Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions

Abstract
This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.
Description
INDEX
I. Background of the Invention
II. Summary of the Invention
III. Brief Description of the Figures
V. Detailed Description of the Invention





    • A. Definitions

    • B. Stimulation of CTL and HTL responses against HBV

    • C. Binding Affinity of Peptide Epitopes for HLA Molecules

    • D. Peptide Epitope Binding Motifs and Supermotifs
      • 1. HLA-A1 supermotif
      • 2. HLA-A2 supermotif
      • 3. HLA-A3 supermotif
      • 4. HLA-A24 supermotif
      • 5. HLA-B7 supermotif
      • 6. HLA-B27 supermotif
      • 7. HLA-B44 supermotif
      • 8. HLA-B58 supermotif
      • 9. HLA-B62 supermotif
      • 10. HLA-A1 motif
      • 11. HLA-A2.1 motif
      • 12. HLA-A3 motif
      • 13. HLA-A11 motif
      • 14. HLA-A24 motif
      • 15. HLA-DR-1-4-7 supermotif
      • 16. HLA-DR3 motifs

    • E. Enhancing Population Coverage of the Vaccine

    • F. Immune Response Stimulating Peptide Analogs

    • G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif or Motif Containing Peptides

    • H. Preparation of Peptide Epitopes

    • I. Assays to Detect T-Cell Responses

    • J. Use of Peptide Epitopes for Evaluating Immune Responses

    • K. Vaccine Compositions
      • 1. Minigene Vaccines
      • 2. Combinations of CTL Peptides with Helper Peptides

    • L. Administration of Vaccines for Therapeutic or Prophylactic Purposes

    • M. Kits





V. Examples
VI. Claims
VII. Abstract
I. BACKGROUND OF THE INVENTION

Chronic infection by hepatitis B virus (HBV) affects at least 5% of the world's population and is a major cause of cirrhosis and hepatocellular carcinoma (Hooffnagle, J., N. Engl. J. Med. 323:337, 1990; Fields, B. and Knipe, D., In: Fields Virology 2:2137, 1990). The World Health Organization lists hepatitis B as a leading cause of death worldwide, close behind chronic pulmonary disease, and more prevalent than AIDS. Chronic HBV infection can range from an asymptomatic carrier state to continuous hepatocellular necrosis and inflammation, and can lead to hepatocellular carcinoma.


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 directly or the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.


Several studies have emphasized the association between self-limiting acute hepatitis and multispecific CTL responses (Penna, A. et al., J. Exp. Med. 174:1565, 1991; Nayersina, R. et al., J. Immunol. 150:4659, 1993). Spontaneous and interferon-related clearance of chronic HBV infection is also associated with the resurgence of a vigorous CTL response (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764, 1994). In all such cases the CTL responses are polyclonal, and specific for multiple viral proteins including the HBV envelope, core and polymerase antigens. By contrast, in patients with chronic hepatitis, the CTL activity is usually absent or weak, and antigenically restricted.


The crucial role of CTL in resolution of HBV infection has been further underscored by studies using HBV transgenic mice. Adoptive transfer of HBV-specific CTL into mice transgenic for the HBV genome resulted in suppression of virus replication. This effect was primarily mediated by a non-lytic, lymphokine-based mechanism (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764, 1994; Guidotti, L. G., Guilhot, S., and Chisari, F. V. J. Virol. 68:1265, 1994; Guidotti, L. G. et al., J. Virol. 69:6158, 1995; Gilles, P. N., Fey, G., and Chisari, F. V., J. Virol. 66:3955, 1992).


As is the case for HLA class I restricted responses, HLA class II restricted T cell responses are usually detected in patients with acute hepatitis, and are absent or weak in patients with chronic infection (Chisari, F. V. and Ferrari, C., Annu. Rev. Immunol. 13:29, 1995). HLA Class II responses are tied to activation of helper T cells (HTLs) Helper T lymphocytes, which recognize Class II HLA molecules, may directly contribute to the clearance of HBV infection through the secretion of cytokines which suppress viral replication (Franco, A. et al., J. Immunol. 159:2001, 1997). However, their primary role in disease resolution is believed to be mediated by inducing activation and expansion of virus-specific CTL and B cells.


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.


Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines. The epitopes for inclusion in such a vaccine are to be selected from conserved regions of viral or tumor-associated antigens, in order to reduce the likelihood of escape mutants. The advantage of an epitope-based approach over the use of whole antigens is that 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. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.


Additionally, with an epitope-based vaccine approach, there is an ability to combine selected epitopes (CTL and HTL) and additionally 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.


However, one of the most formidable obstacles to the development of broadly efficacious epitope-based immunotherapeutics has been the extreme polymorphism 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. 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.


Furthermore, as described herein in greater detail, a need has existed 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 whereby the natural immune responses noted in self-limiting acute hepatitis, or of spontaneous clearance of chronic HBV infection is induced 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.


The information provided in this section is intended to disclose the presently understood state of the art as of the filing date of the present application. Information is included in this section which was generated subsequent to the priority date of this application. Accordingly, background in this section is not intended, in any way, to delineate the priority date for the invention.


II. SUMMARY OF THE INVENTION

This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.


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 (CTL and HTL), 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 polymorphism 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.


Furthermore, as described herein in greater detail, a need has existed 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 of the invention are selected by a process whereby protein sequences of known antigens are evaluated for the presence of motif or supermotif-bearing epitopes. Peptides corresponding to a motif- or supermotif-bearing epitope are then synthesized and tested for the ability to bind to the HLA molecule that recognizes the selected motif. Those peptides that bind at an intermediate or high affinity i.e., an IC50 (or a KD value) of 500 nM or less for HLA class I molecules or 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptides are selected for inclusion in vaccine compositions.


