected using a Crrelease assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3. Demonstration of recall T cell responses from immune individuals who have recovered from infection, and/or from chronically infected patients (Reherinann, B. et al., J Exp. Med. 181: 1047, 1995; Doolan, D. L. et al., immunity 7: 97,1997 ; Bertoni, R. et al., J Clin. Invest. 100: 5 03,1997 ; Threlkeld, S. C. et al., J linmunol. 159: 1648,1997 ; Diepolder, H. M. et al., J Virol. 71: 6011, 1997). In applying this strategy, recall responses were detected by culturing PBL from subjects that had been naturally exposed to the antigen, for instance through infection, and thus had generated an immune response"naturally". PBL from subjects were cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APQ to allow activation of "memory"T cells, as compared to"naive"Tcells. At the end of the culture period, T cell activity is detected using assays for T cell activity including 5 1 Cr release involving peptide-sensitized targets, T cell proliferation or lymphokine release.

In one aspect, the peptides of the present invention are derived from the HBV core protein (HBc).

In another embodiment the peptides are derived from the HBV envelope antigen (HBenv).

In yet another embodiment the peptides are derived from the HBV polymerase protein (HBpol).

The peptides of the present invention contain at least 9 contiguous HBV amino acid sequence residues and are derived from an HBc sequence, an HBenv sequence and/or an HBpol sequence.

It may be desirable to optimize peptides of the invention to a length of more than nine amino acid residues, commensurate in size with endogenously processed viral peptides that are bound to MHC class I molecules on the cell surface. 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. Peptides can have a size such that actually more than one peptide as described above appears in the sequence of the peptide (multiple repears or combinations).

The peptides in the present invention can contain modifications such as mutations, subject to the condition that the modification not destroy the biological activity of the peptides as herein described. Modification of the structure of the peptide can be of particular interest in order to achieve broader HLA binding capacity.

The peptides of the invention can also be 8 or 7 amino acid residues long while still maintaining substantially all of the biological activity of the large peptide. By biological activity is meant the ability to bind an appropriate MHC molecule and induce a cytotoxic T lymphocyte response against HBV antigen or antigen mimetic.

The terms"homologous","homolog thereof',"substantially homologous", and "substantial homology"as used herein denote a sequence of amino acids having at least 50%, 60%, 70%, 80%, 90%, 95% identity wherein one sequence is compared to a reference sequence of 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 HBV regions as described hereafter.

Additional amino acids can be added to the termini of an oligopeptide or peptide to provide for ease of linking peptides one to another, for coupling to a carrier, support or a larger peptide or for modifying the physical or chemical properties of the peptide or oligopeptide, and the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the C-or N-terminus of the peptide or oligopeptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal- NH2 acylation, e. g., acetylation, or thioglycolic acid amidation, terminal-carboxy amidation, e. g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

It will be understood that the HBV peptides of the present invention or homologues thereof which have cytotoxic T lymphocyte stimulating activity may be modified as necessary to provide certain other desired attributes, e. g. , improved pharmacological characteristics, while increasing or at least retaining substantially the biological activity of the unmodified peptide. For instance, the peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, for example, the addition or deletion of amino acids on either the amino terminal or carboxy terminal end, or both, of peptides derived from the sequences disclosed herein. The peptides may be modified to substantially enhance the CTL inducing activity, such that the modified peptide analogs have CTL activity greater than a peptide of the wild-type sequence. For example, it may be desirable to increase the hydrophobicity of the N- terminal of a peptide, particularly where the second residue of the N-terminal is hydrophobic and is implicated in binding to the HLA restriction molecule. By increasing hydrophobicity at the N- terminal, the efficiency of the presentation to T cells may be increased.

Therefore, the peptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes provide for certain advantages in their use. By conservative substitutions is meant replacing an amino acid <BR> <BR> residue with another which is biologically and/or chemically similar, e. g. , one hydrophobic residue for another, or one polar residue for another. Usually, the portion of the sequence which is intended to substantially mimic an HBV cytotoxic T lymphocyte stimulating epitope will not differ by more than about 20% from the sequence of at least one subtype of HBV, except where additional amino acids may be added at either terminus for the purpose of modifying the <BR> <BR> physical or chemical properties of the peptide for, e. g. , ease of linking or coupling, and the like.

Where regions of the peptide sequences are found to be polymorphic among HBV subtypes, it may be desirable to vary one or more particular amino acids to more effectively mimic differing cytotoxic T-lymphocyte epitopes of different HBV strains or subtypes.

Within the peptide sequences identified by the present invention, there are residues (or those which are substantially functionally equivalent) which allow the peptide to retain their biological activity, i. e., the ability to stimulate a class t-restricted cytotoxic T-lymphocytic response against HBV infected cells or cells which express HBV antigen. These residues can be identified by single amino acid substitutions, deletions, or insertions. In addition, the contributions made by the side chains of the residues can be probed via a systematic scan with a specified amino acid (e. g., Ala). Peptides which tolerate multiple substitutions generally incorporate such substitutions as small, relatively neutral molecules, e. g., Ala, Gly, Pro, or similar residues. The number and types of residues which can be substituted, added or subtracted will depend on the spacing necessary between the essential epitopic points and <BR> <BR> certain conformational and functional attributes which are sought (e. g. , hydrophobicity vs. hydrophilicity). If desired, increased binding affinity of peptide analogues to its MHC molecule for presentation to a cytotoxic T-lymphocyte can also be achieved by such alterations. Generally, any spacer substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid steric and charge interference which might disrupt binding.

In a preferred embodiment, the peptides of the invention provide a broad coverage to different HLA-types. Each peptide provides suffcient coverage to HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3.

In another embodiment it may be desirable to join two or more peptides in a composition.

Said peptides are known herein as"combinations". 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, which peptides can be combined in a"cocktail"to provide enhanced immunogenicity for cytotoxic T lymphocyte responses. Peptides of one region can be combined with peptides of other HBV regions, from the same or different HBV protein, particularly when a second or subsequent peptide has a MHC restriction element different from the first. This composition 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.

In another embodiment peptides of the invention can be combined with weak binding peptides to ensure adequate numbers of cross-reactive cellular binders.

In yet another-embodiment one peptide can bind with high affinity to one or more HLA- antigens and with weaker affinity to other antigens.

The peptides of the invention can be combined via linkage to form polymers (multimers), or can be formulated in a composition without linkage, as an admixture. Where the same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are <BR> <BR> presented. When the peptides differ, e. g. , a cocktail representing different HBV subtypes, different epitopes within a subtype, different HLA restriction specificities, a peptide which contains T helper epitopes, heteropolymers with repeating units are provided. In addition to covalent linkages, noncovalent linkages capable of forming intermolecular and intrastructural bonds are included.

Linkages for homo-or hetero-polymers or for coupling to carriers can be provided in a variety of ways. For example, cysteine residues can be added at both the amino-and carboxy- termini, where the peptides are covalently bonded via controlled oxidation of the cysteine residues. Also useful are a large number of heterobifunctional agents which generate a disulfide link at one functional group end and a peptide link at the other, including N-succidimidyl-3- (2- pyridyidithio) proprionate (SPDP). This reagent creates a disulfide linkage between itself and a cysteine residue in one protein and an amide linkage through the amino on a lysine or other free amino group in the other. A variety of such disulfide/amide forming agents are known. See, for example, Immun. Rev. 62: 185 (1982), which is incorporated herein by reference. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether forming agents are commercially available and include reactive esters of 6- maleimidocaproic acid, 2 bromoacetic acid, 2-iodoacetic acid, 4- (N- maleimidomethyl) cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt.

A particularly preferred coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC). It will be understood that linkage should not substantially interfere with <BR> <BR> either of the linked groups to function as described, e. g. , as an HBV cytotoxic T cell determinant, peptide analogs, or T helper determinant.

The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and <BR> <BR> 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 <BR> <BR> 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 <BR> <BR> Spring Harbor, N. Y. (1982), and Ausubel et al., (ed. ) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York (1987), and U. S. Pat. Nos. 4,237, 224,4, 273,875, 4,431, 739,4, 363,877 and 4,428, 941, for example, whose disclosures are each incorporated herein by reference. Thus, combination peptides which comprise one or more peptide sequences of the invention can be used to present the HBV cytotoxic T cell determinants. For example, a recombinant core protein is prepared in which the HBc 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 peptide is used which incorporates several T cell epitopes.

As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103: 3185 (1981), modification can be made simply by substituting the appropriate base (s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.

Compositions are provided which comprise a peptide of the invention formulated with an additional peptide, a liposom, an adjuvant and/or a pharmaceutically acceptable carrier. Thus, pharmaceutical compositions can be used in methods of treating acute HBV infection, particularly in an effort to prevent the infection from progressing to a chronic or carrier state.

Methods for treating chronic HBV infection and HBV carrier states are also provided, where the pharmaceutical compositions are administered to infected individuals in amounts sufficient to stimulate immunogenically effective cytotoxic T cell responses against HBc epitopes. For treating these infections it may be particularly desirable to combine the peptides which induce MHC class I restricted cytotoxic T lymphocyte responses against HBV antigen with other peptides or proteins that induce immune response to other HBV antigens, such as HBsAg.

To treat individuals with chronic or carrier state infections the compositions may be administered in repeated dosages over a prolonged period of time, as necessary, to resolve or substantially mitigate the infection and/or shedding of virus.

Vaccine compositions for preventing HBV infection, particularly chronic HBV infection, are also provided. The vaccine compositions comprise an immunogenically effective amount of a HBV nucleocapsid peptide which induces a MHC class I restricted cytotoxic T lymphocyte response. To achieve enhanced protection against HBV, the vaccine can further comprise components which elicit a protective antibody response to HBV envelope antigen.

Of particular relevance for vaccines may also be"nested epitopes". Nested epitopes occur where at least two epitopes overlap in a given polypeptide. Such a polypeptide can comprise both HLA class I and HLA class 11 epitopes. If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non- native epitope. Of particular concern is a junctional. epitope that is a"dominant epitope."A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

Vaccine compositions comprising CTL peptides of the invention can be modified to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity. For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.

Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e. g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero-or homo-oligomer.

When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes tO or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

One skilled in the art will appreciate that suitable methods of administering a compound to a mammal for the treatment and/or prevention of an acute or chronic case of HBV, for example, which would be useful in a method of the present invention are available. Although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described methods provided herein are merely exemplary and are in no way limiting.

Generally, the peptides of the present invention as described above will be administered in a pharmaceutical composition to an individual already infected with HBV. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective cytotoxic T lymphocyte response to HBV and to cure or at least partially arrest its symptoms and/or complications. An amount adequate to accomplish this is defined as a"therapeutically or prophylactically effective dose"which is also an"immune response provoking amount."Amounts <BR> <BR> effective for a therapeutic or prophylactic use will depend on, e. g. , the stage and severity of the disease being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the peptide composition, method of administration, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound or stimulated CTL's and the desired physiological effect.

It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.

The present inventive method typically will involve the administration of about 0. 1 ug to about 50 mg of one or more of the compounds described above per kg body weight of the individual. For a 70 kg patient, dosages of from about 10 ug to about 100 mg of peptide would be more commonly used, followed by booster dosages from about 1 ug to about 1 mg of peptide over weeks to months, depending on a patient's CTL response, as determined by measuring HBV- specific CTL activity in PBLs obtained from the patient. For the reintroduction of stimulated CTL's, which were derived from the patient, typically a dose would range upward from 1 % of the number of cells removed up to all of them.

It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

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

For therapeutic use, administration should begin at the first sign of HBV infection or shortly after diagnosis in cases of acute infection, and continue until at least symptoms are substantially abated and for a period thereafter. In well established and chronic cases, loading doses followed by maintenance or booster doses may be required. The elicitation of an effective cytotoxic T lymphocyte response to HBV during treatment of acute hepatitis will minimize the possibility of subsequent development of chronic hepatitis, HBV carrier stage, and ensuing hepatocellular carcinoma.

Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals, the majority of whom are capable of resolving the infection naturally. For those individuals susceptible (or predisposed) to developing chronic infection, the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, for instance by using the diagnostic procedures described herein, the composition can be targeted to them, minimizing need for administration to a larger population.

The peptide compositions can also be used for the treatment of chronic hepatitis and to stimulate the immune system of carriers to substantially reduce or even eliminate virus-infected cells. Those with chronic hepatitis can be identified as testing positive for virus from about 3-6 months after infection. 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 stimulate effectively a cytotoxic T cell response. Thus, for treatment and/or prevention of chronic hepatitis, a representative dose is in the range of about 1 ug to 1,000 mg, preferably about 5 ug to 100 mg for a 70 kg patient per dose.

Administration should continue until at least clinical symptoms or laboratory indicators indicate that the HBV infection has been eliminated or substantially abated and for a period thereafter.

Immunizing doses followed by maintenance or booster doses at established intervals, e. g. , from one to four weeks, may be required, possibly for a prolonged period of time, as necessary to resolve the infection. For the treatment of chronic and carrier HBV infection, it may be desirable to combine the CTL peptides with peptides or proteins that induce immune response to a combination of HBV antigens.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration and generally comprise a pharmaceutical acceptable carrier and an amount of the active ingredient sufficient to reverse or prevent the bad effects of acute or chronic HBV infection, for example. The carrier may be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration.

Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p- toluenesulphonic acids, and arylsulphonic, for example.

The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.

The choice of excipient will be determined in part by the particular epitope and epitope formulation chosen, as well as by the particular method used to administer the composition.

Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

Preferably, the pharmaceutical compositions are administered parenterally, e. g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution of the cytotoxic T-lymphocyte stimulatory peptides dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions.

Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds. ,<BR> pages 238-250, (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed. , pages 622- 630 (1986). Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2, 2-dimethyl-1, 3-dioxolane-4-methanol, ethers, such as poly (ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glycerid, or an acetylated fatty acid glycerid with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) <BR> <BR> amphoteric detergents such as, for example, alkyl-. beta. -aminopropionates, and 2-alkyl- imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.

The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the present invention for application to skin.

Formulations suitable for oral administration require extra considerations considering the peptidyl nature of the epitopes and the likely breakdown thereof if such compounds are administered orally without protecting them from the digestive secretions of the gastrointestinal tract. Such a formulation can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutical acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

The molecules and/or peptides of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. For aerosol administration, the cytotoxic T-lymphocyte stimulatory peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0. 01 %-20% by weight, preferably 1 %-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0. 1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included <BR> <BR> as desired, e. g. , lecithin for intranasal delivery. These aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa.

Additionally, the compounds and polymers useful in the present inventive methods may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

In some embodiments, it may be desirable to include in the pharmaceutical composition at least one component that primes CTL generally. Lipids have been identified that are capable of priming CTL in vivo against viral antigens, e. g., tripalmitoyl-S-glycerylcysteinly-seryl-serine (P3 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). Peptides of the present invention can be coupled to P3 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 P3 CSS conjugated to a peptide that <BR> <BR> displays an appropriate epitope, e. g. , certain NS3 epitopes, the two compositions can be combined to elicit more effectively both humoral and cell-mediated responses to HBV infection.

The concentration of cytotoxic T-lymphocyte stimulatory peptides of the present invention <BR> <BR> in the pharmaceutical formulations can vary widely, i. e. , from less than about 1 %, usually at or at least about 10% to as much as 20 to 50% or more by weight, and will be selected primarily by <BR> <BR> fluid volumes, viscosities, etc. , in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of peptide. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (17th ed., Mack Publishing Company, Easton, Pa. , 1985).

It will be appreciated by one of ordinary skill in the art that, in addition to the aforedescribed pharmaceutical compositions, the compounds of the present inventive method may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes serve to target the peptides to a particular tissue, such as lymphoid tissue or HBV-infected hepatic cells. Liposomes can also be used to increase the half-life of the peptide composition. Liposomes useful in the present invention include emulsions, foams, micelies, insoluble monolayers, liquid crystals, phospholipid dispersions, lamella layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposom, <BR> <BR> alone or in conjunction with a molecule which binds to, e. g. , a receptor, prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled with a desired peptide of the invention can be directed to the site of lymphoid or hepatic cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposom size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, <BR> <BR> for example, Szoka et al., Ann. Rev. Biophys. Bioeng. , 9,467 (1980), and U. S. Pat. Nos.

4,235, 871,4, 501,728, 4,837, 028 and 5,019, 369. For targeting to the immune cells, a ligand to be incorporated into the liposom can include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposom suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the peptide being delivered, the stage of disease being treated, etc.

In another aspect the present invention is directed to vaccines that contain as an active ingredient an immunogenically effective amount of a cytotoxic T-lymphocyte stimulating peptide, as described herein. The peptide (s) may be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or cytotoxic T cells that react with different antigenic determinants of HBV. Useful carriers are well known in the art, and include, e. g., keyhold limpet hemocyanin, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamin acids such as poly (D-lysine : D-glutamic acid), and the like.

The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum or materials well known in the art. And, as mentioned above, cytotoxic T lymphocyte responses can be primed by conjugating peptides of the invention to lipids, such as P3 CSS. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of cytotoxic T-lymphocytes specific for HBV antigen, and the host becomes at least partially immune to HBV infection, or resistant to developing chronic HBV infection.

Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of HBV infection to enhance the patient's own immune response capabilities. Such an amount is defined to be a"immunogenically effective dose"or a "prophylactically effective dose."In this use, the precise amounts again depend on the patient's <BR> <BR> state of health and weight, the mode of administration, the nature of the formulation, etc. , but generally range from about 1. 0 ug to about 500 mg per 70 kilogram patient, more commonly from about 50 ug to about 200 mg per 70 kg of body weight. The peptides are administered to individuals of an appropriate HLA type.

In some instances, it may be desirable to combine the peptide vaccines of the invention with vaccines directed at neutralizing antibody responses to HBV, particularly to HBV envelope and/or core antigens. Such a vaccine may be composed of, for example, recombinant HBV env- and/or neucleocapsid-encoded antigens or purified plasma preparations obtained from HBV- infected individuals.

A combination vaccine directed to prophylaxis or treatment of both HBV and HCV is also contemplated in the present invention. Such a combination vaccine includes antigenic determinants that reflect those of either or both of the B and C hepatitis viruses. For examples of HBV vaccines that can be formulated with the HCV-directed 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. The vaccines can be combined and administered concurrently, or as separate preparations.

For therapeutic or immunization purposes, the peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the HBV peptides of the invention. Upon introduction into an acutely or chronically HBV-infected host or into a non- infected host, the recombinant vaccinia virus expresses the HBV peptide and thereby elicits a host cytotoxic T lymphocyte response to HBV. Vaccinia vectors and methods useful in <BR> <BR> immunization protocols are described in, e. g. , U. S. Pat. No. 4,722, 848. Another vector is BCG (bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature, 351,456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e. g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.

The compositions and methods of the claimed invention may be employed for ex vivo therapy, whereina portion of a patient's lymphocytes are removed, challenged with a stimulating dose of a peptide of the present invention, and the resultant stimulated CTL's are returned to the patient. Accordingly, in more detail, ex vivo therapy as used herein concerns the therapeutic or immunogenic manipulations that are performed outside the body on lymphocytes or other target cells that have been removed from a patient. Such cells are then cultured in vitro with high doses of the subject peptides, providing a stimulatory concentration of peptide in the cell medium far in excess of levels that could be accomplished or tolerated by the patient. Following treatment to stimulate the CTLs, the cells are returned to the host, thereby treating the HBV infection. The host's cells may also be exposed to vectors that carry genes encoding the peptides, as described above. Once transfected with the vectors, the cells may be propagated in vitro or returned to the patient. The cells that are propagated in vitro may be returned to the patient after reaching a predetermined cell density.