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


The invention also includes an embodiment comprising a method for monitoring immunogenic activity of a vaccine for HBV in a patient having a known HLA-type, the method comprising incubating a T lymphocyte sample from the patient with a peptide composition comprising an HBV epitope consisting essentially of an amino acid sequence described in Tables VI to Table XX or Table XXII which binds the product of at least one HLA allele present in said patient, and detecting for the presence of a T lymphocyte that binds to the peptide. In a preferred embodiment, the peptide comprises a tetrameric complex.


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 allele-specific 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.


As will be apparent from the discussion below, other methods and embodiments are also contemplated. Further, novel synthetic peptides produced by any of the methods described herein are also part of the invention.





III. BRIEF DESCRIPTION OF THE FIGURES


FIG. 1: FIG. 1 provides a graph of total frequency of genotypes as a function of the number of HBV candidate epitopes bound by HLA-A and B molecules, in an average population.



FIG. 2: FIG. 2 Illustrates the Position of Peptide Epitopes in Experimental Model Minigene Constructs





IV. DETAILED DESCRIPTION OF THE INVENTION

The peptides and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to HBV either by stimulating the production of CTL or HTL responses. The peptides, which are derived directly or indirectly from native HBV amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to HBV. The complete polyprotein sequence from HBV and its variants can be obtained from Genbank. Peptides can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of HBV as will be clear from the disclosure provided below.


The peptides of the invention have been identified in a number of ways, as will be discussed below. Further, analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with multiple HLA antigens to provide broader population coverage than prior vaccines.


IV.A. DEFINITIONS

The invention can be better understood with reference to the following definitions, which are listed alphabetically.


“Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.


A “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.


A “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen. (See, e.g., Sercarz, et al., Annu. Rev. Immunol. 11:729766 (1993)) Such a response is cross-reactive in vitro with an isolated peptide epitope.


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 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, T cell receptor or HLA molecule.


“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).


An “HLA supertype or family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype family, and HLA xx-like supertype molecules (where xx denotes a particular HLA type) are synonyms.


Throughout this disclosure, results are expressed in terms of “IC50's.” IC50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate KD values. It should be noted that IC50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC50 of a given ligand.


Assays for determining binding are described in detail in PCT publications WO 94/20127 and WO 94/03205. Alternatively, binding is expressed relative to a reference peptide. As a particular assay becomes more, or less, sensitive, the IC50's of the peptides tested may change somewhat. However, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC50 of the reference peptide increases 10-fold, the IC50 values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC50, relative to the IC50 of a standard peptide.


Binding may also be determined using other assays including, for example, inhibition of antigen presentation (Sette et al., J. Immunol. 141:3893, 1991), in vitro assembly assays (Townsend et al., Cell 62:285, 1990), measures of dissociations rates (Parker et al., J. Immunol. 149:1896-1904, 1992), and FACS-based assays using mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol. 21:2963, 1991).


As used herein, high affinity with respect to HLA class I molecules is defined as binding with an IC50 or KD value of less than 50 nM; intermediate affinity is binding with an IC50 (or KD) of between about 50 and about 500 nM. High affinity with respect to binding to HLA class II molecules is defined as binding with an IC50 or KD value of less than 100 nM; intermediate affinity is binding with an IC50 or KD of between about 100 and about 1000 nM.


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 which comprises an allele-specific motif or supermotif such that the peptide 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 phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.


“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 the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York, 1993.


The term “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.


A “negative binding residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) of a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule. Any residue that is not “deleterious” is a “non-deleterious” residue.


The term “peptide” is used interchangeably with “oligopeptide” 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 α-amino and carboxyl groups of adjacent amino acids. The preferred CTL-inducing oligopeptides 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. The preferred HTL-inducing oligopeptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.


“Pharmaceutically acceptable” refers to a non-toxic, inert, and physiologically compatible composition.


A “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment, the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9 residue peptide in accordance with the invention. The primary anchor positions for each motif and supermotif are set forth in Table I. For example, analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.


“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.


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


The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.


A “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position. The secondary anchor residues are said to occur at “secondary anchor positions.” A secondary anchor residue can be identified as a residue which is present at a higher frequency among high affinity binding peptides, or a residue otherwise associated with high affinity binding. For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.


A “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.


A “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. A supermotif-bearing epitope is preferably is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.


“Synthetic peptide” refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.


The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for the amino acids are shown below.

















Single Letter Symbol
Three Letter Symbol
Amino Acids









A
Ala
Alanine



C
Cys
Cysteine



D
Asp
Aspartic Acid



E
Glu
Glutamic Acid



F
Phe
Phenylalanine



G
Gly
Glycine



H
His
Histidine



I
Ile
Isoleucine



K
Lys
Lysine



L
Leu
Leucine



M
Met
Methionine



N
Asn
Asparagine



P
Pro
Proline



Q
Gln
Glutamine



R
Arg
Arginine



S
Ser
Serine



T
Thr
Threonine



V
Val
Valine



W
Trp
Tryptophan



Y
Tyr
Tyrosine










IV.B. STIMULATION OF CTL AND HTL RESPONSES AGAINST HBV

The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our new understanding of the immune system we have generated efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to HBV infection in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of the technology is provided.


A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A., and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are described here and set forth in Tables I, II, and III (see also, e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994). Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate allele-specific residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present (Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991).


Accordingly, the definition of class I and class II allele-specific HLA binding motifs or class I supermotifs allows identification of regions within a protein that have the potential of binding particular HLA antigens (see also e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Kast, W. M. et al., J. Immunol., 152:3904, 1994).


Furthermore, a variety of assays to quantify the affinity of interaction between peptide and HLA have also been established. Such assays include, for example, measures of IC50 values, inhibition of antigen presentation (Sette et al., J. Immunol. 141:3893, 1991), in vitro assembly assays (Townsend et al., Cell 62:285, 1990), measures of dissociations rates (Parker et al., J. Immunol. 149:1896-1904, 1992), and FACS-based assays using mutated cells, such as RMA.S (Melief, et al., Eur. J. Immunol. 21:2963, 1991).