In one method, in vitro CTL responses to HBV are induced by incubating in tissue culture a patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (a HBV infected cell). To optimize the in vitro conditions for the generation of specific cytotoxic T cells, the culture of stimulator cells is typically maintained in an appropriate serum-free medium. Peripheral blood lymphocytes are isolated conveniently following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp. In one embodiment, the appropriate APC's are incubated with about 10-100. mu. M of peptide in serum-free media for four hours under appropriate culture conditions. The peptide-loaded APC are then incubated with the responder cell populations in vitro for 5 to 10 days under optimized culture conditions.

Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of HBV antigen as further discussed below.

Specifically, the MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class 1. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides.

The induction of CTL in vitro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC. Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary CTL responses.

Because mutant cell lines do not exist for every MHC allele, it may be advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest. The use of non-transformed, non-infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies. Typically, prior to incubation of the APCs with the CTLp to be activated, an amount of antigenic peptide is added to the APC or stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the APCs. Resting or precursor CTLs are then incubated in culture with the appropriate APCs for a time period sufficient to activate the CTLs. Preferably, the CTLs are activated in an antigen- specific manner. The ratio of resting or precursor CTLs to APCs may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the described treatment modality is used. Preferably, however, the CTL: APC ratio is in the range of about 30: 1 to 300: 1. The CTL/APC may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CTL.

Activated CTL may be effectively separated from the APC using one of a variety of known methods. For example, monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the CTL (or a segment thereof) may be utilized to bind their appropriate complementary ligand. Antibody-tagged molecules may then be extracted from the admixture via appropriate means, e. g. , via well-known immunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of the activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells.

The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 x 10 6 to about 1 x 10 12, more preferably about 1 x 10 8 to about 1 x 10", and even more preferably, about 1 x 10 9 to about 1 x 10 10 activated CD8+ cells are utilized for adult humans, compared to about 5 x 10 6 to about 5 x 10 7 cells used in mice.

Methods of reintroducing cellular components are known in the art and include procedures such as those exemplified in U. S. Pat. No. 4,844, 893 to Honsik, et al. and U. S. Pat.

No. 4,690, 915 to Rosenberg. For example, administration of activated CTLs via intravenous infusion is typically appropriate.

In one aspect of the invention, HLA class I and class 11 binding peptides as described herein can be used as reagents to evaluate an immune response. The immune response to be evaluated is induced by using as an immunogen any agent that may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope (s) to be employed as the reagent. The peptide reagent need not be used as the immunogen. Assay systems that are used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.

Peptides of the invention are also used as reagents to evaluate immune recall <BR> <BR> responses. (see, e. g. , Bertoni et al., J Clin. Invest. 100: 503-513,1997 and Penna et al., J Exp.<BR> <P>Med. 174: 1565-1570, 1991. ) For example, patient PBMC samples from individuals infected with HPV are analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity.

The peptides are also used as reagents to evaluate the efficacy of a vaccine.

PBMCs obtained from a patient vaccinated with an immunogen are analyzed using, for example, either of the methods described above. The patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis. The immunogenicity of the vaccine is indicated by the presence of HPV epitope- specific CTLs and/or HTLs in the PBMC sample.

The peptides of the invention are also be used to make antibodies, using techniques well known in the art (see, e. g. Current protocols in immunology, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose HPV infection. Such antibodies include those that <BR> <BR> recognize a peptide in the context of an HLA molecule, i. e. , antibodies that bind to a peptide- MHC complex.

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

The peptide and nucleic acid compositions of this invention can be provided in kit- form together with instructions for vaccine administration. Typically the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration. An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-1 2 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.

Epitopes in accordance with the present invention induce an immune response. Immune responses with these epitopes can be induced by administering the epitopes in various forms, such as peptides, as nucleic acids, and as viral vectors comprising nucleic acids that encode one or more epitopes of the invention. Upon administration of peptide-based epitope forms, immune responses can be induced by direct loading of an epitope onto an empty HLA molecule that is expressed on a cell, and via internalization of the epitope and processing via the HLA class I pathway; in either event, the HLA molecule expressing the epitope was then able to interact with and induce a CTL response. Peptides can be delivered directly or using such agents as liposomes. They can additionally be delivered using ballistic delivery, in which the peptides are typically in a crystalline form. When DNA is used to induce an immune response, it is administered either as naked DNA, generally in a dose range of approximately 1-5mg, or via the ballistic"gene gun"delivery, typically in a dose range of approximately 10-100 tg. The DNA can be delivered in a variety of conformations, e. g., linear, circular etc. Various viral vectors have also successfully been used that comprise nucleic acids which encode epitopes in accordance with the invention.

Accordingly compositions in accordance with the invention exist in several forms.

One composition in accordance with the invention comprises a plurality of peptides. This plurality or cocktail of peptides is generally admixed with one or more pharmaceutically acceptable excipients. The peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides. The peptides can be analogs of naturally occurring epitopes. The peptides can comprise artificial amino acids and/or chemical modifications such as addition of a surface active molecule, e. g., lipidation ; acetylation, glycosylation, biotinylation, phosphorylation etc. The peptides can be CTL or HTL epitopes. In a preferred embodiment the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope.

The HTL epitope can be naturally or non-naturally. The number of distinct epitopes in an embodiment of the invention is generally a whole unit integer from one through one hundred fifty.

An additional embodiment of a composition in accordance with the invention comprises a <BR> <BR> polypeptide multi-epitope construct, i. e. , a polyepitopic peptide. Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another. The polyepitopic peptides can be linear or non-linear, e. g., multivalent. These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, Ranking amino acids, or chemical modifications between adjacent epitope units. The polyepitopic construct can be a heteropolymer or a homopolymer. The polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-150. The polyepitopic construct can comprise CTL and/or HTL epitopes. One or more of the epitopes in <BR> <BR> the construct can be modified, e. g. , by addition of a surface active material, e. g. a lipid, or chemically modified, e. g., acetylation, etc. Moreover, bonds in the multiepitopic construct can be other than peptide bonds, e. g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.

Alternatively, a composition in accordance with the invention comprises construct which <BR> <BR> comprises a series, sequence, stretch, etc. , of amino acids that have homology to i. e.. corresponds to or is contiguous with) to a native sequence. This stretch of amino acids comprises at least one subsequence of amino acids that, if cleaved or isolated from the longer series of amino acids, functions as an HLA class 1 or HLA class 11 epitope in 1 5 accordance with the invention. In this embodiment, the peptide sequence is modified, so as to become a construct as defined herein, by use of any number of techniques known or to be provided in the art. The polyepitopic constructs can contain homology to a native sequence in any whole unit integer increment from 70-100%.

A further embodiment of a composition in accordance with the invention is an antigen presenting cell that comprises one or more epitopes in accordance with the invention. The antigen presenting cell can be a"professional"antigen presenting cell, such as a dendritic cell.

The antigen presenting cell can comprise the epitope of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more individual epitopes or with one or more peptides that comprise multiple epitopes, by nucleic acid administration such as ballistic nucleic acid delivery or by other techniques in the art for administration of nucleic acids, including vector-based, e. g. viral vector, delivery of nucleic acids.

Further embodiments of compositions in accordance with the invention comprise nucleic acids that encode one or more peptides of the invention, or nucleic acids which encode a polyepitopic peptide in accordance with the invention. As appreciated by one of ordinary skill in the art, various nucleic acids compositions will encode the same peptide due to the redundancy of the genetic code. Each of these nucleic acid compositions falls within the scope of the present invention. This embodiment of the invention comprises DNA or RNA, and in certain embodiments a combination of DNA and RNA. It is to be appreciated that any composition comprising nucleic acids that will encode a peptide in accordance with the invention or any other peptide based composition in accordance with the invention, falls within the scope of this invention.

It is to be appreciated that peptide-based forms of the invention (as well as the nucleic acids that encode them) can comprise analogs of epitopes of the invention generated using priniciples already known, or to be known, in the art. Principles related to analoging are now known in the art. Generally the compositions of the invention are isolated or purified.

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

The examples herein are provided to illustrate the invention but not to limit its scope.

Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent application cited herein are hereby incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1. Schematic overview of the information generated by steps 1-4 of a method as used in the present invention.

FIGURE 2. Flow chart of a method as used in the present invention.

FIGURE 3. Drawing of the 43 lowest energy peptides resulting from the VSV-8 docking. The crystallographically determined structure is presented by the sticks model. Black color is used for the main-chain atoms and gray for the side-chain atoms. Only"heavy" (non-H) atoms are shown.

The viewpoint is from the"side"of the peptide with the N-terminus at the left. In the complex, the peptide is buried within the MHC aiaz domain, with the as-helix in front, the a1-helix at the back and the 8-sheet at the bottom; the upper part of the peptide is solvent accessible. The MHC receptor itself, while present during docking, is not shown in the figure.

FIGURE 4. Comparison between crystallographic temperature factors and theoretical structure variation. The average B-factors for the main-chain atoms of each residue of the peptide LLFGYPVYV, obtained from the PDB entry 1DUZ (c-chain) are compared with the standard deviation on the main-chain RMSD, observed in the ensemble of docked structures. The docking experiment itself is described in EXAMPLE 2 of the present invention.

FIGURE 5. Distribution of the number of docking solutions. All nonapeptides derived from the HPV E6 and E7 proteins were docked to the A*0201 receptor according to the protocol described in EXAMPLE 2 of the present invention. Each experiment yielded a set of receptor- compatible structures, ranging from 0 to 500. This diagram shows the distribution of docking solutions. 27 peptides were found to be incompatible with the receptor (inset). The main reason was the presence of either a bulky (R, Y, F) or a main-chain restricting (P) side-chain at position P2.

FIGURE 6. Probability distribution of the root-mean-square deviation (RMSD) between the backbone atoms of any two peptide main-chain structures of the {MHC/PmC} ensemble described in EXAMPLE 3 of the present invention.

FIGURE 7. Distribution of predicted average binding energies of HPV E6 and E7 peptides to HLA A*0201. Results are obtained as described in EXAMPLE 4 of the present invention. The energies do not include an entropical component. FIGURE 8. Correlation between experimental and predicted affinities for 15 peptides from HPV E6 and E7 that are known to bind to HLA A*0201. Results are obtained as described in EXAMPLE 4 of the present invention. Panel (a), scores obtained from average binding energies only. Panel (b), scores obtained by including the entropical component. Two peptides (sequences indicated) were considered as outliers and their scores were not included in the regression analysis.

EXAMPLES EXAMPLE 1. Peptide docking In the present example, we describe the flexible docking of the octapeptide VSV-8 (peptide p = RGYVYQGL) to murine MHC class I H-2Kb (Fremont, D. H. et al., (1995) Proc. Natl.

Acad. Sci. USA 92,2479-2483). The following experimental conditions were used.

1. Peptide build-up : Tyr-P5 was chosen as the root residue because of its potential to form multiple contacts with the binding groove on the MHC. Elongation proceeded first towards the C-and then towards the N-terminal end, in the following manner :--y->--YQ-->--YQG- >----YQGL >---VYQGL >--YVYQGL >-GYVYQGL > RGYVYQGL.

2. Peptide translations: the peptide was systematically displaced to each of 79 translational offsets at relative distances of 1.0, 2.0 and 4.0 A from the initial position.

3. Rotations: at each translational offset, discrete yet full-space rotation was performed over 84 rotational configurations.

4. Conformations: for the peptide residues Tyr-P3, Val-P4, Tyr-P5 and Gln-P6 the rotamer library contained 47 main-chain conformations; for Gly-P2 and Gly-P7 there were 125 rotamers and for the N-and C-terminal residues Arg-P1 and Leu-P8 there were 12.

5. Peptide and receptor side-chain conformations: side-chain conformations were retrieved from the backbone-dependent rotamer library described in Desmet et a/. (1997). On average, there were 16 side-chain rotamers per residue. In addition to the 8 peptide residues, 28 receptor residues were assigned as flexible during the docking.

6. Force field: all-atom CHARMM force field comprising terms for bond stretching, bond angle bending, a periodic function for the torsion angles, a Lennard-Jones potential for the non- bonded atom pairs, a 10-12 potential for hydrogen bonds and a coulombic function for charged atoms. A distance-dependent dielectric constant was used (E=rjJ, where rjj is the distance between two atoms i and j ; Warshel, A. and Levitt, M. (1976) J. Mol. BioL 103,227-249.

7. Water molecules : this experiment was performed in the presence of 9 crystallographically determined buried water molecules that were considered as part of the protein.

8. Partial-peptide conformations (fragments) were accepted for further elongation while using a relative energy threshold of 10 kcal mol'. In this experiment, final full-length peptides were accepted using. the same threshold.

9. The docking algorithm terminated spontaneously and successfully after having elongated in a combinatorial fashion, i. e. residue by residue, all partial peptides to their full length.

The docking of the VSV-8 peptide to MHC class I H-2Kb finally yielded a {MHC/pfU@ ensemble of 323 full-peptide configurations within an energy interval of 10 kcal mol~1 (see TABLE 9). For this purpose, 1,117, 957 partial peptide fragments had been processed during buildup.

TABLE 9: VSV-8 docking: Column 1: fragment length (number of residues) ; column 2: fragment sequence in one-letter code; column 3: total number of generated configurations for fragments of the corresponding length ; column 4: number of accepted configurations; column 5: acceptance ratio in % ; column 6: binding energy of the lowest-energy fragment (kcal mol~1) ; column 6: incremental binding energy (kcal mol~1) : length peptide #conf #accep % accep E_best JE_best ----Y---311, 892 920 0.29-24. 4-24.4 2----YQ--43, 240 2,074 4.80-43. 8-19.4 3----YQG-259, 250 13,081 5.05-51. 2-7.4 4----YQGL 156,972 289 0.18-73. 9-22.7 5---VYQGL 13,583 1,064 7.83-82. 0-8.1 6--YVYQGL 50,008 1,148 2.30-109. 5-27.5 7-GYVYQGL 143,500 11,626 8.10-120. 1-10.6 8 RGYVYQGL 139,512 323 0.23-147. 1-27.0 sum or average : 1,117, 957 30, 525 2.73-18. 4 Importantly, the docking algorithm rebuilds all side-chain conformations completely from scratch each time a partial or full peptide configuration is generated. In the present example this was accomplished by a dead-end elimination (DEE) method. In total, 1,117, 957 separate DEE side-chain placement operations were performed, i. e. one for each peptide fragment. This approach might be described as an elegant way to decouple the side-chain modeling from the main-chain construction. It enormously reduces the space to be searched and yet avoids any potential bias from incorrectly positioned or frozen side-chains. As a possible alternative to the DEE method, the present inventors refer to the recently published FASTER method (Desmet et a/., 2002). In general, any method for side-chain placement may be applicable. Prediction accuracy may actually form a lesser problem in view of the fact that the modeling of side-chains is repeated completely in step 3 of a method of the present invention. (But then only for the final full-length peptides, i. e. in the present example only 323 full structures instead of more than one million partial structures).

In summary, Table 1 shows that the acceptance ratio of partial peptide fragments was as low as 30,525 out of a total of 1,117, 957 examined fragments or 2.73%. Higher acceptance ratios were observed when extending a fragment by a weakly restrained residue type, such as Gly at position P2. Yet, the combinatorial buildup did not lead to an explosion of fragments.

Of the 323 final structures within an energy interval of 10 kcal mol-1, 43 had a binding energy within 5 kcal mol'1 above the lowest (-147.1 kcal mol~1) and are displayed in FIGURE 3.

Compared with the experimental structure of the complex, the lowest-energy peptide had a main-chain RMSD of only 0.56 A. For the 43 displayed structures the average RMSD was 0.89 0.27 A and for all 323 results it was 1.01 0.39 A. The anchor residues Tyr-P3, Tyr-P5 and Leu- P8 were correctly packed into their complementary pockets (Fremont, D. H. et a/., (1992) Science 257,919-927). The side-chain of Leu-P8 adopted two different conformational states.

Other apparently bi-stable conformations were observed for Gln-P6 and Arg-P1 (FIGURE 3).

The side-chain conformation of Gln-P6 was clearly coupled to the conformation of the MHC residues Glu-152 and Arg-155. Interestingly, the alternative conformation for these two residues has also been crystallographically observed, namely in the structure of the same H-2Kb receptor complexe with the nonapeptide SEV-9 (Fremont et al., 1992). This illustrates the importance of taking into account at least some limited flexibility for the side-chains of the receptor.

EXAMPLE 2. Systematic docking of viral peptides This example illustrates the performance of the docking algorithm described in EXAMPLE 1 in an application to large-scale docking. The purpose of this example is to demonstrate that the algorithm remains useful not only for studying selected cases that are known to form high-affinity complexes, but also for handling a large number of diverse peptides derived from a common protein source. Some features of such a collection are (i) that the set of peptides is not biased with respect to the presence of anchor residues and (ii) that the majority of peptides are most likely non-binders. Attention is paid to the computational requirements of the method, to statistics of the simulated structures and to potential difficulties in large-scale docking. This example also illustrates the preferred embodiment of steps 1 and 2 of a method of the present invention, i. e. MHC model preparation and flexible docking, respectively. In addition, we have performed a clustering analysis on the different observed peptide binding modes in order to study the (theoretical) variability of the main-chain of a peptide in a complex.

The test case was constructed as follows.

1. MHC receptor type/subtype: class 1, A*0201 2. PDB structure for model preparation: 1 DUZ a-chain 3. List of peptides to be docked: all nonameric (9-residue) peptides that can be derived from the human papillomavirus type 18 (HPV-18) E6 and E7 proteins, i. e. 150 and 97 peptides, respectively. Experimental binding affinities for the same set are available from the literature (Rudolf, M. P. et al., (2001) Clin. Cancer Res. 7, 788s-795s) 4. Docking conditions: force field and rotamer library are identical to Example 1.

Translations were limited to 26 relative displacements over 0.5 A from the original position. No rotational moves were allowed. All crystallographic water molecules were removed. The peptide residue P1 was selected as the root residue, thus elongation of fragments occurred from the N- to the C-terminus. The relative energy threshold for accepting partial peptide fragments was made dependent on the fragment length : 7,7, 10,13, 15,15, 15,13 and 10 for lengths 1-9, respectively. This was necessary because partial peptides of intermediate length tended to form many tight but false interactions with the receptor (class I nonapeptides typically bulge out in the middle ; Fremont et a/., 1992).

The selection of the PDB structure 1 DUZ to construct the MHC template model was decided on basis of its high crystallographic resolution (1.8 A). The whole PDB entry (chains a-e) were refined by 200 steps steepest descent energy minimization. Next, chains a (MHC) and c (peptide sequence LLFGYPVYV) were extracted. The only PDB information regarding the peptide that was retained upon docking were the coordinates of the backbone N, Ca and C atoms of residue P1. Prior to docking, each peptide was initialized by rebuilding it in an extended conformation with standard bond lengths and angles. The N, Ca and C atoms at residue P1 of the initialized peptide were fitted onto those observed in the PDB structure. Next, the peptide of the PDB file was removed. The MHC receptor together with the initialized peptide formed the starting situation for docking. A number of trial dockings were then performed using the"self'peptide LLFGYPVYV in order to determine the optimal settings for the relative energy thresholds of partial peptides of different length (values given supra, see: 4. Docking conditions). These trial experiments also served to reduce, in a safe way, the number of flexibly treated receptor side- chains: of the initial 29 side-chains in contact with the peptide, only 14 were finally kept flexible for they had a significant influence on the final ensemble of predicted structures (a7, a63, a66, a70, a73, a80, a84, a97, a99, a114, a116, a143, a146 and a159). With these settings, an ensemble of 210 structures was obtained for the A*0201/LLFGYPVYV complex. All peptide conformations compared well with the known crystallographic structure: the backbone RMSD ranged from 0.75 to 1.81 A, with an average of 1.08 0.20 A. A good correlation was observed between the crystallographic temperature factors and the structural variation exhibited by the ensemble of docked structures (Figure 4). The B-factors, averaged over the main-chain atoms of each peptide residue, appeared to follow well the standard deviation on the main-chain RMSD with the crystallographic structure, abbreviated as SD (RMSD). The latter was taken as a measure of the theoretical flexibility of the peptide main-chain. A somewhat larger than expected flexibility was observed for Gly-P4, which was due to a high degree of torsional freedom of the peptide planes flanking P4. A surprisingly high flexibility was also observed for Pro-P6: the Ca-Ca vector of this residue displayed a relatively large rotational variation over around the peptide's principal axis. Yet, this theoretical result appears to be fully justified on basis of the experimental B-factors. Also, the general correlation between both parameters suggests that the computed ensemble reflects the real dynamic behavior of the bound peptide. Given these satisfactory results, it was concluded that the experimental settings were correctly chosen. The latter were applied in all next docking experiments.

The large-scale docking of all HPV E6 and E7 peptides was performed in an automated fashion. The jobs were distributed over a cluster of four SGI Origin 200 computers, each equipped with four 270 MHz R12000 processors and 4 GB of memory. The average computational time needed per job was 8.7 CPU-hours, but some terminated almost immediately (0.01 CPU-h) or took a very long time (113.6 CPU-h). Typically, the docking of peptides containing large side-chains (Phe, Tyr, Arg) or Pro at position P2 tended to terminate before reaching their full length (FIGURE 5. Analysis showed that the P2 residue of these peptides could be accommodated only in "non-standard"conformations, for sterical reasons.

Rudolf et al. (2001) published experimental affinity data for peptides derived from the HPV E6 and E7 sequences and binding to HLA A*0201. Fifteen out of the 247 displayed IC50 values ranging from 3 to 943 nM. These peptides can thus be classified as strong or moderate binders to HLA A*0201. All other possible E6 and E7 peptides had ICso values higher than 1000 nM and can be termed weak or non-binders. Interestingly, many of the binding peptides had amino acid residues at positions P2 and P9 (the so-called primary anchor positions) that were non-typical for binding to HLA A*0201. For example, the top-ranked peptide, FAFKDLFW (with Ala at position P2 instead of Leu, fie or Met) displayed an IC50 value of only 3 nM. The peptide FKDLFVVYR (with Lys at P2 and Arg at P9) being a very non-typical peptide, still had an IC50 value of 500 nM. Two other binding peptides also had a non-typical aromatic residue at position P2, namely LYNLLIRCL and LFLNTLSFV. Especially for these peptides it was interesting to investigate the behavior of the docking algorithm.

It can be seen from Figure 5 that none of the docking experiments failing to extend the peptide to its full length (26 out of 247 in total) concerned binding peptides (15 out of 247). Even the two binding peptides containing Tyr or Phe at position P2 could be successfully docked (the LYNLLIRCL and LFLNTLSFV docking resulted in 8 and 13 solutions, respectively), in contrast to many other peptides containing an aromatic side-chain at that position (Figure 5). The FKDLFVVYR peptide could also be successfully docked (30 solutions) in spite of its bulky Arg side-chain at P9. In general, large side-chains at the primary anchors P2 and P9 had the effect of reducing the number of docking solutions due to sterical restraints. For some peptides, all of which are weak or non-binders, this led to premature termination of the docking process.

Another important observation was that the binding peptides had, on average, a much higher number of docking solutions than the non/weak binders. Binding peptides were represented by about twice as much solutions as non/weak binders (on average: 91 vs. 42 solutions, respectively). Similarly, only 3 of the 15 binders (20%) had less than 25 solutions whereas there were 132 of the 232 (57%) with less than 25 solutions among the non/weak binders. A logical conclusion is that the number of solutions obtained from the peptide docking experiments provides an indication of true conformational flexibility of a peptide within the MHC binding groove. This is consistent with the fundamental entropical principle stating that the higher the number of micro-states for a given macro-state (in this case the bound state) the higher will be the probability of that state. This example also illustrates the importance of working with ensembles of structures, rather than with a single modeled structure, to study the binding properties of MHC/peptide complexes.

EXAMPLE 3. Construction of a generic MHC/peptide database An embodiment of the present invention is a method wherein the binding of one or more peptides is studied by applying an advanced database approach. As explained in the detailed description of the invention, such a database may be compiled from experimental (preferably X- ray) or theoretical (preferably docked) structures. A database obtained from known 3D structures has the advantage of being based on validated structural information but may suffer from the lack of such data, especially for certain MHC subtypes for which no complex structure has been solved. Even for well-represented subtypes, like the MHC class I HLA A*0201 allotype, there may be a strong bias towards particular observed peptide binding modes whereas many other feasible conformations are not yet represented in the Protein Databank. Consequently, in order to avoid problems related to a lack of experimental structures, the present inventors prefer to generate a database of MHCIP,, structures by systematically docking a large number of peptides of different sequence. Evidently, this can be done separately for different MHC subtypes and for peptides of different length. In this example we illustrate the construction of an {MHClPmC} ensemble for nonameric peptides oriented within the binding groove of HLA A*0201 (represented by PDB code 1 DUZ, chain a).

The docking experiments were performed in an identical way to the experiments described in Example 2. A set of 180 nonameric peptide sequences to be docked was established in a pseudo-random fashion as follows. The present inventors have selected combinations of typical anchor residues at positions P2 and P9, i. e. Leu, Ile and Met at P2 and Leu, Ile and Val at P9. At all other positions, residue types were selected in a fully random fashion from the set of naturally occurring amino acids. This means that each of the 3x3=9 possible P2/P9 combinations was represented by 180/9=20 sequences with randomized residues at positions P1 and P3-P8. This procedure was followed to avoid the docking of peptides that cannot bind to the HLA A*0201 model because of incompatible anchor residues. At the same time, the randomization was assumed to generate sufficient variation in the peptide sequences to ensure a broad and unbiased sampling of the conformational space.

All but one docking experiments terminated in a successful way, i. e. only one simulation (of the peptide p = DIGVHKWVV) terminated before the peptide was extended to its full length. All other simulations yielded a number of MHClpmc solutions ranging from 1 to 500 (a user-set hard limit) and with an average of 22 per peptide. The total number of MHcipmc structures was 3951.

All docking results were then pooled into one global {MHGlPmc} ensemble, the side- chains were stripped off and the coordinates of the main-chain atoms of each peptide structure were stored in a suitable format in a database. This completed the construction of a generic database collection of M/C/Pmc structures, applicable for studying the binding of nonapeptides to the MHC class I HLA A*0201 subtype.

The ensemble was afterwards further analyzed with respect to the spatial distribution of peptide conformations in the {MHClPm} ensemble. A suitable parameter to analyze this distribution is the peptide backbone root-mean-square deviation (RMSD) between different Pmc structures in the ensemble. FIGURE 6 shows the probability distribution of finding two main- chain structures having a certain RMSD. From the integrated probability curve it is seen that for any selected Pmc structure the expected number of other structures with an RMSD < 0.5 A is only about 0.3% of the total population. This shows that there is very limited, if any, redundancy among the members of the ensemble. The probability of an RMSD < 1 A raises to 0.062 or 6.2%. With respect to modeling side-chains on backbones, a difference in RMSD of up to 1 A can be expected to yield similar results. In other words, the further modeling of a peptide sequence onto each Pmc structure will be statistically performed onto 0. 062x3951 or about 250 relatively correct structures. This situation offers the possibility of a further clustering of the ensemble and/or the averaging of the results from different side-chain placements. Furthermore, the width of the probability distribution (-3 A) suggests that a great variety of different binding modes, some of which may be required for specific peptides, are represented in the ensemble.

From these results, the inventors concluded that the database approach forming an embodiment of the present invention may be very useful to predict the binding properties of a peptide within an MHC binding groove.

EXAMPLE 4. Application of a scoring function to predict affinities A property of an MHC/peptide complex is the affinity of the peptide for the MHC molecule. In accordance with the structure-based approach of the present invention, the binding affinity is predominantly derived from information related to the three-dimensional structure of a modeled complex. For this purpose, a so-called scoring function is required which translates structural information into one or more contributions that are expected to correlate with experimental affinity. Different contributions may be combined, for example added up, in order to provide a qualitative or quantitative score for an MHC/peptide complex of interest. By extension, different scores for different complexes may be computed, for example to rank different peptides according to their predicted affinity for a given MHC.

This example is included to illustrate a practical implementation of an embodiment of the present invention. This example is further included to demonstrate that the incorporation of an entropical contribution derived from an ensemble of modeled complex structures, rather than from a single modeled or experimental structure, significantly enhances the quality of predicted affinities. Said incorporation of an entropical component is in agreement with both Eqs. [1] and [5] of the present invention.

The results of the docking experiments described in example 2, more specifically the computer simulated binding of all HPV E6/E7 peptides to the HLA A*0201 receptor, have been further analyzed so as to eventually predict the affinity of the peptides. We recall that each of these docking experiments yielded an ensemble of MHclpms solutions, in accordance with a second step (MHC/peptide main-chain construction) of an embodiment of the present invention.

These ensembles have been further processed in accordance with a third step (MHC/full peptide construction) and a fourth step (MHC/peptide affinity assessment) of an embodiment of the present invention.

First, the side-chains of each MHClpmc structure in each ensemble were rebuilt by applying the DEE method of De Maeyer et a/. (2000). Side-chains of the MHC receptor that were flexibly treated were the same as during the docking experiments described in Example 2 (14 in total). In order to reduce the effects from discrete rotameric placement of the side-chains, an additional modeling step was performed on each DEE-modeled structure: the full structures were further refined by 50 steps of steepest descent energy minimization to optimize local contacts. This resulted in the final set of ensembles {MHCIpful i, e. one ensemble of full complex structures for each peptide p. These data formed the major source of structure-related input information for a fourth step of an embodiment of the present invention.

Since complex formation involves a physico-chemical reaction between a receptor and ligand molecule from the unbound to the bound state, the binding process is driven by a change in free energy or AG (see Eqs. [3] and [4] ). Consequently, an energetical evaluation of complex structures is preferably complemented by a similar evaluation of models of the unbound molecules. The free MHC receptor was therefore modeled separately by performing DEE side- chain placement with the same 14 flexibly treated side-chains as for the full complexes, followed by 50 steps of steepest descent energy minimization. Structures for the free peptide, on the other hand, were not generated by DEE modeling but by generating maximally extended conformations, also followed by 50 steps of steepest descent energy refinement. The binding energy Ebjnd (p, i) of a solution i from the ensemble generated for a peptide p was calculated using equation [6]: Ebind (P,') = Ecomp) ex (P, )-EMHC-Ep ( ?) [6] where all energy values are the potential energies computed in accordance with the force field, and where Ecomplex (pX EMHC and Ep (p) are the potential energy of the complex, free receptor and free peptide, respectively. Next, the binding energies were averaged over all solutions i for each peptide p so as to obtain the average binding energy <Ebind (P) > for the each ensemble {M-C/pM}. This quantity corresponds to the term <E> in Eq. [1] of the present invention.

Figure 7 shows the distribution of the average binding energies for all predicted peptides.

Peptides that were experimentally found to be good binders by Rudolf et al. (2001) are indicated in black whereas the non-binders are indicated with gray bars. It is clearly seen that the known binders tend to score well in comparison with the non-binders. Yet, both populations are not clearly separated in that several non-binders score better than most of the binders (they can be envisaged as"false positives"). This suggests that the discriminative power of potential energy alone is not strong enough to obtain good separation.

In view of the observation that most of the non-binding peptides had, on average, less MHC/pmC solutions in the docking step (see Example 2), it was investigated whether this factor could be converted into a significant, quantitative contribution of the scoring function. The most significant improvement in separation between binders and non-binders was obtained when adding to the potential energy term a logarithmic term depending on the total number of solutions N contained within each ensemble. Thus, the optimal scoring function F appeared to be of the form FlN)-Ebind (p) -c x In N (p) wherein c is a constant. Interestingly, the theory of statistical mechanics states that the entropy of (microcanonical) ensembles is logarithmically related to the number of micro-states that are energetically accessible. (More specifically, the entropy S equals kg in (N) where. kB is Boltzmann's constant). Thus, it was straightforward to rationalize the logarithmical dependence on the number of solutions as a true reflection the intrinsic conformational flexibility a peptide within a complex. In other words, the number of energetically feasible peptide conformations as derived from the simulations probably correlates in a statistically significant way with the true conformational entropy of a complex.

From the optimization of the separation of binders and non-binders, the best value for parameter c in Eq. [7] was found to be 20 kcal mol'. This value was applied in a further analysis wherein the predicted scores for the 15 binding peptides were directly correlated with the known experimental affinity (Rudolf et a/. (2001) only published quantitative values for the binding peptides). Figure 8 shows a correlation plot between predicted scores and known binding free energies. In Figure 8a the entropical term is turned off (c=0) while in Figure 8b it was set to its optimal value from the previous optimization procedure (c=20). Two peptides (FQQLFLNTL and FLNTLSFVC) showed an aberrant behavior compared to the rest and were considered as outliers. They were not included in the regression analysis. Interestingly, both peptides have a non-typical anchor residue (Gln at P2 of FQQLFLNTL and Cys at P9 of FLNTLSFVC) while their scores appeared to be overestimated. This suggests that an additional correction factor may be desirable for typical anchor residues.

An important observation within the context of the present invention was the markedly better correlation obtained with the scoring function including the entropical term (panel b, R2 = 0.71) compared to the function based exclusively on potential energy (panel a, R2 = 0.19).

Without the entropy component only a very weak correlation could be observed. This is consistent with the distribution plot presented in Figure 7 showing that the energy component itself is practically useful only to identify peptides with a clear suboptimal energetic compatibility with the receptor. Only the combination of potential energy with a term reflecting conformational entropy enabled a good qualitative separation between binding and non-binding peptides.

Furthermore, it enabled the establishing of a quantitative relationship between predicted and experimental affinities. Figure 8b shows the equation that can be used to convert any score value F into a predicted free energy of binding.

EXAMPLE 5: HBV peptide generation using Epibase In order to identify HBV epitopes for an antigen of interest, the HBV core, polymerase and surface protein regions are computer screened and scored by the method of this invention.

The HBV protein is chopped up into all possible nonamers, referred to as peptides hereafter. Each peptide is docked into the groove of an HLA antigen receptor. HLA-receptors used in this method include HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3. Each peptide- receptor docking is scored using a the scoring function as described herein. The scoring function is an indication of the binding affinity of the peptide-receptor complex. A score of less than-30 stands for a high affinity binder. A score between-30 and-25 represents a good binder. The higher the score, the lower the binding affinity.

The scoring results for HBV core peptides binding to multiple antigens, said antigens being HLA-A2, HLA-A24 and HLA-CW4 are represented in Table 5. Table 6 represents the scoring results of HBV surface peptides binding to said multiple antigens. Table 7 represents the scoring results of HBV polymerase peptides for said multiple antigens.

Table 1 represents peptides for the HBV core protein that bind with high affinity to multiple antigens, said antigens being HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3.

Table 2 represents peptides for the HBV surface protein that bind with high affinity to said multiple antigens. Peptides for the HBV polymerase protein that bind with high affinity to said multiple antigens are listed in Table 3. These lists are characterised in that for each particular peptide, at least one HLA-antigen receptor has a high affinity score of lower than-30, while for all other HLA-antigens a binding score between-30 and-25 is obtained. Table 4 represents the top-10 scoring HBV peptides according to this rule.