The present inventors have found that the correlation of binding affinity with immunogenicity is an important factor to be considered when evaluating candidate peptides. Thus, by a combination of motif searches and HLA-peptide binding assays, candidates for epitope-based vaccines have been identified. After determining their binding affinity, additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of antigenicity and immunogenicity. Various strategies can be utilized to evaluate immunogenicity, including:


1) Evaluation of primary T cell cultures from normal individuals (Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedure involves the stimulation of PBL from normal 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 51Cr-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. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 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 51Cr-release 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 (Rehermann, 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. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 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 (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays for T cell activity including 51Cr release involving peptide-sensitized targets, T cell proliferation or lymphokine release.


The following describes the peptide epitopes and corresponding nucleic acids of the invention.


IV.C. BINDING AFFINITY OF PEPTIDE EPITOPES FOR HLA MOLECULES

As indicated herein, 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.


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 mM or less. HTL-inducing peptides preferably include those that have an IC50 or binding affinity value for class II HLA molecules of 1000 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. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, 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 relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors. The correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (Sette, et al., J. Immunol. 153:5586-5592, 1994). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL (peripheral blood lymphocytes) of acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold of approximately 500 nM (preferably an IC50 value of 500 nM or less) determines the capacity of a peptide epitope to elicit a CTL response. These data are true for class I binding affinity measurements for naturally processed peptides and for synthesized T cell epitopes. These data also indicate the important role of determinant selection in the shaping of T cell responses.


An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has also been delineated (Southwood et al. J. Immunology 160:3363-3373, 1998, and U.S. Ser. No. 60/087,192 filed May 29, 1998). In order to define a biologically significant threshold of DR binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e., binding affinities of with an IC50 value of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinities in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.


The binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.


IV.D. PEPTIDE EPITOPE BINDING MOTIFS AND SUPERMOTIFS

In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I, and possibly class II molecules can be classified into a relatively few supertypes characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets.


For HLA molecule pocket analyses, the residues comprising the B and F pockets of HLA class I molecules as described in crystallographic studies (Guo, H. C. et al., Nature 360:364, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991; Madden, D. R., Garboczi, D. N. and Wiley, D. C., Cell 75:693, 1993), have been compiled from the database of Parham, et al. (Parham, P., Adams, E. J., and Arnett, K. L., Immunol. Rev. 143:141, 1995). In these analyses, residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the B pocket, and to determine the specificity for the residue in the second position of peptide ligands. Similarly, residues 77, 80, 81, and 116 were considered to determine the specificity of the F pocket, and to determine the specificity for the C-terminal residue of a peptide ligand bound by the HLA molecule.


Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues required for allele-specific binding to HLA molecules have been identified. The presence of these residues correlates with binding affinity for HLA molecules. The identification of motifs and/or supermotifs that correlate with high and intermediate affinity binding is an important issue with respect to the identification of immunogenic peptide epitopes for the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912, 1994) have shown that motif-bearing peptides account for 90% of the epitopes that bind to allele-specific HLA class I molecules. In this study all possible peptides of 9 amino acids in length and overlapping by eight amino acids (240 peptides), which cover the entire sequence of the E6 and E7 proteins of human papillomavirus type 16, were evaluated for binding to five allele-specific HLA molecules that are expressed at high frequency among different ethnic groups. This unbiased set of peptides allowed an evaluation of the predictive value of HLA class I motifs. From the set of 240 peptides, 22 peptides were identified that bound to an allele-specific HLA molecules with high or intermediate affinity. Of these 22 peptides, 20, (i.e. 91%), were motif-bearing. Thus, this study demonstrates the value of motifs for the identification of peptide epitopes for inclusion in a vaccine: application of motif-based identification techniques eliminates screening of 90% of the potential epitopes.


Such peptide epitopes are identified in the Tables described below. The Tables for the HLA class I epitopes include over 90% of the peptides that will bind to an allele-specific HLA class I molecule with intermediate or high affinity.


Peptides of the present invention may also include epitopes that bind to MHC class II DR molecules. A significant difference between class I and class II HLA molecules is that, although a stringent size restriction exists for peptide binding to class I molecules, a greater degree of heterogeneity in both sizes and binding frame positions of the motif, relative to the N and C termini of the peptide, can be demonstrated for class II peptide ligands. This increased heterogeneity is due to the structure of the class II-binding groove which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of DRB*0101-peptide complexes (see, e.g. Madden, D. R. Ann. Rev. Immunol. 13:587, 1995) showed that the residues occupying position 1 and position 6 of peptides complexed with DRB*0101 engage two complementary pockets on the DRBa*0101 molecules, with the P1 position corresponding to the most crucial anchor residue and the deepest hydrophobic pocket. Other studies have also pointed to the P6 position as a crucial anchor residue for binding to various other DR molecules.


Thus, peptides of the present invention are identified by any one of several HLA-specific amino acid motifs (see, e.g., Tables I-III). If the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens it is referred to as a supermotif. The allele-specific HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.”


The peptide motifs and supermotifs described below provide guidance for the identification and use of peptides in accordance with the invention.


Examples of peptide epitopes bearing the respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif. The Tables include a binding affinity ratio listing for some of the peptide epitopes. The ratio may be converted to IC50 by using the following formula: IC50 of the standard peptide/ratio=IC50 of the test peptide (i.e. the peptide epitope). The IC50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV. The IC50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V. The peptides used as standards for the binding assay are examples of standards; alternative standard peptides can also be used when performing such an analysis.