TABLE 1: HBV Core peptides having best scores for multiple antigens HLA-A2, HLA-A24, HLA- CW4, HLA-A1 and HLA-A3 (X = no score available) : HLA-A2 HLA-A24 HLA-CW4 HLA-A1 HLA-A3 SEQ ID sequence score score score score score 5 PYKEFGATV-32. 978-34. 6274-31. 95 X-17. 39 16 LSFLPSDFF-33. 70-28. 88-30. 00-23. 62-23. 47 23 FFPSVRDLL-39. 53-28. 87-29. 42-24. 90-27. 95 87 NYVNTNMGL-30. 015-32. 1094-33. 87-22. 24-25. 78 95 LKIRQLWWF-39. 23-41. 97-26. 71-26. 30-28. 01 100 LWWFHISCL-36. 52-34. 90-30. 86-27. 27-28. 93 101 WWFHISCLT-29. 32-30. 35-29. 06-17. 59-35. 01 102 WFHISCLTF-33. 46-30. 73-27. 82-29. 77-28. 78 114 TVLEYLVSF-34. 83-33. 77-26. 81-26. 58-22. 53 118 YLVSFGVWI-43. 30-33. 99-31. 35-33. 29 124 VWIRTPPAY-30. 9754-27. 721-32. 51-24. 56-18. 26 131 AYRPPNAPI-33. 0374-27. 2275-28. 81-20. 24-26. 85 TABLE 2: HBV Surface peptides having best scores for multiple antigens HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3 together (X = no score available) : HLA-A2 HLA-A24 HLA-CW4 HLA-A1 HLA-A3 SEQ ID Sequence score score score score score 346 PLLVLQAGF-25. 82-27. 29-31. 6 X X 347 LLVLQAGFF-29. 44-30. 42-30. 65-25. 04-28. 58 348 LVLQAGFFL-31. 05-29. 48-28. 64-28. 72-27. 09 349 VLQAGFFLL-33-31. 82-34. 52-30. 43-31. 21 352 AGFFLLTKI-30. 08-29. 19-30. 63-25. 38-24. 27 s 355 FLLTKILTI-34. 58-30. 77-29. 19-28. 45-31. 23 402 PICPGYRWM-30. 66-31. 7-25. 71-28. 49 414 RFIIFLCIL-27. 91-32. 27-31. 75-35. 45-28. 37 416 IIFLCILLL-33. 14-27. 75-29-28. 09-20. 63 422 LLLCLIFLL-37. 8-32. 55-30. 6-29. 24-31. 87 425 CLIFLLVLL-38. 73-28. 68-28. 97-28. 42-30. 58 443 PLIPGSSTT-33. 27-25. 5-25. 89-25. 35 507 WLSLLVPFV-32. 07-29. 82-26. 03-25. 78-28. 74 510 LLVPFVQWF-32. 16-38. 07-39. 21-33-36. 44 511 LVPFVQWFV-33. 3-29. 13-28. 86-25. 28-33. 5 524 TVWLSVIWM-32. 77-32. 28-28. 64-27. 42-28. 51 526 WLSVIWMMW-29. 96-34. 68-26. 3-33. 43-19. 52 527 LSVIWMMWY-31. 41-44. 35-29. 95-33. 32-28. 02 529 VIWMMWYWG-28. 88-38. 06-29. 91-38. 81-27. 56 531 WMMWYWGPS-26. 68-31. 03-27. 94-31. 59-29. 08 532 MMWYWGPSL-28. 4-35. 04-33. 73-30. 26-32. 72 540 LYNILSPFM-31. 82-36. 92-33. 61-31. 07-31. 68 542 NILSPFMPL-30. 28-27. 99-25. 49-29. 05-26. 78 543 ILSPFMPLL-39. 3-31. 97-32. 68-34. 22-32. 76 547 FMPLLPIFF-29. 9-38. 01-36. 76 x-33. 89 549 PLLPIFFCL-41. 5-35. 24-37. 39-31. 35-39. 04 552 PIFFCLWVY-29. 6-37. 52-32. 42 X X TABLE 3: HBV DNA Polymerase peptides having best scores for multiple antigens HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3 together (X = no score available) : HLA-A2 HLA-A24 HLA-CW4 HLA-A1 HLA-A3 SEQ ID sequence score score score score score 615 LYSSTVPVF-28. 14-32. 19-35. 97-28. 88-26. 19 660 LKLIMPARF-25. 05-38. 02-29. 48-21. 27-33. 54 662 LIMPARFYP-32. 65-36. 27-25. 15-25. 08-28. 97 873 VLSCWWLQF-25. 64-31. 01-25. 52-22. 85-29. 2 1038 NLYVSLMLL-33. 54-26. 23-30. 3-27. 19-26. 39 1039 LYVSLMLLY-27. 12-38. 83-37. 13-33. 52-27. 19 1042 SLMLLYKTY-30. 27-29. 24-29. 02-28. 39-31. 27 1046 LYKTYGWKL-29. 99-31. 98-33. 31-37. 38-25. 98 1060 PIILGFRKI-30. 46-33. 35-Z5. 94-29. 27-29. 42 1069 PMGVGLSPF-26. 47-31. 62-29. 9-30. 53-28. 79 1120 YAAVTNFLL-28. 88-27. 36-30. 44-25. 81-26. 14 1126 FLLSLGIHL-34. 68-29. 49-28. 22-26. 28-28. 99 1144 YSLNFMGYV-26. 65-33. 16-25. 69-27. 62-26. 19 1145 SLNFMGYVI-31. 84-27. 53-28. 56-29. 59-22. 17 1148 FMGYVIGSW-26. 17-28. 02-30. 25 X-31. 73 1191 GLLGFAAPF-30. 17-27u77-28. 04-26. 4-29. 15 1206 ALMPLYACI-34. 26-29. 46-30. 07-28. 17-32. 69 1222 FSPTYKAFL-28. 52-32. 33-26. 22-33. 8-20. 39 1229 FLSKQYMTL-31. 64-28. 94-31. 92-34. 12-32. 23 1329 SFVYVPSAL-25. 5-30. 97-34. 96-32. 67-27. 22 1353 PLLRLPYRP-34. 64-32. 29-27. 05-21. 87-33. 09 TABLE 4: Top 10 scoring HBV peptides for multiple antigens HLA-A2, HLA-A24, HLA-CW4, HLA-A1 and HLA-A3: HLA-A2 HLA-A24 HLA-CW4 HLA-A1 HLA-A3 SEQ ID sequence score score score score score 95 LKIRQLWWF''-39. 23-41. 97-26. 71-26. 30-28. 01 348 LVLQAGFFL (Z)-31. 05-29. 48-28. 64-28. 72-27. 09 524 TVWLSVIWM''-32. 77-32. 28-28. 64-27. 42-28. 51 527 LSVIWMMWY, 41-31. 41-44. 35-29. 95-33. 32-28. 02 540 LYNILSPFM (Z)-31. 82-36. 92-33. 61-31. 07-31. 68 615 LYSSTVPVF''-28. 14-32. 19-35. 97-28. 88-26. 19 1042 SLMLLYKTY (3)-30. 27-29. 24-29. 02-28. 39-31. 27 1120 YAÅVTNFLL (3)-28. 88-27. 36-30. 44-25. 81-26. 14 1222 FSPTYKAFL (3)-28. 52-32. 33-26. 22-33. 8-20. 39 1229 FLSKQYMTL-31. 64-28. 94-31. 92-34. 12-32. 23 (') HBV Core (2) HBV Surface (3) HBV DNA Polymerase TABLE 5: HBV Core binders for at least two different HLA-types chosen from HLA-A2, HLA- A24 and HLA-CW4: SEQ ID Source Sequence HLA-A2 score HLA-A24 score HLA-CW4 score 5 HBV core PYKEFGATV-32. 98-34. 63-31. 95 68 HBV core LATWVGNNL-33. 62-27. 09-24. 69 85 HBV core VVNYVNTNM-33. 71-25. 64-24. 46 130 HBV core PAYRPPNAP-36. 34-25. 11-21. 02 64 HBV core ELMTLATWV-35. 73-24. 28-22. 25 47 HBV core HCSPHHTAL-34. 66-28. 87-17. 39 8 HBV core EFGATVELL-30. 12-25. 34-25. 12 60 HBV core LCWGELMTL-32. 44-26. 71-21. 01 138 HBV core PILSTLPET-33. 46-23. 24-21. 6 108 HBV core LTFGRETVL-34. 05-22. 57-20. 62 65 HBV core LMTLATWVG-33. 64-21. 88-21. 62 58 HBV core AILCWGELM-21. 92-22. 11 57 HBV core QAILCWGEL-30. 37-24. 78-20. 68 132 HBV core YRPPNAPIL-28. 52-26. 4-18. 29 83 HBV core DLVVNYVNT-32. 73-18. 49-20. 55 66 HBV core MTLATWVGN-32. 11-22. 14-17. 18 129 HBV core PPAYRPPNA-34. 47-19. 33-17. 57 139 HBV core ILSTLPETT-33. 61-17. 3-19. 79 84 HBV core LVVNYVNTN-28. 79-19. 19-20. 06 136 HBV core NAPILSTLP-35. 74-17. 25-13. 03 7 HBV core KEFGATVEL-26. 45-20. 3-16. 77 61 HBV core CWGELMTLA-26. 13-20. 15-16. 81 134 HBV core PPNAPILST-33. 03-12. 89-16. 93 135 HBV core PNAPILSTL-38. 02 X-24. 71 10 HBV core GATVELLSF-27. 29-21. 01-14. 18 25 HBV core PSVRDLLDT-31. 46-17. 99-12. 84 163 HBV core PRRRRSQSP-27. 38-22. 63-11. 11 42 HBV core LKSPEHCSP-25. 82-19. 31-15. 53 79 HBV core PASRDLVVN-28. 62-17. 48-14. 36 9 HBV core FGATVELLS-26. 12-16. 34-15. 77 70 HBV core TWVGNNLED-28. 46-13. 77-15. 43 80 HBV core ASRDLVVNY-27. 43-14. 03-15. 73 43 HBV core KSPEHCSPH-29. 2-19. 96-7. 81 62 HBV core WGELMTLAT-22. 56-20. 87-12. 89 152 HBV core RGRSPRRRT-26. 3-24. 47-4. 74 161 HBV core PSPRRRRSQ-27. 97-18. 55-8. 85 148 HBV core WRRRGRSP-28. 87-17. 98-7. 86 63 HBV core GELMTLATW-24. 24-16. 99-13. 48 3 HBV core IDPYKEFGA-23. 99-15. 77-14. 22 46 HBV core EHCSPHHTA-25. 9-17. 27-10. 45 71 HBV core WVGNNLEDP-22. 98-16. 07-14. 2 59 HBV core ILCWGELMT-32. 29-19. 8 4 HBV core DPYKEFGAT-21. 16-13. 47-17. 14 6 HBV core YKEFGATVE-14. 82-20. 52-16. 25 82 HBV core RDLVVNYVN-21. 28-20. 16-9. 64 55 HBVcore LRQAILCWG-23. 87-15. 76 1-10. 7 154 HBV core RSPRRRTPS-25. 72-19. 33-3. 71 67 HBV core TLATWVGNN-29. 82 X-18. 81 81 HBV core SRDLVVNYV-19. 72-12. 68-15. 25 56 HBV core RQAILCWGE-18. 34-18. 96-7. 96 153 HBV core GRSPRRRTP-22. 91-15. 85-5. 82 48 HBV core CSPHHTALR-19. 82-18. 03-6. 26 159 HBV core RTPSPRRRR-23. 83-15. 15-4. 95 156 HBV core PRRRTPSPR-20. 37-17. 18-6. 13 171 HBV core PRRRRSQSR-21. 68-16. 21-5. 55 167 HBV core RSQSPRRRR-20. 83-17. 34-2. 59 69 HBV core ATWVGNNLE-23. 82-15. 69 TABLE 6: HBV Surface binders for at least two different HLA-types chosen from HLA-A2, HLA- A24 and HLA-CW4: SEQ ID Source Sequence HLA-A2 score HLA-A24 score HLA-CW4 score 546 HBV surface PFMPLLPIF-24. 85-42. 86-45. 84 540 HBV surface LYNILSPFM-31. 82-36. 92-33. 61 552 HBV surface PIFFCLWVY-29. 6-37. 52-32. 42 414 HBV surface RFIIFLCIL-27. 91-32. 27-31. 75 347 HBV surface LLVLQAGFF-29. 44-30. 42-30. 65 348 HBV surface LVLQAGFFL-31. 05-29. 48-28. 86 402 HBV surface PICPGYRWM-30. 66-31. 7-25. 71 437 HBV surface GMLPVCPLI-34. 21-24. 93-26. 27 346 HBV surface PLLVLQAGF-25. 82-27. 29-31. 6 443 HBV surface PLIPGSSTT-33. 27-25. 5-25. 89 280 HBV surface AMQWNSTTF-19. 36-30. 33-34. 43 542 HBV surface NILSPFMPL-30. 28-27. 99-25. 49 342 HBV surface GLLGPLLVL-33. 13-26. 6-23. 4 224 HBV surface FGPGFTPPH-26. 05-26. 01-30. 99 350 HBV surface LQAGFFLLT-37. 68-25. 32-19. 7 539 HBV surface SLYNILSPF-29. 27-23. 57-29. 34 183 HBV surface LGFFPDHQL-24. 11-27. 24-30. 48 7 547 HBV surface FMPLLPIFF-29. 9-26. 28-24. 99 227 HBV surface GFTPPHGGL-21. 56-31. 46-27. 59 544 HBV surface LSPFMPLLP-36. 01-23. 96-19. 89 01 551 HBV surface LPIFFCLWV-25. 8-24. 68-28. 15 541 HBV surface YNILSPFMP-26. 45-25. 3-26. 69 182 HBV surface PLGFFPDHQ-27. 43-27. 01-23. 23 343 HBV surface LLGPLLVLQ-32. 06-22. 7-22. 4 228 HBV surface FTPPHGGLL-24. 91-28. 78-23. 39 300 HBV surface LYFPAGGSS-19. 17-27. 9-29. 93 340 HBV surface ASGLLGPLL-30. 7-24. 02-22. 18 410 HBV surface MCLRRFIIF-23. 5-31. 8-20. 79 -2 299 HBV surface ALYFPAGGS-26. 95-22. 93-24. 71 245 HBV surface ILTSVPAAP-29. 18-21. 88-23. 1 538 HBV surface PSLYNILSP-25. 89-22. 24-25. 58 -25. 58 246 HBV surface LTSVPAAPP-26. 47-20. 5-26. 6 265 HBV surface QPTPLSPPL-27. 07-16. 61-29. 09 298 HBV surface RALYFPAGG-22. 02-30. 67-19. 16 238 HBV surface WSPQAQGIL-22. 75-29. 24-19. 69 220 HBV surface GVGAFGPGF-25. 82-22. 13-23. 56 318 HBV surface TVSAISSIL-30. 46-21. 04-19. 33 429 HBV surface LLVLLDYQG-27. 64-24. 26-18. 11 248 HBV surface SVPAAPPPA-25. 24-17. 64-26. 75 223 HBV surface AFGPGFTPP-19. 51-24. 49-25. 04 413 HBV surface RRFIIFLCI-22. 95-29. 43-16. 18 296 HBV surface RVRALYFPA-25. 1-24. 41-l8, gg 185 HBV surface FFPDHQLDP-18. 41-26. 92-23. 04 314 HBV surface PAQNTVSAI-20. 92-25. 33-22 339 HBV surface IASGLLGPL-26. 67-22. 5-19. 03 250 HBV surface PAAPPPAST-25. 34-22. 55-20. 3 409 HBV surface WMCLRRFII-20. 15-26. 03-21. 51 351 HBV surface QAGFFLLTK-24. 81-21. 78-21. 03 335 HBV surface NMENIASGL-19. 73-25. 57-22. 3 268 HBV surface PLSPPLRDT-27. 31-20. 68-19. 59 247 HBV surface TSVPAAPPP-29. 19-21. 02-16. 8 281 HBV surface MQWNSTTFH-20. 98-25. 66-20. 01 235 HBV surface LLGWSPQAQ-24. 96-21. 23-20. 02 -20-02 545 HBV surface SPFMPLLPI-29. 19-15. 44-21. 23 325 HBV surface ILSKTGDPV-25. 43-20. 5-19. 46 548 HBV surface MPLLPIFFC-27. 34-18-20. 03 218 HBV surface KVGVGAFGP-26. 92-19. 94-18. 39 26. 92 9 341 HBV surface SGLLGPLLV-28. 74-18. 46-17. 46 221 HBV surface VGAFGPGFT-24. 54-21. 42-18. 23 317 HBV surface NTVSAISSI-22. 32-20. 46-21. 37 372 HBV surface TSLNFLGGT-20. 41-25. 54-17. 16 -17. 16 373 HBV surface SLNFLGGTP-24. 53-18. 11-20. 44 222 HBV surface GAFGPGFTP-24. 2-19. 01-19. 85 344 HBV surface LGPLLVLQA-27. 15-20. 68-15. 05 338 HBV surface NIASGLLGP-23. 82-20. 29-17. 87 177 HBV surface LSVPNPLGF-21. 53-20. 96-18. 95 175 HBV surface TNLSVPNPL-23. 33-20. 18-17. 84 311 HBV surface TVSPAQNTV-23. 08-21. 11-17. 16 241 HBV surface QAQGILTSV-21. 42-22. 16-17. 7 244 HBV surface GILTSVPAA-24. 59-19. 17-16. 97 332 HBV surface PVPNMENIA-20. 79-20. 41-19. 31 415 HBV surface FIIFLCILL-34. 24-9. 44-16. 76 179 HBV surface VPNPLGFFP-26. 22-17. 2-16. 05 _l7. 2 05 292 HBV surface LQDPRVRAL-24. 98-20. 18-12. 74 178 HBV surface SVPNPLGFF-24. 04-21. 45-11. 03 03 461 HBV surface TPAQGTSMF-15. 73-17. 39-23. 25 412 HBV surface LRRFIIFLC-19. 99-19. 14-16. 15 217 HBV surface HKVGVGAFG-16. 51-25. 75-12. 46 406 HBV surface GYRWMCLRR-21. 65-18. 45-14. 15 368 HBV surface DSWWTSLNF-16. 67-20. 76-16. 21 401 HBV surface PPICPGYRW-23. 41-11. 91-17. 13 375 HBV surface NFLGGTPVC-10. 44-19. 52-20. 16 442 HBV surface CPLIPGSST-23. 43-6-12. 87 459 HBV surface CTTPAQGTS-15. 78-12. 77-13. 45 439 HBV surface LPVCPLIPG-23. 47 5. 53-13. 22 396 HBV surface SPTCCPPIC-15. 78-5. 19-4. 83 371 HBV surface WTSLNFLGG-15. 63-18. 6 1000 TABLE 7: HBV DNA Polymerase binders for at least two different HLA-types chosen from HLA-A2, HLA-A24 and HLA-CW4: SEQ ID Source Sequence HLA-A2 score HLA-A24 score HLA-CW4 score 1039 HBV DNA pol LYVSLMLLY-27. 12-38. 83-37. 13 1046 HBV DNA pol LYKTYGWKL-29. 99-31. 98-33. 31 662 HBV DNA pol LIMPARFYP-32. 65-36. 27-25. 15 1353 HBV DNA pol PLLRLPYRP-34. 64-32. 29-27. 05 668 HBV DNA pol FYPNVTKYL-24. 77-37. 14-31. 5 660 HBV DNA pol LKLIMPARF-25. 05-38. 02-29. 48 1229 HBV DNA pol FLSKQYMTL-31. 64-28. 94-31. 92 1049 HBV DNA pol TYGWKLHLY-16. 88-39. 96-33. 