To obtain the peptide epitope sequences listed in each Table, protein sequence data from twenty HBV strains (HPBADR, HPBADR1CG, HPBADRA, HPBADRC, HPBADRCG, HPBCGADR, HPBVADRM, HPBADW, HPBADW1, HPBADW2, HPBADW3, HPBADWZ, HPBHEPB, HPBVADW2, HPBAYR, HPBV, HPBVAYWC, HPBVAYWCI, NAD HPBVAYWE) were evaluated for the presence of the designated supermotif or motif. Peptide epitopes were also selected on the basis of their conservancy. A criterion for conservancy requires that the entire sequence of a peptide be totally conserved in 75% of the sequences available for a specific protein. The percent conservancy of the selected peptide epitopes is indicated on the Tables. The frequency, i.e. the number of strains of the 20 strains in which the peptide sequence was identified, is also shown. The “1st position” column in the Tables designates the amino acid position of the HBV protein that corresponds to the first amino acid residue of the epitope. “Number of amino acids” indicates the number of residues in the epitope sequence.


HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

The primary anchor residues of the HLA class I peptide epitope supermotifs and motifs delineated below are summarized in Table I. The HLA class I motifs set out in Table I(a) are those most particularly relevant to the invention claimed here. Primary and secondary anchor positions are summarized in Table II. Allele-specific HLA molecules that comprise HLA class I supertype families are listed in Table VI.


IV.D1. HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, M, or F) primary anchor residue in position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind to the A1 supermotif (i.e., the HLA-A1 supertype) is comprised of at least A*0101, A*2601, A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M. et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997.). Other allele-specific HLA molecules predicted to be members of the A1 superfamily are shown in Table VI. Peptides binding to each of the individual HLA proteins can be modulated by substitutions at primary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the A1 supermotif are set forth on the attached Table VII.


IV.D.2. HLA-A2 Supermotif

Primary anchor specificities for allele-specific HLA A2.1 molecules (Falk et al., Nature 351:290-296, 1991; Hunt et al., Science 255:1261-1263, 1992) and cross-reactive binding within the HLA A2 family (Fruci et al., Human Immunol. 38:187-192, 1993; Tanigaki et al., Human Immunol. 39:155-162, 1994) have been described. The present inventors have defined additional primary anchor residues that determine cross-reactive binding to multiple allele-specific HLA A2 molecules (Del Guercio et al., J. Immunol. 154:685-693, 1995). The HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.


The corresponding family of HLA molecules (i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, a*0214, A*6802, and A*6901. Other allele-specific HLA molecules predicted to be members of the A2 superfamily are shown in Table VI. As explained in detail below, binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise an A2 supermotif are set forth on the attached Table VIII. The motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.


IV.D.3. HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by the presence in peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope (e.g., in position 9 of 9-mers). Exemplary members of the corresponding family of HLA molecules (the HLA-A3 supertype) that bind the A3 supermotif include at least: A*0301, A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA molecules predicted to be members of the A3 superfamily are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the A3 supermotif are set forth on the attached Table IX.


IV.D.4. HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) residue as a primary anchor in position 2, and a hydrophobic (Y, F, L, I, V, or M) residue as primary anchor at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind to the A24 supermotif (i.e., the A24 supertype) includes at least A*2402, A*3001, and A*2301. Other allele-specific HLA molecules predicted to be members of the A24 superfamily are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the A24 supermotif are set forth on the attached Table X.


IV.D.5. HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of at least twenty six HLA-B proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995; Hill, et al., Nature 360:434, 1992; Rammensee, et al., Immunogenetics 41:178, 1995). Other allele-specific HLA molecules predicted to be members of the B7 superfamily are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that contain the B7 supermotif are set forth on the attached Table XI.


IV.D.6. HLA-B27 Supermotif

The HLA-B27 supermotif is characterized by the presence in peptide ligands of a positively charged (R, H, or K) residue as a primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue as a primary anchor at the C-terminal position of the epitope. Exemplary members of the corresponding family of HLA molecules that bind to the B27 supermotif (i.e., the B27 supertype) include at least B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301. Other allele-specific HLA molecules predicted to be members of the B27 superfamily are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the B27 supermotif are set forth on the attached Table XII.


IV.D.7. HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as a primary anchor at the C-terminal position of the epitope. Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (i.e., the B44 supertype) include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006, B*4402, B*4403, and B*4006. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions; preferably choosing respective residues specified for the supermotif.


IV.D.8. HLA-B58 Supermotif

The HLA-B58 supermotif is characterized by the presence in peptide ligands of a small aliphatic residue (A, S, or T) as a primary anchor residue at position 2, and an aromatic or hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor residue at the C-terminal position of the epitope. Exemplary members of the corresponding family of HLA molecules that bind to the B58 supermotif (i.e., the B58 supertype) include at least: B*1516, B*1517, B*5701, B*5702, and B*5801. Other allele-specific HLA molecules predicted to be members of the B58 superfamily are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the B58 supermotif are set forth on the attached Table XIII.


IV.D.9. HLA-B62 Supermotif

The HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or a hydrophobic aliphatic residue (L, V, M, or I) as a primary anchor in position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor at the C-terminal position of the epitope. Exemplary members of the corresponding family of HLA molecules that bind to the B62 supermotif (i.e., the B62 supertype) include at least: B*1501, B*1502, B*1513, and B5201. Other allele-specific HLA molecules predicted to be members of the B62 superfamily are shown in Table VI.


Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary anchor positions, preferably choosing respective residues specified for the supermotif.


Representative peptide epitopes that comprise the B62 supermotif are set forth on the attached Table XIV.


IV.D.10. HLA-A1 Motif

The allele-specific HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope. An alternative allele-specific A1 motif (i.e., a “submotif”) is characterized by a primary anchor residue at position 3 rather than position 2. This submotif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3, and a Y as a primary anchor residue at the C-terminal position of the epitope. Peptide binding to HLA A1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.


Representative peptide epitopes that comprise either A1 motif are set forth on the attached Table XV. Those epitopes comprising T, S, or M at position 2 and Y at the C-terminal position are also included in the listing of HLA-A1 supermotif-bearing peptide epitopes listed in Table VII.