39 1038 HBV DNA pol NLYVSLMLL-33. 54-26. 23-30. 3 1060 HBV DNA pol PIILGFRKI-30. 46-33. 35-25. 94 987 HBV DNA pol FYHLPLHPA-23. 16-33. 62-31. 88 1042 HBV DNA pol SLMLLYKTY-30. 27-29. 24-29. 02 1069 HBV DNA pol PMGVGLSPF-26. 47-31. 62-29. 9 1145 HBV DNA pol SLNFMGYVI-31. 84-27. 53-28. 56 1233 HBV DNA pol QYMTLYPVA-20. 19-35. 46-32. 05 667 HBV DNA pol RFYPNVTKY-18. 09-38. 83-29. 83 1120 HBV DNA pol YAAVTNFLL-28. 88-27. 36-30. 44 661 HBV DNA pol KLIMPARFY-27. 79-35. 11-23. 62 1347 HBV DNA pol RLGLYRPLL-31. 7-33. 34-20. 8 1144 HBV DNA pol YSLNFMGYV-26. 65-33. 16-25. 69 1388 HBV DNA pol PLHVAWRPP-28. 57-32. 17-24. 18 1041 HBV DNA pol VSLMLLYKT-28. 62-31. 23-24. 98 1148 HBV DNA pol FMGYVIGSW-26. 17-28. 02-30. 25 986 HBV DNA pol AFYHLPLHP-17. 79-35. 44-30. 56 556 HBV DNA pol LSYQHFRKL-24. 46-36. 08-23. 23 684 HBV DNA pol PYYPEHVVN-18. 03-33. 44-31. 24 1062 HBV DNA pol ILGFRKIPM-33. 44-23. 64-25. 62 1043 HBV DNA pol LMLLYKTYG-27. 51-30. 8-24. 2 663 HBV DNA pol IMPARFYPN-26. 68-27. 97-27. 72 873 HBV DNA pol VLSCWWLQF-25. 64-31. 01-25. 52 1150 HBV DNA pol GYVIGSWGT-25. 91-29. 08-27. 14 1070 HBV DNA pol MGVGLSPFL-33. 38-25. 18-23. 5 1044 HBV DNA pol MLLYKTYGW-27. 85-24. 73-29. 1 692 HBV DNA pol NHYFQTRHY-17. 41-37. 8-25. 94 1272 HBV DNA pol FVSPLPIHT-32. 37-25. 95-22. 59 1066 HBV DNA pol RKIPMGVGL-23. 93-32. 16-24. 56 1020 HBV DNA pol IINHQHGTM-20. 89-32. 03-27. 71 1141 HBV DNA pol RWGYSLNFM-15. 46-28. 91-35. 93 682 HBV DNA pol IKPYYPEHV-29. 46-29. 19-21. 54 1074 HBV DNA pol LSPFLLAQF-28. 65-23. 74-27. 5 670 HBV DNA pol PNVTKYLPL-25. 68-28. 67-25. 51 1037 HBV DNA pol RNLYVSLML-26. 31-31. 79-21. 73 621 HBV DNA pol PVFNPEWQT-25. 13-31. 5-22. 99 982 HBV DNA pol DVSAAFYHL-25. 99-29. 1-24. 07 555 HBV DNA pol PLSYQHFRK-22. 87-29. 45-26. 76 984 HBV DNA pol SMFYHLPL-27. 64-27. 33-24. 09 1227 HBV DNA po) KAFLSKQYM-24. 29-28. 25-26. 35 1071 HBV DNA pol GVGLSPFLL-31. 69-24. 95-22. 08 677 HBV DNA pol PLDKGIKPY-21. 37-33. 95-23. 37 1361 HBV DNA pol PTTGRTSLY-22. 25-30. 66-25. 61 992 HBV DNA pol LHPAAMPHL-27. 95-27. 8-22. 62 989 HBV DNA pol HLPLHPAAM-24. 94-26. 24-27. 12 952 HBV DNA pol NTRVSWPKF-20. 26-33. 31-24. 72 1122 HBV DNA pol AVTNFLLSL-30. 57-23. 97-23. 52 1143 HBV DNA pol GYSLNFMGY-23. 5-28. 02-26. 47 1045 HBV DNA pol LLYKTYGWK-27. 36-26. 07-24. 51 1118 HBV DNA pol SLYAAVTNF-25. 7-19. 92-32. 12 1214 HBV DNA pol IQAKQAFTF-18. 79-30. 43-28. 45 1234 HBV DNA pol YMTLYPVAR-25. 62-26. 94-25. 04 627 HBV DNA pol WQTPSFPDI-25. 24-31. 36-20. 68 665 HBV DNA pol PARFYPNVT-25. 6-28. 82-22. 72 687 HBV DNA pol PEHVVNHYF-16. 02-29. 27-31. 74 1239 HBV DNA pol PVARQRPGL-24. 1-29. 61-23. 29 1268 HBV DNA pol MRGTFVSPL-27. 77-24. 69-24. 47 1230 HBV DNA pol LSKQYMTLY-26. 69-25 : 69-24. 54 1270 HBV DNA pol GTFVSPLPI-30. 07-25. 57-21. 16 1354 HBV DNA pol LLRLPYRPT-29. 25-26. 81-20. 47 749 HBV DNA pol SFCPQSPGI-21. 63-26. 86-27. 91 1048 HBV DNA pol KTYGWKLHL-25. 82-28. 58-21. 99 999 HBV DNA pol HLLVGSSGL-27. 38-23. 94-25. 06 985 HBV DNA pol AAFYHLPLH-24. 88-24. 65-26. 78 954 HBV DNA pol RVSWPKFAV-25. 98-26. 33-23. 73 696 HBV DNA pol QTRHYLHTL-25. 22-27. 77-22. 95 1151 HBV DNA pol YVIGSWGTW-27. 27-26. 2-22. 41 1385 HBV DNA pol FASPLHVAW-26. 67-24. 45-24. 75 991 HBV DNA pol PLHPAAMPH-24. 97-26. 72-24. 08 890 HBV DNA pol YCLSHIVNL-24. 81-25. 99-24. 84 1199 HBV DNA poi FTQCGYPAL-23. 33-26. 7-25. 52 1040 HBV DNA pol YVSLMLLYK-28. 5-24. 9-22. 09 1231 HBV DNA pol SKQYMTLYP-26. 53-28. 21-20. 73 1232 HBV DNA pol KQYMTLYPV-27. 66-24. 94-22. 55 601 HBV DNA pol VSIPWTHKV-26. 22-27. 71-20. 75 988 HBV DNA pol YHLPLHPAA-23. 71-26. 62-24. 26 1164 HBV DNA IVQNFKLCF-23. 07-22. 98-28 1377 HBV DNA pol SHLPDRVHF-19. 97-35. 9-18. 17 1260 HBV DNA pol GLAIGHQRM-23. 43-28. 43-22. 12 891 HBV DNA pol CLSHIVNLI-28. 52-22. 75-22. 67 664 HBV DNA pol MPARFYPNV-22. 26-23. 54-28. 03 799 HBV DNA pol RIHPSPWGT-24. 89-28. 19-20. 47 955 HBV DNA pol VSWPKFAVP-27. 95-24. 47-21 1349 HBV DNA pol GLYRPLLRL-28. 25-20. 97-24. 02 1209 HBV DNA pol PLYACIQAK-24. 92-24. 19-24. 06 1210 HBV DNA pol LYACIQAKQ-21. 49-26. 51-24. 98 1271 HBV DNA pol TFVSPLPIH-19. 85-27. 89-25. 18 755 HBV DNA pol PGILPRSSV-28. 38-25. 72-18. 68 1350 HBV DNA pol LYRPLLRLP-27. 55-24. 02-21. 17 681 HBV DNA pol GIKPYYPEH-24. 92-27. 91-19. 87 757 HBV DNA pol ILPRSSVGP-27. 72-22. 74-22. 13 678 HBV DNA pol LDKGIKPYY-20. 02-30. 59-21. 88 802 HBV DNA pol PSPWGTVGV-24. 46-26. 14-21. 86 1198 HBV DNA pol PFTQCGYPA-18. 55-27. 62-26. 06 1050 HBV DNA pol YGWKLHLYS-23. 02-27. 72-21. 44 1092 HBV DNA pol RAFPHCLAF-19. 4-29. 04-23. 68 1138 HBV DNA pol KTKRWGYSL-20. 96-29. 51-21. 33 1310 HBV DNA pol YTSFPWLLG-25. 22-23. 9-22. 53 689 HBV DNA pol HVVNHYFQT-20. 92-27. 6-22. 91 646 HBV DNA pol KQFVGPLTV-25. 78-25. 43-20. 12 1061 HBV DNA pol IILGFRKIP-25. 15-27. 89-18. 19 1146 HBV DNA pol LNFMGYVIG-24. 54-25. 63-20. 76 1073 HBV DNA pol GLSPFLLAQ-30. 54-19, 7-20. 41 1098 HBV DNA pol LAFSYMDDV-23. 94-22-24. 5 1235 HBV DNA pol MTLYPVARQ-24. 12-23. 35-22. 28 1224 HBV DNA pol PTYKAFLSK-22. 48-26-21. 16 1382 HBV DNA pol RVHFASPLH-19. 75-33. 04-16. 81 1052 HBV DNA pol WKLHLYSHP-21. 13-30. 56-17. 8 871 HBV DNA pol GSVLSCWWL-24v57-29. 14-15. 62 644 HBV DNA pol RCKQFVGPL-19. 54-30. 25-19. 47 1124 HBV DNA pol TNFLLSLGI-29. 16-23. 33-16. 76 674 HBV DNA pol KYLPLDKGI-15. 03-26. 56-27. 65 1173 HBV DNA pol RKLPVNRPI-17. 87-29. 65-21. 6 1343 HBV DNA pol PSRGRLGLY-20. 24-27. 23-21. 54 1064 HBV DNA pol GFRKIPMGV-21. 35-23. 94-23. 69 669 HBV DNA pol YPNVTKYLP-22. 61-22. 27-24. 09 983 HBV DNA pol VSAAFYHLP-23. 91-27. 23-17. 75 1047 HBV DNA pol YKTYGWKLH-16. 61-29. 44-22. 83 1317 HBV DNA pol LGCAANWIL-23. 13-23. 58-22. 11 1237 HBV DNA pol LYPVARQRP-17. 63-29. 51-21. 56 1072 HBV DNA pol VGLSPFLLA-29. 24-22. 26-17. 13 1152 HBV DNA pol VIGSWGTWP-24. 49-23. 92-20. 22 800 HBV DNA pol IHPSPWGTV-22. 75-23. 74-22. 06 679 HBV DNA pol DKGIKPYYP-22. 98-25. 82-19. 4 607 HBV DNA pol HKVGNFTGL-20. 07-26. 15-21. 67 1188 HBV DNA pol RIVGLLGFA-22. 12-26. 42-19. 16 1267 HBV DNA pol RMRGTFVSP-23. 52-25. 54-18. 43 1035 HBV DNA pol CSRNLYVSL-23. 75-27. 36-16. 37 750 HBV DNA pol FCPQSPGIL-20. 84-26. 9-19. 56 675 HBV DNA pol YLPLDKGIK-19. 12-23. 12-25. 01 878 HBV DNA pol WLQFRNSKP-21. 78-26. 89-18. 46 1156 HBV DNA pol WGTWPQDHI-18. 56-25. 79-22. 37 838 HBV DNA pol AYSPISTSK-16. 81-24. 7-25. 17 1142 HBV DNA pol WGYSLNFMG-19. 78-26. 52-20. 25 812 HBV DNA pol PSGSGHTHI-18. 58-28. 48-19. 2 1108 HBV DNA pol LGAKSVQHL-21. 19-23. 99-21. 04 1383 HBV DNA pol VHFASPLHV-24. 78-21. 75-19. 68 737 HBV DNA pol LVFQTSKRH-21. 78-26. 28-18. 15 1115 HBV DNA pol HLESLYAAV-25. 94-20. 28-19. 95 1157 HBV DNA pol GTWPQDHIV-25. 3-22. 83-18. 04 1082 HBV DNA pol FTSAICSVV-23. 77-21. 63-20. 65 1149 HBV DNA pol MGYVIGSWG-24. 18-24. 15-17. 69 630 HBV DNA pol PSFPDIHLQ-20. 79-25. 15-19. 98 705 HBV DNA pol WKAGILYKR-20. 61-28. 31-16. 94 804 HBV DNA pol PWGTVGVEP-24. 35-23. 43-17. 96 1200 HBV DNA pol TQCGYPALM-22. 58-23. 34-19. 79 996 HBV DNA pol AMPHLLVGS-24. 12-21. 34-19. 9 1236 HBV DNA pol TLYPVARQR-24. 93-21. 13-19. 17 709 HBV DNA pol ILYKRESTH-21. 56-23. 64-19. 88 1112 HBV DNA pol SVQHLESLY-23. 58-25. 39-16. 02 1348 HBV DNA pol LGLYRPLLR-27. 04-24. 73-13. 11 1093 HBV DNA pol AFPHCLAFS-20. 3-23. 38-21. 05 625 HBV DNA pol PEWQTPSFP-21. 46-21. 96-21. 3 1185 HBV DNA pol VCQRIVGLL-22. 49-24. 6-17. 5 1269 HBV DNA pol RGTFVSPLP-25. 93-22. 23-16. 35 763 HBV DNA pol VGPCIQSQL-21. 74-22. 81-19. 2 1290 HBV DNA pol RSRSGANLI-22. 72-25. 2-15. 64 1311 HBV DNA pol TSFPWLLGC-23. 87-22. 89-16. 78 1256 HBV DNA pol PTGWGLAIG-22. 12-23. 28-18. 04 1255 HBV DNA pol TPTGWGLAI-26. 36-18. 49-18. 32 722 HBV DNA pol CGSPYSWEQ-20. 25-22. 89-19. 8 995 HBV DNA pol AAMPHLLVG-23. 77-21. 49-17. 23 914 HBV DNA pol RTPRTPARV-23. 47-23. 41-15. 16 836 HBV DNA pol TAAYSPIST-23. 79-22. 27-15. 89 1013 HBV DNA pol RLSSNSRII-22. 84-23. 46-15. 58 1345 HBV DNA pol RGRLGLYRP-16. 96-29. 58-15. 31 1283 HBV DNA pol LLAACFARS-24. 83-19-17. 82 797 HBV DNA pol RARIHPSPW-20. 27-29. 36-11. 95 1128 HBV DNA pol LSLGIHLNP-23. 99-21. 13-16. 38 1063 HBV DNA pol LGFRKIPMG-20. 9-22. 28-18. 23 1019 HBV DNA pol RIINHQHGT-15. 34-26. 54-19. 15 1312 HBV DNA pol SFPWLLGCA-20. 55-20. 07-20. 31 554 HBV DNA pol MPLSYQHFR-18. 75-15. 71-26. 44 1106 HBV DNA pol VVLGAKSVQ-21. 15-21. 33-18. 34 1371 HBV DNA pol DSPSVPSHL-21. 54-23. 01-16. 21 849 HBV DNA pol SSSGHAVEL-23. 52-20. 81-16. 3 856 HBV DNA pol ELHHFPPNS-18. 05-26. 1-16. 47 866 HBV DNA pol RSQSQGSVL-19. 3-25. 04-16. 1 933 HBV DNA pol PHNTTESRL-15. 76-26. 32-18. 33 835 HBV DNA pol RTAAYSPIS-22. 95-21. 91-15. 51 854 HBV DNA pol AVELHHFPP-21. 44-21. 88-17. 02 998 HBV DNA pol PHLLVGSSG-18. 03-25. 11-17. 09 683 HBV DNA pol KPYYPEHVV-20. 97-17. 03-22. 1 876 HBV DNA pol CWWLQFRNS-16. 27-25. 43-18. 38 648 HBV DNA pol FVGPLTVNE-25. 67-18. 35-15. 92 925 HBV DNA pol GVFLVDKNP-22. 12-21. 3-16. 46 1297 HBV DNA pol LIGTDNSVV-24. 11-18. 53-17. 23 619 HBV DNA pol TVPVFNPEW-23. 32-18. 39-18. 14 1315 HBV DNA pol WLLGCAANW-20. 87-20. 01-18. 97 1346 HBV DNA pol GRLGLYRPL-24. 02-22. 4-13. 42 1355 HBV DNA pol LRLPYRPTT-25. 98-23. 65-10. 11 617 HBV DNA pol SSTVPVFNP-20. 9-21. 12-17. 62 1169 HBV DNA pol KLCFRKLPV-22. 88-20. 24-16. 43 1000 HBV DNA pol LLVGSSGLS-24. 58-17. 82-16. 95 1116 HBV DNA pol LESLYAAVT-25. 54-19. 02-14. 45 981 HBV DNA pol LDVSAAFYH-21. 57-19. 63-17. 48 566 HBV DNA pol LLDDEAGPL-22. 75-17. 55-18. 31 1107 HBV DNA pol VLGAKSVQH-20. 35-21. 03-17. 23 1168 HBV DNA pol FKLCFRKLP-17. 21-27. 11-14. 04 1298 HBV DNA pol IGTDNSVVL-22. 04-17. 24-19. 01 828 HBV DNA pol CLHQSAVRT-20. 8-21. 57-15. 76 1254 HBV DNA pol ATPTGWGLA-22. 43-19. 87-15. 71 1246 HBV DNA pol GLCQVFADA-24. 13-19. 8-14. 04 570 HBV DNA pol EAGPLEEEL-20. 91-19. 81-16. 92 1273 HBV DNA pol VSPLPIHTA-23. 06-19. 29-15. 21 1121 HBV DNA pol AAVTNFLLS-24. 66-18. 44-14. 28 1257 HBV DNA pol TGWGLAIGH-23. 07-19. 57-14. 41 629 HBV DNA pol TPSFPDIHL-24. 48-11. 98-19. 99 833 HBV DNA pol AVRTAAYSP-24. 64-18. 29-13. 33 888 HBV DNA pol SEYCLSHIV-21-32-20. 31-14. 52 1184 HBV DNA pol KVCQRIVGL-22. 2-18. 85-15. 01 872 HBV DNA pol SVLSCWWLQ-22. 08-19. 34-14. 19 837 HBV DNA pol AAYSPISTS-21. 36-16. 87-17. 23 618 HBV DNA pol STVPVFNPE-24. 35-13. 84-17. 14 1362 HBV DNA pol TTGRTSLYA-22. 55-20. 44-12. 3 752 HBV DNA pol PQSPGILPR-22. 72-19. 23-12. 94 609 HBV DNA pol VGNFTGLYS-20. 93-18. 51-15. 33 680 HBV DNA pol KGIKPYYPE-21. 58-16. 21-16. 85 1097 HBV DNA pol CLAFSYMDD-22. 77-13. 39-18. 4 935 HBV DNA pol NTTESRLVV-20. 76-21. 57-12. 06 718 HBV DNA pol SASFCGSPY-20. 94-12. 7-20. 45 792 HBV DNA pol GSGSIRARI-20. 83-20. 13-12. 79 645 HBV DNA pol CKQFVGPLT-21. 3-19. 31-13. 01 1123 HBV DNA pol VTNFLLSLG-21. 55-16. 43-14. 99 721 HBV DNA pol FCGSPYSWE-20. 29-17. 47-14. 46 1003 HBV DNA pol GSSGLSRYV-22. 93-18. 55-10. 68 658 HBV DNA pol RRLKLIMPA-22. 27-24. 91-4. 76 671 HBV DNA pol NVTKYLPLD-20. 82-14. 66-16. 34 972 HBV DNA pol LSSNLSWLS-20. 58-14. 42-16. 72 821 HBV DNA pol CASSSSSCL-20. 54-17. 48-13. 61 898 HBV DNA pol LIEDWGPCA-21. 37-16. 25-13. 47 756 HBV DNA pol GILPRSSVG-21. 86-16. 66-12. 35 1001 HBV DNA pol LVGSSGLSR-20. 42-18. 31-11. 79 892 HBV DNA pol LSHIVNLIE-20. 28-18. 39-11. 01 1352 HBV DNA pol RPLLRLPYR-20. 61-19. 62-8. 21 912 HBV DNA pol RIRTPRTPA-20. 26-21. 82-4. 24 1068 HBV DNA pol IPMGVGLSP-20. 78 X-17. 39 EXAMPLE 6 : In vitro testing of peptides for binding to HLA A peptide binding to HLA is tested using the procedure described by Melief in S. H. van der Burg et al. 1995. Human Immunology. 44,189-198. The strategy of the protocol is hereby described in short. The peptide-binding assay employs native cell-bound MHC class I molecules on HLA-homozygous B cell line. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment. They are replaced by fluorescein (FL)-labeled reference peptides that bind to HLA class I molecules. The cells are incubated with FL-labeled reference peptides together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACScan analysis.