IV.D.11. HLA-A2.1 Motif

An allele-specific HLA-A2.1 motif was first determined to be characterized by the presence in peptide ligands of L or M as a primary anchor residue in position 2, and L or V as a primary anchor residue at the C-terminal position of a 9 amino acid epitope (Falk et al., Nature 351:290-296, 1991). Furthermore, the A2.1 motif was determined to further comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (Hunt et al., Science 255:1261-1263, Mar. 6, 1992). Additionally, the A2.1 allele-specific motif has been found to comprise a T at the C-terminal position (Kast et al., J. Immunol. 152:3904-3912, 1994). Subsequently, the A2.1 allele-specific motif has been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M as a primary anchor residue at the C-terminal position of the epitope. Thus, the HLA-A2.1 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope. The preferred and tolerated residues that characterize the primary anchor positions of the HLA-A2.1 motif are identical to the preferred residues of the A2 supermotif. (for reviews of relevant data, see, e.g., Del Guercio et al., J. Immunol. 154:685-693, 1995; Sidney et al., Immunol. Today 17:261-266, 1996; Sette and Sidney, Curr. Opin. in Immunol. 10:478-482, 1998). Secondary anchor residues that characterize the A2.1 motif have additionally been defined as disclosed herein. These are disclosed in Table II. Peptide binding to HLA-A2.1 molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.


Representative peptide epitopes that comprise an A2.1 motif are set forth on the attached Table VII. The A2.1 motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.


IV.D.12 HLA-A3 Motif

The allele-specific HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, Y, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope. Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.


Representative peptide epitopes that comprise the A3 motif are set forth on the attached Table XVI. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX.


IV.D.13. HLA-A11 Motif

The allele-specific HLA-A11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2, and K, R, Y, or H as a primary anchor residue at the C-terminal position of the epitope. Peptide binding to HLA-A 11 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.


Representative peptide epitopes that comprise the A11 motif are set forth on the attached Table XVII; peptide epitopes comprising the A3 allele-specific motif are also present in this Table because of the extensive overlap between the A3 and A11 motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX.


IV.D.14. HLA-A24 Motif

The allele-specific HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2, and F, L, I, or W as a primary anchor residue at the C-terminal position of the epitope. Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the motif.


Representative peptide epitopes that comprise the A24 motif are set forth on the attached Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif-bearing peptide epitopes.


Motifs Indicative of Class II HTL Inducing Peptide Epitopes

The primary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs delineated below are summarized in Table III.


IV.D.15. HLA DR-14-7 Supermotif

Motifs have also been identified for peptides that bind to three common HLA class II allele-specific HLA molecules: HLA DRB1*0401, DRB1*0101, and DRB1*0701. Collectively, the common residues from these motifs delineate the HLA DR-1-4-7 supermotif. Peptides that bind to these DR molecules carry a supermotif characterized by a large aromatic or hydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residue in position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as a primary anchor residue in position 6 of the epitope. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified. These are set forth in Table III. Peptide binding to HLA-DR4, DR1, and/or DR7 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.


Conserved peptide epitopes (i.e. 75% conservancy in the 20 HBV strains used for the analysis), corresponding to a nine residue core comprising the DR-1-4-7 supermotif (wherein position 1 of the motif is at position I of the nine residue core) are set forth in Table XIXa (see, e.g., Madden, Annu. Rev. Immunol. 13:587-622, 1995). Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core, are also shown in section “a” of the Table. Cross-reactive binding data for the exemplary 15-residue supermotif-bearing peptides denoted by a peptide number are shown in Table XIXb.


IV.D.16. HLA DR3 Motifs

Two alternative motifs (i.e., submotifs) characterize peptide epitopes that bind to HLA-DR3 molecules. In the first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope.


The alternative DR3 submotif provides for lack of the large, hydrophobic residue at anchor position 1, and/or lack of the negatively charged or amide-like anchor residue at position 4, by the presence of a positive charge at position 6 towards the carboxyl terminus of the epitope. Thus, for the alternative allele-specific DR3 motif (submotif DR3B): L, I, V, M, F, Y, A, or Y is present at anchor position 1; D, N, Q, E, S, or T is present at anchor position 4; and K, R, or H is present at anchor position 6. Peptide binding to HLA-DR3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.


Conserved peptide epitopes (i.e., sequences that are 75% conservaned in the 20 HBV strains used for the analysis), corresponding to a nine residue core comprising the DR3A submotif (wherein position 1 of the motif is at position I of the nine residue core) set forth in Table XXa. Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a conserved nine residue core, are also shown in section “a” of the Table. Table XXb shows binding data of the exemplary DR3 submotif A-bearing peptides denoted by a peptide number.


Conserved peptide epitopes (i.e., 75% conservancy in the 20 HBV strains used for the analysis), corresponding to a nine residue core comprising the DR3B submotif and respective exemplary 15-mer peptides comprising the DR3 submotif-B epitope are set forth in Table XXc. Table XXd shows binding data of the exemplary DR3 submotif B-bearing peptides denoted by a peptide number.


Each of the HLA class I or class II peptide epitopes set out in the Tables herein are deemed singly to be an inventive aspect of this application. Further, it is also an inventive aspect of this application that each peptide epitope may be used in combination with any other peptide epitope.


IV.E. Enhancing Population Coverage of 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 HLA alleles which, when considered in total, are present in most of the population. Table XXI lists the overall frequencies of the HLA class I supertypes in various ethnicities (Table XXIa) and the combined population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXIb). The A2-, A3-, and B7 supertypes are each present on the average of over 40% in each of these five major ethnic groups. Coverage in excess of 80% is achieved with a combination of these supermotifs. These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides. Although the population coverage reached with these three main peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearing peptides.


The B44-, A1-, and A24-supertypes are present, on average, in a range from 25% to 40% of these major ethnic populations (Table XXIa). While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency>25% in at least one major ethnic group (Table XXIa). Table XXIB summarizes the estimated combined prevalence in five major ethnic groups of HLA supertypes that have been identified. The incremental coverage obtained by the inclusion of A1, -A24-, and B44-supertypes to the A2, A3, and B7 coverage, or all of the supertypes described herein is shown. By including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups.


The data presented herein, together with the previous definition of the A2-, A3-, and B7-supertypes, indicates that all antigens, with the possible exception of A29, B8, and B46, can be classified into a total of nine HLA supertypes. Focusing on the six most common supertypes affords population coverage greater than 98% for all major ethnic populations.


IV.F. IMMUNE RESPONSE STIMULATING PEPTIDE ANALOGS

Although peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always complete and in such cases procedures to further increase cross-reactivity of peptides can be useful; such procedures can also be used to modify other properties of the peptides. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed. More specifically, peptides which exhibit the broadest cross-reactivity patterns, (both amongst the known T cell epitopes, as well as the more extended set of peptides that contain the appropriate supermotifs), can be produced in accordance with the teachings herein.


The strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules. The motifs or supermotifs are defined by having primary anchors, though secondary anchors can also be modified. Analog peptides can be created by substituting amino acids residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif. Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.


For a number of the motifs or supermotifs in accordance with the invention, residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind to the respective motif or supermotif (Tables II and III). Accordingly, removal of residues that are detrimental to binding can be performed in accordance with the present invention. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of analyzed peptides, the incidence of cross-reactivity increases from 22% to 37% (see, e.g., Sidney, J. et al., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide). An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, residues associated with high affinity binding to multiple alleles within a superfamily are inserted.


To ensure that an analog peptide, when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated. It will be desirable to use as antigen presenting cells, cells that have been either infected, or transfected with the appropriate genes, or, in the cae of class II epitopes only, cells that have been pusled with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.


Another embodiment of the invention to ensure adequate numbers of cross-reactive cellular binders is to create analogs of weak binding peptides. Class I peptides exhibiting binding affinities of 500-50000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype. The analog peptides can then be tested for crossbinding activity.


Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in a liquid environment. This substitution may occur at any position of the peptide epitope. For example, a cysteine (C) can be substituted out in favor of α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (Review: A. Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999). Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide epitope, i.e. at either anchor or non-anchor positions.


In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few immunodominant determinants (Zinkemagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been recognized that immunodominance (Benacerraf, et al., Science 175:273-279, 1972) could be explained by either the ability of a given epitope to selectively bind a particular HLA protein (determinant selection theory) (Vitiello, et al., J. Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or being selectively recognized by the existing TCR (T cell receptor) specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OF SELFNONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310, 1982). It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict immunogenicity, which of the many potential determinants will be presented as immunodominant (Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).


The concept of dominance and subdominance is relevant to immunotherapy of both infectious diseases and cancer. For example, in the course of chronic viral disease, recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol. 7:524-531, (1995)). In the case of cancer and tumor antigens, CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these, times.


In particular, it has been noted that a significant number of epitopes derived from known non-viral tumor associated antigens (TAA) bind HLA class I with intermediate affinity (IC50 in the 50-500 nM range). For example, it has been found that 8 of 15 known TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nM range. (These data are in contrast with estimates that 90% of known viral antigens that were recognized as peptides bound HLA with IC50 of 50 nM or less, while only approximately 10% bound in the 50-500 nM range (Sette, et al., J. Immunol., 153:558-5592 (1994)). In the cancer setting this phenomenon is probably due to elimination, or functional inhibition of the CTL recognizing several of the highest binding peptides, presumably because of T cell tolerization events.


Without intending to be bound by theory, it is believed that because T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow extant T cells to be recruited, which will then lead to a therapeutic response. However, the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide. Thus, a need exists to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide-based vaccines and therapeutic agents.


Representative analog peptides are set forth in Table XXII. The Table indicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate. The information in the “Fixed Nomenclature” column indicates the residues substituted at the indicated position numbers for the respective analog.


IV.G. COMPUTER SCREENING OF PROTEIN SEQUENCES FROM DISEASE-RELATED ANTIGENS FOR SUPERMOTIF OR MOTIF CONTAINING PEPTIDES

Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well. Generally, the identified sequences will be from a pathogenic organism or a tumor-associated peptide. For example, the target molecules considered herein include all of the HBV proteins (e.g. surface, core, polymerase, and X).


In cases where the sequence of multiple variants of the same target protein are available, peptides may also be selected on the basis of their conservancy. A presently preferred criterion for conservancy defines that the entire sequence of a peptide be totally conserved in 75% of the sequences evaluated for a specific protein; this definition of conservancy has been employed herein.


It is important that the selection criteria utilized for prediction of peptide binding are as accurate as possible, to correlate most efficiently with actual binding. Prediction of peptides that bind, for example, to HLA-A*0201, on the basis of the presence of the appropriate primary anchors, is positive at about a 30% rate (Ruppert, J. et al. Cell 74:929, 1993). However, by analyzing an extensive peptide-HLA binding database, the present inventors have developed a number of allele specific polynomial algorithms that dramatically increase the predictive value over identification on the basis of the presence of primary anchor residues alone. These algorithms take into account not only the presence or absence of the correct primary anchors, but also consider the positive or deleterious presence of secondary anchor residues (to account for the impact of different amino acids at different positions). The algorithms are essentially based on the premise that the overall affinity (or AG) of peptide-HLA interactions can be approximated as a linear polynomial function of the type:





ΔG=a1i×a2i×a3i . . . ×ani


where aij is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. An important assumption of this method is that the effects at each position are essentially independent of each other. This assumption is justified by studies that demonstrated that peptides are bound to HLA molecules and recognized by T cells in essentially an extended conformation. Derivation of specific algorithm coefficients has been described in Gulukota et al. (Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997).