Some measurements are listed herein by way of illustration TABLE 10A Sequence Cso (nM) FLPSDFFPS 0. 22 STLPETTVV 0. 75 ATVELLSFL 0.94 AILCWGELM 0. 99 ELMTLATWV 1. 07 LEYLVSFGV 4. 49 WGELMTLAT Non-binder TABLE 10B : Binding studies of selected peptides to MHC class I HLA-A2 and HLA-A24 types: ICso (pM) Source SEQ ID NO Peptide HLA-A2 HLA-A24 HBV core 23 FFPSVRDLL 71. 4825 0. 0150 HBV core 95 LKIRQLWWF 18. 8511 0. 7800 HBV DNA pol 1222 FSPTYKAFL 16. 1506 11. 3900 HBV DNA pol 660 LKLIMPARF 30. 6375 6. 2000 HBV DNA pol 1119 LYAAVTNFL 39. 0800 0. 0280 HBV DNA pol 1046 LYKTYGWKL 88. 7750 0. 0590 HBV DNA pol 615 LYSSTVPVF 143. 9750 0. 0110 HBV DNA pol 1329 SFVYVPSAL 38. 3575 0. 3600 HBV DNA pol 1042 SLMLLYKTY 19. 5867 45. 0000 HBV DNA pol 873 VLSCWWLQF 4. 7750 0. 3200 HBV DNA pol 1120 YAAVTNFLL 0. 5500 0. 1350 HBV DNA pol 1144 YSLNFMGYV 0. 8925 9. 9000 HBV surface 511 LVPFVQWFV 0. 5133 0. 8500 HBV surface 540 LYNILSPFM 28. 3175 0. 2500 HBV surface 524 TVWLSVIWM 15. 9550 2. 4200 HBV surface 526 WLSVIWMMW 35. 9400 1. 2800 HBV surface 531 WMMWYWGPS 7. 7000 3. 2400 EXAMPLE 7 : In vitro testing of peptides for CTL activity CTL response is measured using the 51 Cr release response methodology as described in S. H. van der Burg et al. (1995), AIDS 9,121. SEQ ID 1 MDIDPYKEF SEQ ID 41 ALKSPEHCS SEQ ID 81 SRDLVVNYV SEQ ID 2 DIDPYKEFG SEQ ID 42 LKSPEHCSP SEQ ID 82 RDLVVNYVN SEQ ID 3 IDPYKEFGA SEQ ID 43 KSPEHCSPH SEQ ID 83 DLVVNYVNT SEQ ID 4 DPYKEFGAT SEQ ID 44 SPEHCSPHH SEQ ID 84 LVVNYVNTN SEQ ID 5 PYKEFGATV SEQ ID 45 PEHCSPHHT SEQ ID 5 VVNYVNTNM SEQ ID 6 YKEFGATVE SEQ ID 46 EHCSPHHTA SEQ ID 86 VNYVNTNMG SEQ ID 7 KEFGATVEL SEQ ID 47 HCSPHHTAL SEQ ID 87 NYVNTNMGL SEQ ID 8 EFGATVELL SEQ ID 48 CSPHHTALR SEQ ID 88 YVNTNMGLK SEQ ID 9 FGATVELLS SEQ ID 49 SPHHTALRQ SEQ ID 89 VNTNMGLKI SEQ ID 10 GATVELLSF SEQ ID 50 PHHTALRQA SEQ ID 90 NTNMGLKIR SEQ ID 11 ATVELLSFL SEQ ID 51 HHTALRQAI SEQ ID 91 TNMGLKIRQ SEQ ID 12 TVELLSFLP SEQ ID 52 HTALRQAIL SEQ ID 92 NMGLKIRQL SEQ ID 13 VELLSFLPS SEQ ID 53 TALRQAILC SEQ ID 93 MGLKIRQLW SEQ ID 14 ELLSFLPSD SEQ ID 54 ALRQAILCW SEQ ID 94 GLKIRQLWW SEQ ID 15 LLSFLPSDF SEQ ID 55 LRQAILCWG SEQ ID 95 LKIRQLWWF SEQ ID 16 LSFLPSDFF SEQ ID 56 RQAILCWGE SEQ ID 96 KIRQLWWFH SEQ ID 17 SFLPSDFFP SEQ ID 57 QAILCWGEL SEQ ID 97 IRQLWWFHI SEQ ID 18 FLPSDFFPS SEQ ID 58 AILCWGELM SEQ ID 98 RQLWWFHIS SEQ ID 19 LPSDFFPSV SEQ ID 59 ILCWGELMT SEQ ID 99 QLWWFHISC SEQ ID 20 PSDFFPSVR SEQ ID 60 LCWGELMTL SEQ ID 100 LWWFHISCL SEQ ID 21 SDFFPSVRD SEQ ID 61 CWGELMTLA SEQ ID 101 WWFHISCLT SEQ ID 22 DFFPSVRDL SEQ ID 62 WGELMTLAT SEQ ID 102 WFHISCLTF SEQ ID 23 FFPSVRDLL SEQ ID 63 GELMTLATW SEQ ID 103 FHISCLTFG SEQ ID 24 FPSVRDLLD SEQ ID 64 ELMTLATWV SEQ ID 104 HISCLTFGR SEQ ID 25 PSVRDLLDT SEQ ID 65 LMTLATWVG SEQID105 ISCLTFGRE SEQ ID 26 SVRDLLDTV SEQ ID 66 MTLATWVGN SEQ ID 106 SCLTFGRET SEQ ID 27 VRDLLDTVS SEQ ID 67 TLATWVGNN SEQ ID 107 CLTFGRETV SEQ ID 28 RDLLDTVSA SEQ ID 68 LATWVGNNL SEQ ID 108 LTFGRETVL SEQ ID 29 DLLDTVSAL SEQ ID 69 ATWVGNNLE SEQ ID 109 TFGRETVLE SEQ ID 30 LLDTVSALY SEQ ID 70 TWVGNNLED SEQ ID 110 FGRETVLEY SEQ ID 31 LDTVSALYR SEQ ID 71 WVGNNLEDP SEQ ID 111 GRETVLEYL SEQ ID 32 DTVSALYRE SEQ in 72 VGNNLEDPA SEQ ID 112 RETVLEYLV SEQ ID 33 TVSALYREA SEQ ID 73 GNNLEDPAS SEQ ID 113 ETVLEYLVS SEQ ID 34 VSALYREAL SEQ ID 74 NNLEDPASR SEQ ID 114 TVLEYLVSF SEQ ID 35 SALYREALK SEQ ID 75 NLEDPASRD SEQ ID 115 VLEYLVSFG SEQ ID 36 ALYREALKS SEQ ID 76 LEDPASRDL SEQ ID 116 LEYLVSFGV SEQ ID 37 LYREALKSP SEQ ID 77 EDPASRDLV SEQ ID 117 EYLVSFGVW SEQ ID 38 YREALKSPE SEQ ID 78 DPASRDLVV SEQ ID 118 YLVSFGVWI SEQ ID 39 REALKSPEH SEQ ID 79 PASRDLVVN SEQ ID 119 LVSFGVWIR SEQ ID 40 EALKSPEHC SEQ ID 80 ASRDLVVNY SEQ ID 120 VSFGVWIRT SEQ ID 121 SFGVWIRTP SEQ ID 161 PSPRRRRSQ SEQ ID 201 NPDWDLNPH SEQ ID 122 FGVWIRTPP SEQ ID 162 SPRRRRSQS SEQ ID 202 PDWDLNPHK SEQ ID 123 GVWIRTPPA SEQ ID 163 PRRRRSQSP SEQ ID 203 DWDLNPHKD SEQ ID 124 VWIRTPPAY SEQ ID 164 RRRRSQSPR SEQ ID 204 WDLNPHKDN SEQ ID 125 WIRTPPAYR SEQ ID 165 RRRSQSPRR SEQ ID 205 DLNPHKDNW SEQ ID 126 IRTPPAYRP SEQ ID 166 RRSQSPRRR SEQ ID 206 LNPHKDNWP SEQ ID 127 RTPPAYRPP SEQ ID 167 RSQSPRRRR SEQ ID 207 NPHKDNWPD SEQ ID 128 TPPAYRPPN SEQ ID 168 SQSPRRRRS SEQ ID 208 PHKDNWPDA SEQ ID 129 PPAYRPPNA SEQ ID 169 QSPRRRRSQ SEQ ID 209 HKDNWPDAH SEQ ID 130 PAYRPPNAP SEQ ID 170 SPRRRRSQS SEQ ID 210 KDNWPDAHK SEQ ID 131 AYRPPNAPI SEQ ID 171 PRRRRSQSR SEQ ID 211 DNWPDAHKV SEQ ID 132 YRPPNAPIL SEQ ID 172 RRRRSQSRE SEQ ID 212 NWPDAHKVG SEQ ID 133 RPPNAPILS SEQ ID 173 MGTNLSVPN SEQ ID 213 WPDAHKVGV SEQ ID 134 PPNAPILST SEQ ID 174 GTNLSVPNP SEQ ID 214 PDAHKVGVG SEQ ID 135 PNAPILSTL SEQ ID 175 TNLSVPNPL SEQ ID 215 DAHKVGVGA SEQ ID 136 NAPILSTLP SEQ ID 176 NLSVPNPLG SEQ ID 216 AHKVGVGAF SEQ ID 137 APILSTLPE SEQ ID 177 LSVPNPLGF SEQ ID 217 HKVGVGAFG SEQ ID 138 PILSTLPET SEQ ID 178 SVPNPLGFF SEQ ID 218 KVGVGAFGP SEQ ID 139 ILSTLPETT SEQ ID 179 VPNPLGFFP SEQ ID 219 VGVGAFGPG SEQ ID 140 LSTLPETTV SEQ ID 180 PNPLGFFPD SEQ ID 220 GVGAFGPGF SEQ ID 141 STLPETTVV SEQ ID 181 NPLGFFPDH SEQ ID 221 VGAFGPGFT SEQ ID 142 TLPETTVVR SEQ ID 182 PLGFFPDHQ SEQ ID 222 GAFGPGFTP SEQ ID 143 LPETTVVRR SEQ ID 183 LGFFPDHQL SEQ ID 223 AFGPGFTPP SEQ ID 144 PETTVVRRR SEQ ID 184 GFFPDHQLD SEQ ID 224 FGPGFTPPH SEQ ID 145 ETTVVRRRG SEQ ID 185 FFPDHQLDP SEQ ID 225 GPGFTPPHG SEQ ID 146 TTVVRRRGR SEQ ID 186 FPDHQLDPA SEQ ID 226 PGFTPPHGG SEQ ID 147 TWRRRGRS SEQ ID 187 PDHQLDPAF SEQ ID 227 GFTPPHGGL SEQ ID 148 VVRRRGRSP SEQ ID 188 DHQLDPAFK SEQ ID 228 FTPPHGGLL SEQ ID 149 VRRRGRSPR SEQ ID 189 HQLDPAFKA SEQ ID 229 TPPHGGLLG SEQ ID 150 RRRGRSPRR SEQ ID 190 QLDPAFKAN SEQ ID 230 PPHGGLLGW SEQ ID 151 RRGRSPRRR SEQ ID 191 LDPAFKANS SEQ ID 231 PHGGLLGWS SEQ ID 152 RGRSPRRRT SEQ ID 192 DPAFKANSE SEQ ID 232 HGGLLGWSP SEQ ID 153 GRSPRRRTP SEQ ID 193 PAFKANSEN SEQ ID 233 GGLLGWSPQ SEQ ID 154 RSPRRRTPS SEQ ID 194 AFKANSENP SEQ ID 234 GLLGWSPQA SEQ ID 155 SPRRRTPSP SEQ ID 195 FKANSENPD SEQ ID 235 LLGWSPQAQ SEQ ID 156 PRRRTPSPR SEQ ID 196 KANSENPDW SEQ ID 236 LGWSPQAQG SEQ ID 157 RRRTPSPRR SEQ ID 197 ANSENPDWD SEQ ID 237 GWSPQAQGI SEQ ID 158 RRTPSPRRR SEQ ID 198 NSENPDWDL SEQ ID 238 WSPQAQGIL SEQ ID 159 RTPSPRRRR SEQ ID 199 SENPDWDLN SEQ ID 239 SPQAQGILT SEQ ID 160 TPSPRRRRS SEQ ID 200 ENPDWDLNP SEQ ID 240 PQAQGILTS SEQ ID 241 QAQGILTSV SEQ ID 281 MQWNSTTFH SEQ D 321 AISSILSKT SEQ ID 242 AQGILTSVP SEQ ID 282 QWNSTTFHQ SEQ ID 322 ISSILSKTG SEQ ID 243 QGILTSVPA SEQ ID 283 WNSTTFHQT SEQ ID 323 SSILSKTGD SEQ ID 244 GILTSVPAA SEQ ID 284 NSTTFHQTL SEQ ID 324 SILSKTGDP SEQ ID 245 ILTSVPAAP SEQ ID 285 STTFHQTLQ SEQ ID 325 ILSKTGDPV SEQ ID 246 LTSVPAAPP SEQ ID 286 TTFHQTLQD SEQ ID 326 LSKTGDPVP SEQ ID 247 TSVPAAPPP SEQ ID 287 TFHQTLQDP SEQ ID 327 SKTGDPVPN SEQ ID 248 SVPAAPPPA SEQ ID 288 FHQTLQDPR SEQ ID 328 KTGDPVPNM SEQ ID 249 VPAAPPPAS SEQ ID 289 HQTLQDPRV SEQ ID 329 TGDPVPNME SEQ ID 250 PAAPPPAST SEQ ID 290 QTLQDPRVR SEQ ID 330 GDPVPNMEN SEQ ID 251 AAPPPASTN SEQ ID 291 TLQDPRVRA SEQ ID 331 DPVPNMENI SEQ ID 252 APPPASTNR SEQ ID 292 LQDPRVRAL SEQ ID 332 PVPNMENIA SEQ ID 253 PPPASTNRQ SEQ ID 293 QDPRVRALY SEQ ID 333 VPNMENIAS SEQ ID 254 PPASTNRQS SEQ ID 294 DPRVRALYF SEQ ID 334 PNMENIASG SEQ ID 255 PASTNRQSG SEQ ID 295 PRVRALYFP SEQ ID 335 NMENIASGL SEQ ID 256 ASTNRQSGR SEQ ID 296 RVRALYFPA SEQ ID 336 MENIASGLL SEQ ID 257 STNRQSGRQ SEQ ID 297 VRALYFPAG SEQ ID 337 ENIASGLLG SEQ ID 258 TNRQSGRQP SEQ ID 298 RALYFPAGG SEQ ID 338 NIASGLLGP SEQ ID 259 NRQSGRQPT SEQ ID 299 ALYFPAGGS SEQ ID 339 IASGLLGPL SEQ ID 260 RQSGRQPTP SEQ ID 300 LYFPAGGSS SEQ ID 340 ASGLLGPLL SEQ ID 261 QSGRQPTPL SEQ ID 301 YFPAGGSSS SEQ ID 341 SGLLGPLLV SEQ ID 262 SGRQPTPLS SEQ ID 302 FPAGGSSSG SEQ ID 342 GLLGPLLVL SEQ ID 263 GRQPTPLSP SEQ ID 303 PAGGSSSGT SEQ ID 343 LLGPLLVLQ SEQ ID 264 RQPTPLSPP SEQ ID 304 AGGSSSGTV SEQ ID 344 LGPLLVLQA SEQ ID 265 QPTPLSPPL SEQ ID 305 GGSSSGTVS SEQ ID 345 GPLLVLQAG SEQ ID 266 PTPLSPPLR SEQ ID 306 GSSSGTVSP SEQ ID 346 PLLVLQAGF SEQ ID 267 TPLSPPLRD SEQ ID 307 SSSGTVSPA SEQ ID 347 LLVLQAGFF SEQ ID 268 PLSPPLRDT SEQ ID 308 SSGTVSPAQ SEQ ID 348 LVLQAGFFL SEQ ID 269 LSPPLRDTH SEQ ID 309 SGTVSPAQN SEQ ID 349 VLQAGFFLL SEQ ID 270 SPPLRDTHP SEQ ID 310 GTVSPAQNT SEQ ID 350 LQAGFFLLT SEQ ID 271 PPLRDTHPQ SEQ ID 311 TVSPAQNTV SEQ ID 351 QAGFFLLTK SEQ ID 272 PLRDTHPQA SEQ ID 312 VSPAQNTVS SEQ ID 352 AGFFLLTKI SEQ ID 273 LRDTHPQAM SEQ ID 313 SPAQNTVSA SEQ ID 353 GFFLLTKIL SEQ ID 274 RDTHPQAMQ SEQ ID 314 PAQNTVSAI SEQ ID 354 FFLLTKILT SEQ ID 275 DTHPQAMQW SEQ ID 315 AQNTVSAIS SEQ ID 355 FLLTKILTI SEQ ID 276 THPQAMQWN SEQ ID 316 QNTVSAISS SEQ ID 356 LLTKILTIP SEQ ID 277 HPQAMQWNS SEQ ID 317 NTVSAISSI SEQ ID 357 LTKILTIPQ SEQ ID 278 PQAMQWNST SEQ ID 318 TVSAISSIL SEQ ID 358 TKILTIPQS SEQ ID 279 QAMQWNSTT SEQ ID 319 VSAISSILS SEQ ID 359 KILTIPQSL SEQ ID 280 AMQWNSTTF SEQ ID 320 SAISSILSK SEQ ID 360 ILTIPQSLD SEQ ID 361 LTIPQSLDS SEQ ID 401 PPICPGYRW SEQ ID 441 VCPLIPGSS SEQ ID 362 TIPQSLDSW SEQ ID 402 PICPGYRWM SEQ ID 442 CPLIPGSST SEQ ID 363 IPQSLDSWW SEQ ID 403 ICPGYRWMC SEQ ID 443 PLIPGSSTT SEQ ID 364 PQSLDSWWT SEQ ID 404 CPGYRWMCL SEQ ID 444 LIPGSSTTS SEQ ID 365 QSLDSWWTS SEQ ID 405 PGYRVgMCLR SEQ ID 445 IPGSSTTST SEQ ID 366 SLDSWWTSL SEQ ID 406 GYRWMCLRR SEQ ID 446 PGSSTTSTG SEQ ID 367 LDSWWTSLN SEQ ID 407 YRWMCLRRF SEQ ID 447 GSSTTSTGP SEQ ID 368 DSWWTSLNF SEQ ID 408 RWMCLRRFI SEQ ID 448 SSTTSTGPC SEQ ID 369 SWWTSLNFL SEQ ID 409 WMCLRRFII SEQ ID 449 STTSTGPCK SEQ ID 370 WWTSLNFLG SEQ ID 410 MCLRRFIIF SEQ ID 450 TTSTGPCKT SEQ ID 371 WTSLNFLGG SEQ ID 411 CLRRFIIFL SEQ ID 451 TSTGPCKTC SEQ ID 372 TSLNFLGGT SEQ ID 412 LRRFIIFLC SEQ ID 452 STGPCKTCT SEQ ID 373 SLNFLGGTP SEQ ID 413 RRFIIFLCI SEQ ID 453 TGPCKTCTT SEQ ID 374 LNFLGGTPV SEQ ID 414 RFIIFLCIL SEQ ID 454 GPCKTCTTP SEQ ID 375 NFLGGTPVC SEQ ID 415 FIIFLCILL SEQ ID 455 PCKTCTTPA SEQ ID 376 FLGGTPVCL SEQ ID 416 IIFLCILLL SEQ ID 456 CKTCTTPAQ SEQ ID 377 LGGTPVCLG SEQ ID 417 IFLCILLLC SEQ ID 457 KTCTTPAQG SEQ ID 378 GGTPVCLGQ SEQ ID 418 FLCILLLCL SEQ ID 458 TCTTPAQGT SEQ ID 379 GTPVCLGQN SEQ ID 419 LCILLLCLI SEQ ID 459 CTTPAQGTS SEQ ID 380 TPVCLGQNS SEQ ID 420 CILLLCLIF SEQ ID 460 TTPAQGTSM SEQ ID 381 PVCLGQNSQ SEQ ID 421 ILLLCLIFL SEQ ID 461 TPAQGTSMF SEQ ID 382 VCLGQNSQS SEQ ID 422 LLLCLIFLL SEQ ID 462 PAQGTSMFP SEQ ID 383 CLGQNSQSQ SEQ ID 423 LLCLIFLLV SEQ ID 463 AQGTSMFPS SEQ ID 384 LGQNSQSQI SEQ ID 424 LCLIFLLVL SEQ ID 464 QGTSMFPSC SEQ ID 385 GQNSQSQIS SEQ ID 425 CLIFLLVLL SEQ ID 465 GTSMFPSCC SEQ ID 386 QNSQSQISS SEQ ID 426 LIFLLVLLD SEQ ID 466 TSMFPSCCC SEQ ID 387 NSQSQISSH SEQ ID 427 IFLLVLLDY SEQ ID 467 SMFPSCCCT SEQ ID 388 SQSQISSHS SEQ ID 428 FLLVLLDYQ SEQ ID 468 MFPSCCCTK SEQ ID 389 QSQISSHSP SEQ ID 429 LLVLLDYQG SEQ ID 469 FPSCCCTKP SEQ ID 390 SQISSHSPT SEQ ID 430 LVLLDYQGM SEQ ID 470 PSCCCTKPM SEQ ID 391 QISSHSPTC SEQ ID 431 VLLDYQGML SEQ ID 471 SCCCTKPMD SEQ ID 392 ISSHSPTCC SEQ ID 432 LLDYQGMLP SEQ ID 472 CCCTKPMDG SEQ ID 393 SSHSPTCCP SEQ ID 433 LDYQGMLPV SEQ ID 473 CCTKPMDGN SEQ ID 394 SHSPTCCPP SEQ ID 434 DYQGMLPVC SEQ ID 474 CTKPMDGNC SEQ ID 395 HSPTCCPPI SEQ ID 435 YQGMLPVCP SEQ ID 475 TKPMDGNCT SEQ ID 396 SPTCCPPIC SEQ ID 436 QGMLPVCPL SEQ ID 476 KPMDGNCTC SEQ ID 397 PTCCPPICP SEQ ID 437 GMLPVCPLI SEQ ID 477 PMDGNCTCI SEQ ID 398 TCCPPICPG SEQ ID 438 MLPVCPLIP SEQ ID 478 MDGNCTCIP SEQ ID 399 CCPPICPGY SEQ ID 439 LPVCPLIPG SEQ ID 479 DGNCTCIPI SEQ ID 400 CPPICPGYR SEQ ID 440 PVCPLIPGS SEQ ID 480 GNCTCIPIP SEQ ID 481 NCTCIPIPS SEQ ID 521 LSPTVWLSV SEQ ID 561 FRKLLLLDD SEQ ID 482 CTCIPIPSS SEQ ID 522 SPTVWLSVI SEQ ID 562 RKLLLLDDE SEQ ID 483 TCIPIPSSW SEQ ID 523 PTVWLSVIW SEQ ID 563 KLLLLDDEA SEQ ID 484 CIPIPSSWA SEQ ID 524 TVWLSVIWM SEQ ID 564 LLLLDDEAG SEQ ID 485 IPIPSSWAF SEQ ID 525 VWLSVIWMM SEQ ID 565 LLLDDEAGP SEQ ID 486 PIPSSWAFA SEQ ID 526 WLSVIWMMW SEQ ID 566 LLDDEAGPL SEQ ID 487 IPSSWAFAK SEQ ID 527 LSVIWMMWY SEQ ID 567 LDDEAGPLE SEQ ID 488 PSSWAFAKY SEQ ID 528 SVIWMMWYW SEQ ID 568 DDEAGPLEE SEQ ID 489 SSWAFAKYL SEQ ID 529 VIWMMWYWG SEQ ID 569 DEAGPLEEE SEQ ID 490 SWAFAKYLW SEQ ID 530 IWMMWYWGP SEQ ID 570 EAGPLEEEL SEQ ID 491 WAFAKYLWE SEQ ID 531 WMMWYWGPS SEQ ID 571 AGPLEEELP SEQ ID 492 AFAKYLWEW SEQ ID 532 MMWYWGPSL SEQ ID 572 GPLEEELPR SEQ ID 493 FAKYLWEWA SEQ ID 533 MWYWGPSLY SEQ ID 573 PLEEELPRL SEQ ID 494 AKYLWEWAS SEQ ID 534 WYWGPSLYN SEQ ID 574 LEEELPRLA SEQ ID 495 KYLWEWASV SEQ ID 535 YWGPSLYNI SEQ ID 575 EEELPRLAD SEQ ID 496 YLWEWASVR SEQ ID 536 WGPSLYSIL SEQ ID 576 EELPRLADE SEQ ID 497 LWEWASVRF SEQ ID 537 GPSLYNILS SEQ ID 577 ELPRLADEG SEQ ID 498 WEWASVRFS SEQ ID 538 PSLYNILSP SEQ ID 578 LPRLADEGL SEQ ID 499 EWASVRFSW SEQ ID 539 SLYNILSPF SEQ ID 579 PRLADEGLN SEQ ID 500 WASVRFSWL SEQ ID 540 LYNILSPFM SEQ ID 580 RLADEGLNH SEQ ID 501 ASVRFSWLS SEQ ID 541 YNILSPFMP SEQ ID 581 LADEGLNHR SEQ ID 502 SVRFSWLSL SEQ ID 542 NILSPFMPL SEQ ID 582 ADEGLNHRV SEQ ID 503 VRFSWLSLL SEQ ID 543 ILSPFMPLL SEQ ID 583 DEGLNHRVA SEQ ID 504 RFSWLSLLV SEQ ID 544 LSPFMPLLP SEQ ID 584 EGLNHRVAE SEQ ID 505 FSWLSLLVP SEQ ID 545 SPFMPLLPI SEQ ID 585 GLNHRVAED SEQ ID 506 SWLSLLVPF SEQ ID 546 PFMPLLPIF SEQ ID 586 LNHRVAEDL SEQ ID 507 WLSLLVPFV SEQ ID 547 FMPLLPIFF SEQ ID 587 NHRVAEDLN SEQ ID 508 LSLLVPFVQ SEQ ID 548 MPLLPIFFC SEQ ID 588 HRVAEDLNL SEQ ID 509 SLLVPFVQW SEQ ID 549 PLLPIFFCL SEQ ID 589 RVAEDLNLG SEQ ID 510 LLVPFVQWF SEQ ID 550 LLPIFFCLW SEQ ID 590 VAEDLNLGN SEQ ID 511 LVPFVQWFV SEQ ID 551 LPIFFCLWV SEQ ID 591 AEDLNLGNP SEQ ID 512 VPFVQWFVG SEQ ID 552 PIFFCLWVY SEQ ID 592 EDLNLGNPN SEQ ID 513 PFVQWFVGL SEQ ID 553 IFFCLWVYI SEQ ID 593 DLNLGNPNV SEQ ID 514 FVQWFVGLS SEQ ID 554 MPLSYQHFR SEQ ID 594 LNLGNPNVS SEQ ID 515 VQWFVGLSP SEQ ID 555 PLSYQHFRK SEQ ID 595 NLGNPNVSI SEQ ID 516 QWFVGLSPT SEQ ID 556 LSYQHFRKL SEQ ID 596 LGNPNVSIP SEQ ID 517 WFVGLSPTV SEQ ID 557 SYQHFRKLL SEQ ID 597 GNPNVSIPW SEQ ID 518 FVGLSPTVW SEQ ID 558 YQHFRKLLL SEQ ID 598 NPNVSIPWT SEQ ID 519 VGLSPTVWL SEQ ID 559 QHFRKLLLL SEQ ID 599 PNVSIPWTH SEQ ID 520 GLSPTVWLS SEQ ID 560 HFRKLLLLD SEQ ID 600 NVSIPWTHK SEQ ID 601 VSIPWTHKV SEQ ID 641 IVDRCKQFV SEQ ID 681 GIKPYYPEH SEQ ID 602 SIPWTHKVG SEQ ID 642 VDRCKQFVG SEQ ID 682 IKPYYPEHV SEQ ID-603 IPWTHKVGN SEQ ID-643 DRCKQFVGP SEQ ID 683 KPYYPEHVV SEQ ID 604 PWTHKVGNF SEQ ID 644 RCKQFVGPL SEQ ID 684 PYYPEHWN SEQ ID 605 WTHKVGNFT SEQ ID 645 CKQFVGPLT SEQ ID 685 YYPEHVVNH SEQ ID 606 THKVGNFTG SEQ ID 646 KQFVGPLTV SEQ ID 686 YPEHVVNHY SEQ ID 607 HKVGNFTGL SEQ ID 647 QFVGPLTVN SEQ ID 687 PEHVVNHYF SEQ ID 608 KVGNFTGLY SEQ ID 648 FVGPLTVNE SEQ ID 688 EHVVNHYFQ SEQ ID 609 VGNFTGLYS SEQ ID 649 VGPLTVNEN SEQ ID 689 HWNHYFQT SEQ ID 610 GNFTGLYSS SEQ ID 650 GPLTVNENR SEQ ID 690 WNHYFQTR SEQ ID 611 NFTGLYSST SEQ ID 651 PLTVNENRR SEQ ID 691 VNHYFQTRH SEQ ID 612 FTGLYSSTV SEQ ID 652 LTVNENRRL SEQ ID 692 NHYFQTRHY SEQ ID 613 TGLYSSTVP SEQ ID 653 TVNENRRLK SEQ ID 693 HYFQTRHYL SEQ ID 614 GLYSSTVPV SEQ ID 654 VNENRRLKL SEQ ID 694 YFQTRHYLH SEQ ID 615 LYSSTVPVF SEQ ID 655 NENRRLKLI SEQ ID 695 FQTRHYLHT SEQ ID 616 YSSTVPVFN SEQ ID 656 ENRRLKLIM SEQ ID 696 QTRHYLHTL SEQ ID 617 SSTVPVFNP SEQ ID 657 NRRLKLIMP SEQ ID 697 TRHYLHTLW SEQ ID 618 STVPVFNPE SEQ ID 658 RRLKLIMPA SEQ ID 698 RHYLHTLWK SEQ ID 619 TVPVFNPEW SEQ ID 659 RLKLIMPAR SEQ ID 699 HYLHTLWKA SEQ ID 620 VPVFNPEWQ SEQ ID 660 LKLIMPARF SEQ ID 700 YLHTLWKAG SEQ ID 621 PVFNPEWQT SEQ ID 661 KLIMPARFY SEQ ID 701 LHTLWKAGI SEQ ID 622 VFNPEWQTP SEQ ID 662 LIMPARFYP SEQ ID 702 HTLWKAGIL SEQ ID 623 FNPEWQTPS SEQ ID 663 IMPARFYPN SEQ ID 703 TLWKAGILY SEQ ID 624 NPEWQTPSF SEQ ID 664 MPARFYPNV SEQ ID 704 LWKAGILYK SEQ ID 625 PEWQTPSFP SEQ ID 665 PARFYPNVT SEQ ID 705 WKAGILYKR SEQ ID 626 EWQTPSFPD SEQ ID 666 ARFYPNVTK SEQ ID 706 KAGILYKRE SEQ ID 627 WQTPSFPDI SEQ ID 667 RFYPNVTKY SEQ ID 707 AGILYKRES SEQ ID 628 QTPSFPDIH SEQ ID 668 FYPNVTKYL SEQ ID 708 GILYKREST SEQ ID 629 TPSFPDIHL SEQ ID 669 YPNVTKYLP SEQ ID 709 ILYKRESTH SEQ ID 630 PSFPDIHLQ SEQ ID 670 PNVTKYLPL SEQ ID 710 LYKRESTHS SEQ ID 631 SFPDIHLQE SEQ ID 671 NVTKYLPLD SEQ ID 711 YKRESTHSA SEQ ID 632 FPDIHLQED SEQ ID 672 VTKYLPLDK SEQ ID 712 KRESTHSAS SEQ ID 633 PDIHLQEDI SEQ ID 673 TKYLPLDKG SEQ ID 713 RESTHSASF SEQ ID 634 DIHLQEDIV SEQ ID 674 KYLPLDKGI SEQ ID 714 ESTHSASFC SEQ ID 635 IHLQEDIVD SEQ ID 675 YLPLDKGIK SEQ ID 715 STHSASFCG SEQ ID 636 HLQEDIVDR SEQ ID 676 LPLDKGIKP SEQ ID 716 THSASFCGS SEQ ID 637 LQEDIVDRC SEQ ID 677 PLDKGIKPY SEQ ID 717 HSASFCGSP SEQ ID 638 QEDIVDRCK SEQ ID 678 LDKGIKPYY SEQ ID 718 SASFCGSPY SEQ ID 639 EDIVDRCKQ SEQ ID 679 DKGIKPYYP SEQ ID 719 ASFCGSPYS SEQ ID 640 DIVDRCKQF SEQ ID 680 KGIKPYYPE SEQ ID 720 SFCGSPYSW SEQ ID 721 FCGSPYSWE SEQ ID 761 SSVGPCIQS SEQ ID 801 HPSPWGTVG SEQ ID 722 CGSPYSWEQ SEQ ID 762 SVGPCIQSQ SEQ ID 802 PSPWGTVGV SEQ ID 723 GSPYSWEQD SEQ-ID 763 VGPCIQSQL SEQ ID 803 SPWGTVGVE SEQ ID 724 SPYSWEQDL SEQ ID 764 GPCIQSQLR SEQ ID 804 PWGTVGVEP SEQ ID 725 PYSWEQDLQ SEQ ID 765 PCIQSQLRK SEQ ID 805 WGTVGVEPS SEQ ID 726 YSWEQDLQH SEQ ID 766 CIQSQLRKS SEQ ID 806 GTVGVEPSG SEQ ID 727 SWEQDLQHG SEQ ID 767 IQSQLRKSR SEQ ID 807 TVGVEPSGS SEQ ID 728 WEQDLQHGR SEQ ID 768 QSQLRKSRL SEQ ID 808 VGVEPSGSG SEQ ID 729 EQDLQHGRL SEQ ID 769 SQLRKSRLG SEQ ID 809 GVEPSGSGH SEQ ID 730 QDLQHGRLV SEQ ID 770 QLRKSRLGP SEQ ID 810 VEPSGSGHT SEQ ID 731 DLQHGRLVF SEQ ID 771 LRKSRLGPQ SEQ ID 811 EPSGSGHTH SEQ ID 732 LQHGRLVFQ SEQ) ID 772 RKSRLGPQP SEQ ID 812 PSGSGHTHI SEQ ID 733 QHGRLVFQT SEQ ID 773 KSRLGPQPT SEQ ID 813 SGSGHTHIC SEQ ID 734 HGRLVFQTS SEQ ID 774 SRLGPQPTQ SEQ ID 814 GSGHTHICA SEQ ID 735 GRLVFQTSK SEQ ID 775 RLGPQPTQG SEQ ID 815 SGHTHICAS SEQ ID 736 RLVFQTSKR SEQ ID 776 LGPQPTQGQ SEQ ID 816 GHTHICASS SEQ ID 737 LVFQTSKRH SEQ ID 777 GPQPTQGQL SEQ ID 817 HTHICASSS SEQ ID 738 VFQTSKRHG SEQ ID 778 PQPTQGQLA SEQ ID 818 THICASSSS SEQ ID 739 FQTSKRHGD SEQ ID 779 QPTQGQLAG SEQ ID 819 HICASSSSS SEQ ID 740 QTSKRHGDK SEQ ID 780 PTQGQLAGR SEQ ID 820 ICASSSSSC SEQ ID 741 TSKRHGDKS SEQ ID 781 TQGQLAGRP SEQ ID 821 CASSSSSCL SEQ ID 742 SKRHGDKSF SEQ ID 782 QGQLAGRPQ SEQ ID 822 ASSSSSCLH SEQ ID 743 KRHGDKSFC SEQ ID 783 GQLAGRPQG SEQ ID 823 SSSSSCLHQ SEQ ID 744 RHGDKSFCP SEQ ID 784 QLAGRPQGG SEQ ID 824 SSSSCLHQS SEQ ID 745 HGDKSFCPQ SEQ ID 785 LAGRPQGGS SEQ ID 825 SSSCLHQSA SEQ ID 746 GDKSFCPQS SEQ ID 786 AGRPQGGSG SEQ ID 826 SSCLHQSAV SEQ ID 747 DKSFCPQSP SEQ ID 787 GRPQGGSGS SEQ ID 827 SCLHQSAVR SEQ ID 748 KSFCPQSPG SEQ ID 788 RPQGGSGSI SEQ ID 828 CLHQSAVRT SEQ ID 749 SFCPQSPGI SEQ ID 789 PQGGSGSIR SEQ ID 829 LHQSAVRTA SEQ ID 750 FCPQSPGIL SEQ ID 790 QGGSGSIRA SEQ ID 830 HQSAVRTAA SEQ ID 751 CPQSPGILP SEQ ID 791 GGSGSIRAR SEQ ID 831 QSAVRTAAY SEQ ID 752 PQSPGILPR SEQ ID 792 GSGSIRARI SEQ ID 832 SAVRTAAYS SEQ ID 753 QSPGILPRS SEQ ID 793 SGSIRARIH SEQ ID 833 AVRTAAYSP SEQ ID 754 SPGILPRSS SEQ ID 794 GSIRARIHP SEQ ID 834 VRTAAYSPI SEQ ID 755 PGILPRSSV SEQ ID 795 SIRARIHPS SEQ ID 835 RTAAYSPIS SEQ ID 756 GILPRSSVG SEQ ID 796 IRARIHPSP SEQ ID 836 TAAYSPIST SEQ ID 757 ILPRSSVGP SEQ ID 797 RARIHPSPW SEQ ID 837 AAYSPISTS SEQ ID 758 LPRSSVGPC SEQ ID 798 ARIHPSPWG SEQ ID 838 AYSPISTSK SEQ ID 759 PRSSVGPCI SEQ ID 799 RIHPSPWGT SEQ ID 839 YSPISTSKG SEQ ID 760 RSSVGPCIQ SEQ ID 800) HPSPWGTV SEQ ID 840 SPISTSKGH SEQ ID 841 PISTSKGHS SEQ ID 881 FRNSKPCSE SEQ ID 921 RVTGGVFLV SEQ ID 842 ISTSKGHSS SEQ ID 882 RNSKPCSEY SEQ ID 922 VTGGVFLVD SEQ ID 843 STSKGHSSS SEQ ID 883 NSKPCSEYC SEQ-ID-923 TGGVFLVDK SEQ ID 844 TSKGHSSSG SEQ ID 884 SKPCSEYCL SEQ ID 924 GGVFLVDKN SEQ ID 845 SKGHSSSGH SEQ ID 885 KPCSEYCLS SEQ ID 925 GVFLVDKNP SEQ ID 846 KGHSSSGHA SEQ ID 886 PCSEYCLSH SEQ ID 926 VFLVDKNPH SEQ ID 847 GHSSSGHAV SEQ ID 887 CSEYCLSHI SEQ ID 927 FLVDKNPHN SEQ ID 848 HSSSGHAVE SEQ ID 888 SEYCLSHIV SEQ ID 928 LVDKNPHNT SEQ ID 849 SSSGHAVEL SEQ ID 889 EYCLSHIVN SEQ ID 929 VDKNPHNTT SEQ ID 850 SSGHAVELH SEQ ID 890 YCLSHIVNL SEQ ID 930 DKNPHNTTE SEQ ID 851 SGHAVELHH SEQ ID 891 CLSHIVNLI SEQ ID 931 KNPHNTTES SEQ ID 852 GHAVELHHF SEQ ID 892 LSHIVNLIE SEQ ID 932 NPHNTTESR SEQ ID 853 HAVELHHFP SEQ ID 893 SHIVNLIED SEQ ID 933 PHNTTESRL SEQ ID 854 AVELHHFPP SEQ ID 894 HIVNLIEDW SEQ ID 934 HNTTESRLV SEQ ID 855 VELHHFPPN SEQ ID 895 IVNLIEDWG SEQ ID 935 NTTESRLW SEQ ID 856 ELHHFPPNS SEQ ID 896 VNLIEDWGP SEQ ID 936 TTESRLVVD SEQ ID 857 LHHFPPNSS SEQ ID 897 NLIEDWGPC SEQ ID 937 TESRLVVDF SEQ ID 858 HHFPPNSSR SEQ ID 898 LIEDWGPCA SEQ ID 938 ESRLVVDFS SEQ ID 859 HFPPNSSRS SEQ ID 899 IEDWGPCAE SEQ ID 939 SRLWDFSQ SEQ ID 860 FPPNSSRSQ SEQ ID 900 EDWGPCAEH SEQ ID 940 RLVVDFSQF SEQ ID 861 PPNSSRSQS SEQ ID 901 DWGPCAEHG SEQ ID 941 LVVDFSQFS SEQ ID 862 PNSSRSQSQ SEQ ID 902 WGPCAEHGE SEQ ID 942 VVDFSQFSR SEQ ID 863 NSSRSQSQG SEQ ID 903 GPCAEHGEH SEQ ID 943 VDFSQFSRG SEQ ID 864 SSRSQSQGS SEQ ID 904 PCAEHGEHR SEQ ID 944 DFSQFSRGN SEQ ID 865 SRSQSQGSV SEQ ID 905 CAEHGEHRI SEQ ID 945 FSQFSRGNT SEQ ID 866 RSQSQGSVL SEQ ID 906 AEHGEHRIR SEQ ID 946 SQFSRGNTR SEQ ID 867 SQSQGSVLS SEQ ID 907 EHGEHRIRT SEQ ID 947 QFSRGNTRV SEQ ID 868 QSQGSVLSC SEQ ID 908 HGEHRIRTP SEQ ID 948 FSRGNTRVS SEQ ID 869 SQGSVLSCW SEQ ID 909 GEHRIRTPR SEQ ID 949 SRGNTRVSW SEQ ID 870 QGSVLSCWW SEQ ID 910 EHRIRTPRT SEQ ID 950 RGNTRVSWP SEQ ID 871 GSVLSCWWL SEQ ID 911 HRIRTPRTP SEQ ID 951 GNTRVSWPK SEQ ID 872 SVLSCWWLQ SEQ ID 912 RIRTPRTPA SEQ ID 952 NTRVSWPKF SEQ ID 873 VLSCWWLQF SEQ ID 913 IRTPRTPAR SEQ ID 953 TRVSWPKFA SEQ ID 874 LSCWWLQFR SEQ ID 914 RTPRTPARV SEQ ID 954 RVSWPKFAV SEQ ID 875 SCWWLQFRN SEQ ID 915 TPRTPARVT SEQ ID 955 VSWPKFAVP SEQ ID 876 CWWLQFRNS SEQ ID 916 PRTPARVTG SEQ ID 956 SWPKFAVPN SEQ ID 877 WWLQFRNSK SEQ ID 917 RTPARVTGG SEQ ID 957 WPKFAVPNL SEQ ID 878 WLQFRNSKP SEQ ID 918 TPARVTGGV SEQ ID 958 PKFAVPNLQ SEQ ID 879 LQFRNSKPC SEQ ID 919 PARVTGGVF SEQ ID 959 KFAVPNLQS SEQ ID 880 QFRNSKPCS SEQ ID 920 ARVTGGVFL SEQ ID 960 FAVPNLQSL SEQ ID 961 AVPNLQSLT SEQ ID 1001 LVGSSGLSR SEQ ID 1041 VSLMLLYKT SEQ ID 962 VPNLQSLTN SEQ ID 1002 VGSSGLSRY SEQ ID 1042 SLMLLYKTY SEQ ID 963 PNLQSLTNL SEQ ID 1003 GSSGLSRYV SEQ ID 1043 LMLLYKTYG SEQ ID 964 NLQSLTNLL SEQ ID 1004 SSGLSRYVA SEQ ID 1044 MLLYKTYGW SEQ ID 965 LQSLTNLLS SEQ ID 1005 SGLSRYVAR SEQ ID 1045 LLYKTYGWK SEQ ID 966 QSLTNLLSS SEQ ID 1006 GLSRYVARL SEQ ID 1046 LYKTYGWKL SEQ ID 967 SLTNLLSSN SEQ ID 1007 LSRYVARLS SEQ ID 1047 YKTYGWKLH SEQ ID 968 LTNLLSSNL SEQ ID 1008 SRYVARLSS SEQ ID 1048 KTYGWKLHL SEQ ID 969 TNLLSSNLS SEQ ID 1009 RYVARLSSN SEQ ID 1049 TYGWKLHLY SEQ ID 970 NLLSSNLSW SEQ ID 1010 YVARLSSNS SEQ ID 1050 YGWKLHLYS SEQ ID 971 LLSSNLSWL SEQ ID 1011 VARLSSNSR SEQ ID 1051 GWKLHLYSH SEQ ID 972 LSSNLSWLS SEQ ID 1012 ARLSSNSRI SEQ ID 1052 WKLHLYSHP SEQ ID 973 SSNLSWLSL SEQ ID 1013 RLSSNSRII SEQ ID 1053 KLHLYSHPI SEQ ID 974 SNLSWLSLD SEQ ID 1014 LSSNSRIIN SEQ ID 1054 LHLYSHPII SEQ ID 975 NLSWLSLDV SEQ ID 1015 SSNSRIINH SEQ ID 1055 HLYSHPIIL SEQ ID 976 LSWLSLDVS SEQ ID 1016 SNSRIINHQ SEQ ID 1056 LYSHPIILG SEQ ID 977 SWLSLDVSA SEQ ID 1017 NSRIINHQH SEQ ID 1057 YSHPIILGF SEQ ID 978 WLSLDVSAA SEQ ID 1018 SRIINHQHG SEQ ID 1058 SHPIILGFR SEQ ID 979 LSLDVSAAF SEQ ID 1019 RIINHQHGT SEQ ID 1059 HPIILGFRK SEQ ID 980 SLDVSAAFY SEQ ID 1020 IINHQHGTM SEQ ID 1060 PIILGFRKI SEQ ID 981 LDVSAAFYH SEQ ID 1021 INHQHGTMQ SEQ ID 1061 IILGFRKIP SEQ ID 982 DVSAAFYHL SEQ ID 1022 NHQHGTMQD SEQ ID 1062 ILGFRKIPM SEQ ID 983 VSAAFYHLP SEQ ID 1023 HQHGTMQDL SEQ ID 1063 LGFRKIPMG SEQ ID 984 SAAFYHLPL SEQ ID 1024 QHGTMQDLH SEQ ID 1064 GFRKIPMGV SEQ ID 985 AAFYHLPLH SEQ ID 1025 HGTMQDLHN SEQ ID 1065 FRKIPMGVG SEQ ID 986 AFYHLPLHP SEQ ID 1026 GTMQDLHNS SEQ ID 1066 RKIPMGVGL SEQ ID 987 FYHLPLHPA SEQ ID 1027 TMQDLHNSC SEQ ID 1067 KIPMGVGLS SEQ ID 988 YHLPLHPAA SEQ ID 1028 MQDLHNSCS SEQ ID 1068 IPMGVGLSP SEQ ID 989 HLPLHPAAM SEQ ID 1029 QDLHNSCSR SEQ ID 1069 PMGVGLSPF SEQ ID 990 LPLHPAAMP SEQ ID 1030 DLHNSCSRN SEQ ID 1070 MGVGLSPFL SEQ ID 991 PLHPAAMPH SEQ ID 1031 LHNSCSRNL SEQ ID 1071 GVGLSPFLL SEQ ID 992 LHPAAMPHL SEQ ID 1032 HNSCSRNLY SEQ ID 1072 VGLSPFLLA SEQ ID 993 HPAAMPHLL SEQ ID 1033 NSCSRNLYV SEQ ID 1073 GLSPFLLAQ SEQ ID 994 PAAMPHLLV SEQ ID 1034 SCSRNLYVS SEQ ID 1074 LSPFLLAQF SEQ ID 995 AAMPHLLVG SEQ ID 1035 CSRNLYVSL SEQ ID 1075 SPFLLAQFT SEQ ID 996 AMPHLLVGS SEQ ID 1036 SRNLYVSLM SEQ ID 1076 PFLLAQFTS SEQ ID 997 MPHLLVGSS SEQ ID 1037 RNLYVSLML SEQ ID 1077 FLLAQFTSA SEQ ID 998 PHLLVGSSG SEQ ID 1038 NLYVSLMLL SEQ ID 1078 LLAQFTSAI SEQ ID 999 HLLVGSSGL SEQ ID 1039 LYVSLMLLY SEQ ID 1079 LAQFTSAIC SEQ ID 1000 LLVGSSGLS SEQ ID 1040 YVSLMLLYK SEQ ID 1080 AQFTSAICS SEQ ID 1081 QFTSAICSV SEQ ID 1121 AAVTNFLLS SEQ ID 1161 QDHIVQNFK SEQ ID 1082 FTSAICSVV SEQ ID 1122 AVTNFLLSL SEQ ID 1162 DHIVQNFKL SEQ ID 1083 TSAICSVVR SEQ ID 1123 VTNFLLSLG SEQ ID 1163 HIVQNFKLC SEQ ID 1084 SAICSVVRR SEQ ID 1124 TNFLLSLGI SEQ ID 1164 IVQNFKLCF SEQ ID 1085 AICSVVRRA SEQ ID 1125 NFLLSLGIH SEQ ID 1165 VQNFKLCFR SEQ ID 1086 ICSWRRAF SEQ ID 1126 FLLSLGIHL SEQ ID 1166 QNFKLCFRK SEQ ID 1087 CSVVRRAFP SEQ ID 1127 LLSLGIHLN SEQ ID 1167 NFKLCFRKL SEQ ID 1088 SVVRRAFPH SEQ ID 1128 LSLGIHLNP SEQ ID 1168 FKLCFRKLP SEQ ID 1089 WRRAFPHC SEQ ID 1129 SLGIHLNPN SEQ ID 1169 KLCFRKLPV SEQ ID 1090 VRRAFPHCL SEQ ID 1130 LGIHLNPNK SEQ ID 1170 LCFRKLPVN SEQ ID 1091 RRAFPHCLA SEQ ID 1131 GIHLNPNKT SEQ ID 1171 CFRKLPVNR SEQ ID 1092 RAFPHCLAF SEQ ID 1132 IHLNPNKTK SEQ ID 1172 FRKLPVNRP SEQ ID 1093 AFPHCLAFS SEQ ID 1133 HLNPNKTKR SEQ ID 1173 RKLPVNRPI SEQ ID 1094 FPHCLAFSY SEQ ID 1134 LNPNKTKRW SEQ ID 1174 KLPVNRPID SEQ ID 1095 PHCLAFSYM SEQ ID 1135 NPNKTKRWG SEQ ID 1175 LPVNRPIDW SEQ ID 1096 HCLAFSYMD SEQ ID 1136 PNKTKRWGY SEQ ID 1176 PVNRPIDWK SEQ ID 1097 CLAFSYMDD SEQ ID 1137 NKTKRWGYS SEQ ID 1177 VNRPIDWKV SEQ ID 1098 LAFSYMDDV SEQ ID 1138 KTKRWGYSL SEQ ID 1178 NRPIDWKVC SEQ ID 1099 AFSYMDDVV SEQ ID 1139 TKRWGYSLN SEQ ID 1179 RPIDWKVCQ SEQ ID 1100 FSYMDDVVL SEQ ID 1140 KRWGYSLNF SEQ ID 1180 PIDWKVCQR SEQ ID 1101 SYMDDWLG SEQ ID 1141 RWGYSLNFM SEQ ID 1181 IDWKVCQRI SEQ ID 1102 YMDDVVLGA SEQ ID 1142 WGYSLNFMG SEQ ID 1182 DWKVCQRIV SEQ ID 1103 MDDVVLGAK SEQ ID 1143 GYSLNFMGY SEQ ID 1183 WKVCQRIVG SEQ ID 1104 DDVVLGAKS SEQ ID 1144 YSLNFMGYV SEQ ID 1184 KVCQRIVGL SEQ ID 1105 DVVLGAKSV SEQ ID 1145 SLNFMGYVI SEQ ID 1185 VCQRIVGLL SEQ ID 1106 VVLGAKSVQ SEQ ID 1146 LNFMGYVIG SEQ ID 1186 CQRIVGLLG SEQ ID 1107 VLGAKSVQH SEQ ID 1147 NFMGYVIGS SEQ ID 1187 QRIVGLLGF SEQ ID 1108 LGAKSVQHL SEQ ID 1148 FMGYVIGSW SEQ ID 1188 RIVGLLGFA SEQ ID 1109 GAKSVQHLE SEQ ID 1149 MGYVIGSWG SEQ ID 1189 IVGLLGFAA SEQ ID 1110 AKSVQHLES SEQ ID 1150 GYVIGSWGT SEQ ID 1190 VGLLGFAAP SEQ ID 1111 KSVQHLESL SEQ ID 1151 YVIGSWGTW SEQ ID 1191 GLLGFAAPF SEQ ID 1112 SVQHLESLY SEQ ID 1152 VIGSWGTWP SEQ ID 1192 LLGFAAPFT SEQ ID 1113 VQHLESLYA SEQ ID 1153 IGSWGTWPQ SEQ ID 1193 LGFAAPFTQ SEQ ID 1114 QHLESLYAA SEQ ID 1154 GSWGTWPQD SEQ ID 1194 GFAAPFTQC SEQ ID 1115 HLESLYAAV SEQ ID 1155 SWGTWPQDH SEQ ID 1195 FAAPFTQCG SEQ ID 1116 LESLYAAVT SEQ ID 1156 WGTWPQDHI SEQ ID 1196 AAPFTQCGY SEQ ID 1117 ESLYAAVTN SEQ ID 1157 GTWPQDHIV SEQ ID 1197 APFTQCGYP SEQ ID 1118 SLYAAVTNF SEQ ID 1158 TWPQDHlyQ SEQ ID 1198 PFTQCGYPA SEQ ID 1119 LYAAVTNFL SEQ ID 1159 WPQDHIVQN SEQ ID 1199 FTQCGYPAL SEQ ID 1120 YAAVTNFLL SEQ ID 1160 PQDHIVQNF SEQ ID 1200 TQCGYPALM SEQ ID 1201 QCGYPALMP SEQ ID 1241 ARQRPGLCQ SEQ ID 1281 AELLAACFA SEQ ID 1202 CGYPALMPL SEQ ID 1242 RQRPGLCQV SEQ ID 1282 ELLAACFAR SEQ ID 1203 GYPALMPLY SEQ ID 1243 QRPGLCQVF SEQ ID 1283 LLAACFARS SEQ ID 1204 YPALMPLYA SEQ ID 1244 RPGLCQVFA SEQ ID 1284 LAACFARSR SEQ ID 1205 PALMPLYAC SEQ ID 1245 PGLCQVFAD SEQ ID 1285 AACFARSRS SEQ ID 1206 ALMPLYACI SEQ ID 1246 GLCQVFADA SEQ ID 1286 ACFARSRSG SEQ ID 1207 LMPLYACIQ SEQ ID 1247 LCQVFADAT SEQ ID 1287 CFARSRSGA SEQ ID 1208 MPLYACIQA SEQ ID 1248 CQVFADATP SEQ ID 1288 FARSRSGAN SEQ ID 1209 PLYACIQAK SEQ ID 1249 QVFADATPT SEQ ID 1289 ARSRSGANL SEQ ID 1210 LYACIQAKQ SEQ ID 1250 VFADATPTG SEQ ID 1290 RSRSGANLI SEQ ID 1211 YACIQAKQA SEQ ID 1251 FADATPTGW SEQ ID 1291 SRSGANLIG SEQ ID 1212 ACIQAKQAF SEQ ID 1252 ADATPTGWG SEQ ID 1292 RSGANLIGT SEQ ID 1213 CIQAKQAFT SEQ ID 1253 DATPTGWGL SEQ ID 1293 SGANLIGTD SEQ ID 1214 IQAKQAFTF SEQ ID 1254 ATPTGWGLA SEQ ID 1294 GANLIGTDN SEQ ID 1215 QAKQAFTFS SEQ ID 1255 TPTGWGLAI SEQ ID 1295 ANLIGTDNS SEQ ID 1216 AKQAFTFSP SEQ ID 1256 PTGWGLAIG SEQ ID 1296 NLIGTDNSV SEQ ID 1217 KQAFTFSPT SEQ ID 1257 TGWGLAIGH SEQ ID 1297 LIGTDNSW SEQ ID 1218 QAFTFSPTY SEQ ID 1258 GWGLAIGHQ SEQ ID 1298 IGTDNSWL SEQ ID 1219 AFTFSPTYK SEQ ID 1259 WGLAIGHQR SEQ ID 1299 GTDNSWLS SEQ ID 1220 FTFSPTYKA SEQ ID 1260 GLAIGHQRM SEQ ID 1300 TDNSWLSR SEQ ID 1221 TFSPTYKAF SEQ ID 1261 LAIGHQRMR SEQ ID 1301 DNSWLSRK SEQ ID 1222 FSPTYKAFL SEQ ID 1262 AIGHQRMRG SEQ ID 1302 NSVVLSRKY SEQ ID 1223 SPTYKAFLS SEQ ID 1263 IGHQRMRGT SEQ ID 1303 SVVLSRKYT SEQ ID 1224 PTYKAFLSK SEQ ID 1264 GHQRMRGTF SEQ ID 1304 VVLSRKYTS SEQ ID 1225 TYKAFLSKQ SEQ ID 1265 HQRMRGTFV SEQ ID 1305 VLSRKYTSF SEQ ID 1226 YKAFLSKQY SEQ ID 1266 QRMRGTFVS SEQ ID 1306 LSRKYTSFP SEQ ID 1227 KAFLSKQYM SEQ ID 1267 RMRGTFVSP SEQ ID 1307 SRKYTSFPW SEQ ID 1228 AFLSKQYMT SEQ ID 1268 MRGTFVSPL SEQ ID 1308 RKYTSFPWL SEQ ID 1229 FLSKQYMTL SEQ ID 1269 RGTFVSPLP SEQ ID 1309 KYTSFPWLL SEQ ID 1230 LSKQYMTLY SEQ ID 1270 GTFVSPLPI SEQ ID 1310 YTSFPWLLG SEQ ID 1231 SKQYMTLYP SEQ ID 1271 TFVSPLPIH SEQ ID 1311 TSFPWLLGC SEQ ID 1232 KQYMTLYPV SEQ ID 1272 FVSPLPIHT SEQ ID 1312 SFPWLLGCA SEQ ID 1233 QYMTLYPVA SEQ ID 1273 VSPLPIHTA SEQ ID 1313 FPWLLGCAA SEQ ID 1234 YMTLYPVAR SEQ ID 1274 SPLPIHTAE SEQ ID 1314 PWLLGCAAN SEQ ID 1235 MTLYPVARQ SEQ ID 1275 PLPIHTAEL SEQ ID 1315 WLLGCAANW SEQ ID 1236 TLYPVARQR SEQ ID 1276 LPIHTAELL SEQ ID 1316 LLGCAANWI SEQ ID 1237 LYPVARQRP SEQ ID 1277 PIHTAELLA SEQ ID 1317 LGCAANWIL SEQ ID 1238 YPVARQRPG SEQ ID 1278 IHTAELLAA SEQ ID 1318 GCAANWILR SEQ ID 1239 PVARQRPGL SEQ ID 1279 HTAELLAAC SEQ ID 1319 CAANWILRG SEQ ID 1240 VARQRPGLC SEQ ID 1280 TAELLAACF SEQ ID 1320 AANWILRGT SEQ ID 1321 ANWILRGTS SEQ ID 1344 SRGRLGLYR SEQ ID 1367 SLYADSPSV SEQ ID 1322 NWILRGTSF SEQ ID 1345 RGRLGLYRP SEQ ID 1368 LYADSPSVP SEQ ID 1323 WILRGTSFV SEQ ID 1346 GRLGLYRPL SEQ ID 1369 YADSPSVPS SEQ ID 1324 ILRGTSFVY SEQ ID 1347 RLGLYRPLL SEQ ID 1370 ADSPSVPSH SEQ ID 1325 LRGTSFVYV SEQ ID 1348 LGLYRPLLR SEQ ID 1371 DSPSVPSHL SEQ ID 1326 RGTSFVYVP SEQ ID 1349 GLYRPLLRL SEQ ID 1372 SPSVPSHLP SEQ ID 1327 GTSFVYVPS SEQ ID 1350 LYRPLLRLP SEQ ID 1373 PSVPSHLPD SEQ ID 1328 TSFVYVPSA SEQ ID 1351 YRPLLRLPY SEQ ID 1374 SVPSHLPDR SEQ ID 1329 SFVYVPSAL SEQ ID 1352 RPLLRLPYR SEQ ID 1375 VPSHLPDRV SEQ ID 1330 FVYVPSALN SEQ ID 1353 PLLRLPYRP SEQ ID 1376 PSHLPDRVH SEQ ID 1331 VYVPSALNP SEQ ID 1354 LLRLPYRPT SEQ ID 1377 SHLPDRVHF SEQ ID 1332 YVPSALNPA SEQ ID 1355 LRLPYRPTT SEQ ID 1378 HLPDRVHFA SEQ ID 1333 VPSALNPAD SEQ ID 1356 RLPYRPTTG SEQ ID 1379 LPDRVHFAS SEQ ID 1334 PSALNPADD SEQ ID 1357 LPYRPTTGR SEQ ID 1380 PDRVHFASP SEQ ID 1335 SALNPADDP SEQ ID 1358 PYRPTTGRT SEQ ID 1381 DRVHFASPL SEQ ID 1336 ALNPADDPS SEQ ID 1359 YRPTTGRTS SEQ ID 1382 RVHFASPLH SEQ ID 1337 LNPADDPSR SEQ ID 1360 RPTTGRTSL SEQ ID 1383 VHFASPLHV SEQ ID 1338 NPADDPSRG SEQ ID 1361 PTTGRTSLY SEQ ID 1384 HFASPLHVA SEQ ID 1339 PADDPSRGR SEQ ID 1362 TTGRTSLYA SEQ ID 1385 FASPLHVAW SEQ ID 1340 ADDPSRGRL SEQ ID 1363 TGRTSLYAD SEQ ID 1386 ASPLHVAWR SEQ ID 1341 DDPSRGRLG SEQ ID 1364 GRTSLYADS SEQ ID 1387 SPLHVAWRP SEQ ID 1342 DPSRGRLGL SEQ ID 1365 RTSLYADSP SEQ ID 1388 PLHVAWRPP SEQ ID 1343 PSRGRLGLY SEQ ID 1366 TSLYADSPS TABLE 8 : SEQ ID NOS of HBV peptides