Additional methods to identify preferred peptide sequences, which also make use of specific motifs, include the use of neural networks and molecular modeling programs (Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997; Milik et al., Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995; Buus, S. Curr. Opin. Immunol. 11:209-213, 1999; Brusic, V. et al., Bioinformatics 14:121-130, 1998).


For example, it has been shown that in sets of A*0201 motif peptides, 69% of the peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, will bind A*0201 with an IC50 less than 500 nM (Ruppert, J. et al. Cell 74:929, 1993). These algorithms are also flexible in that cut-off scores may be adjusted to select sets of peptides with greater or lower predicted binding properties, as desired.


In utilizing computer screening to identify peptide epitopes, all protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS’ program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs. As appreciated by one of ordinary skill in the art a large array of software and hardware options are available which can be employed to implement the motifs of the invention relative to known or unknown peptide sequences. The identified peptides will then be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles.


In accordance with the procedures described above, HBV peptides and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX; Table XXII).


IV.H. PREPARATION OF PEPTIDE EPITOPES

Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology, or from natural sources such as native tumors or pathogenic organisms. Peptide epitopes may be synthesized individually or as polyepitopic peptides. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.


The peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. The peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.


Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. When possible, it may be desirable to optimize HLA class I binding peptides of the invention to a length of about 8 to about 13 amino acid residues, preferably 9 to 10. HLA class II binding peptides may be optimized to a length of about 6 to about 25 amino acids in length, preferably to between about 13 and about 20 residues. Preferably, the peptides are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules. Moreover, the identification and preparation of peptides of other lengths can be carried out using the techniques described herein (e.g., the disclosures regarding primary and secondary anchor positions). However, it is also preferred to identify a larger region of a native peptide that encompasses one and preferably two or more epitopes in accordance with the invention. This sequence is selected on the basis that it contains the greatest number of epitopes per amino acid length. It is to be appreciated that epitopes can be present in a frame-shifted manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; each epitope can be exposed and bound by an HLA molecule upon administration of a plurality of such peptides. This larger, preferably multi-epitopic, peptide can then be generated synthetically, recombinantly, or via cleavage from the native source.


The peptides of the invention can be prepared in a wide variety of ways. For the preferred 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 & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co. (1984). Further, individual peptides may be joined using chemical ligation to produce larger peptides.


Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.


As the nucleotide coding sequence for peptides of the preferred lengths 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 and desired nucleic acid base(s) for those that encode 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. Of course, yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.


IV.I. ASSAYS TO DETECT T-CELL RESPONSES

Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response. The preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins in assays using, for example, purified HLA class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules (which lack peptide in their receptor) by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease. Corresponding assays are used for evaluation of HLA class II binding peptides.


Conventional assays utilized to detect CTL responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.


Peripheral blood lymphocytes may be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with peptide and the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the HBV antigen from which the peptide sequence was derived.


More recently, a method has also been devised which allows direct quantification of antigen-specific T cells by staining with Fluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Other relatively recent technical developments include staining for intracellular lymphokines, and interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177, 1998).


HTL activation may also be assessed using such techniques as T cell proliferation and secretion of lymphokines, e.g. IL-2.


Alternatively, immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes. Several transgenic mouse models including mice with human A2.1, A11, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary. Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes. CTL responses may be analyzed using cytotoxicity assays described above. Similarly, HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.


Immunogenic peptide epitopes are set out in Table XXIII.


IV.J. USE OF PEPTIDE EPITOPES AS DIAGNOSTIC AGENTS AND FOR EVALUATING IMMUNE RESPONSES

HLA class I and class II binding peptides as described herein can be used, in one embodiment of the invention, as reagents to evaluate an immune response. The immune response to be evaluated may be induced by using as an immunogen any agent that would potentially result in the production of antigen-specific CTLs or HTLs to the peptide epitope(s) to be employed as the reagent. The peptide reagent is not used as the immunogen.


For example, a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg et al. Science 279:2103-2106, 1998; and Altman et al. Science 174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells. A tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an allele-specific HLA molecules, or supertype molecules, is refolded in the presence of the corresponding HLA heavy chain and β2-microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes.


Peptides of the invention may also be used as reagents to evaluate immune recall responses. (see, e.g., Bertoni et al. J. Clin. Invest. 100:503-513, 1997 and Penna et al. J. Exp. Med. 174:1565-1570, 1991.) For example, patient PBC samples from individuals with acute hepatitis B or who have recently recovered from acute hepatitis B may be analyzed for the presence of HBV antigen-specific CTLs using HBV-specific peptides. A blood sample containing mononuclear cells may be evaluated by cultivating the PBCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population may be analyzed for cytotoxic activity.


The peptides may also be used as reagents to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above. A patient is HLA typed, and appropriate peptide reagents that recognize allele-specific molecules present in that patient may be selected for the analysis. The immunogenicity of the vaccine will be indicated by the presence of HBV epitope-specific CTLs in the PBMC sample.


The peptides of the invention may 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 and Lane, Cold Spring Harbor Laboratory Press, 1989). Such antibodies may be useful as reagents to diagnose HBV infection.


IV.K. VACCINE COMPOSITIONS

Vaccines that contain an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention. Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herein referred to as “vaccine” compositions. Such vaccine compositions can include, for example, lipopeptides (Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptides compositions encapsulated in poly(DL-lactide-co-glycolide) (PLG) microspheres (see, e.g., Eldridge, et al. Molec. Immunol. 28:287-294, 1991: Alonso et al. Vaccine 12:299-306, 1994; Jones et al. Vaccine 13:675-681, 1995), peptide compositions encapsulated in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al. Nature 344:873-875, 1990; Hu et al. Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.


Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptide(s) that can 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 CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targetted for an immune response.


Furthermore, useful carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS).


As disclosed in greater detail herein, upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit when the antigen was tumor-associated.


In some instances it may be desirable to combine the class I peptide vaccines of the invention with vaccines which induce or facilitate neutralizing antibody responses to the target antigen of interest, particularly to viral envelope antigens. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRE™ (Epimmune, San Diego, Calif.) molecule (described in the related U.S. Ser. No. 08/485,218, which is a CIP of U.S. Ser. No. 08/305,871, now U.S. Pat. No. 5,736,142, which is a CIP of abandoned application U.S. Ser. No. 08/121,101.) Furthermore, any of these embodiments can be administered as a nucleic acid mediated modality.


For therapeutic or immunization purposes, the peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors and methods useful in 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. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.


Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular pathogen (infectious agent or tumor antigen) are induced by incubating in tissue culture the patient's CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 14 weeks), in which the precursor cells are activated, mature and expand into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells. Alternatively, dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes will be discussed in greater detail in a following section.


DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) delivery.


Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. It is preferred that each of the following principles are balanced in order to make the selection.


1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV. In other words, it has been observed that in patients who spontaneously clear HBV, that they had generated an immune response to at least 3 epitopes on at least one HBV antigen. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen (see e.g., Rosenberg et al. Science 278:1447-1450).


2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.


3.) Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess population coverage.


4.) When selecting epitopes from cancer-related antigens it is often preferred to select analogs. When selecting epitopes for infectious disease-related antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines (but for cancer-related vaccines as well), are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.


When providing nested epitopes, it is preferable to provide a sequence that has the greatest number of epitopes per provided sequence. A limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, it is important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.


5.) When creating a minigene, as disclosed in greater detail in the following section, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Thus, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that 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.


IV.K.1. Minigene Vaccines

A growing body of experimental evidence demonstrates that a number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention. The use of multi-epitope minigenes is described below and in, e.g. An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201 and A11-restricted epitopes derived from the polymerase, envelope, and core proteins of HBV and HIV, the PADRE™ universal helper T cell (HTL) epitope, and an endoplasmic reticulum-translocating signal sequence was engineered. Immunization of HLA transgenic mice with this plasmid construct result in strong CTL induction responses against the nine epitopes tested, similar to those observed with a lipopeptide of known immunogenicity in humans, and significantly greater than immunization in oil-based adjuvants. Moreover, the immunogenicity of DNA-encoded epitopes in vivo correlated with the in vitro responses of specific CTL lines-against target cells transfected with the DNA plasmid. Thus, these data show that the minigene served to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.


For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that could be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.


The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.


Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.


Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.


Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.


In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.


In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules. Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving CTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases).


Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.


Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.


Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes.


In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.


Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.


IV.K.2. Combinations of CTL Peptides with Helper Peptides


The peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half life, or to enhance immunogenicity.


For instance, the ability of the peptides 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. The use of T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in U.S. Ser. No. 08/820,360, U.S. Ser. No. 08/197,484, U.S. Ser. No. 08/464,234, U.S. Ser. No. 08/464,496, U.S. Ser. No. 08/464,031, abandoned U.S. Ser. No. 08/464,433, and U.S. Ser. No. 08/461,603.


Particularly preferred 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. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.


The CTL peptide epitope may be linked to the HTL 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 CTL epitope or the HTL peptide may be acylated. The HTL peptide epitopes used in the invention can be modified in the same manner as CTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, and malarial circumsporozoite 382-398 and 378-389.


In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences. Examples of amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE), Plasmodium falciparum CS protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.


Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed on the basis of their binding activity to most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVWANTLKAAa, where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type.


HTL peptide epitopes can also be modified to alter their biological properties. For example, peptides comprising HTL epitopes can contain D-amino acids to increase their resistance to proteases and thus extend their serum half-life. Also, the epitope peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity. Specifically, the T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.


In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.


As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. (See, Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.


In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides. However, it is to be noted that modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH2 acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.


IV.L. ADMINISTRATION OF VACCINES FOR THERAPEUTIC OR PROPHYLACTIC PURPOSES

The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent HBV infection. Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk for HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the virus antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.


The vaccine compositions of the invention may also be used purely as prophylactic agents. Vaccine compositions containing the peptide epitopes of the invention are administered to a patient susceptible to, or otherwise at risk for, HBV infection to elicit an immune response against HBV antigens and thus enhance the patient's own immune response capabilities following exposure to HBV. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring the specific activity of CTL and/or HTL-obtained from a sample of the patient's blood.


As noted above, peptides comprising CTL or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide. The manner in which the peptide is contacted with the CTL or HTL is not critical to the invention. For instance, the peptide can be contacted with the CTL or HTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient, or other vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.


For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already infected with HBV. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. 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.


For therapeutic use, administration should generally begin at the first diagnosis of HBV infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.


Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. 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 susceptible individuals are identified prior to or during infection, the composition can be targeted to them, thus minimizing the need for administration to a larger population.


The peptide or other compositions used for the treatment or prophylaxis of HBV infection can be used, e.g., in persons who have not manifested symptoms of disease but who act as a disease vector. In this context, it is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.


The dosage for an initial immunization (i.e., therapeutic or prophylactic administration) generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and/or HTL obtained from the patient's blood. The peptides and compositions of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.


Thus, for treatment of chronic infection, a representative dose is in the range disclosed above, namely where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg, preferably from about 500 μg to about 50,000 μg per 70 kilogram patient. Initial doses followed by boosting doses at established intervals, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively immunize an individual. In the case of chronic infection, administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially abated and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.


The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.


The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.


A human unit dose form of the peptide composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publising Co., Easton, Pa., 1985)


The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or to target selectively to infected cells, as well as to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.


For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.


For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.


For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.


IV.M. KITS

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 instruction for administration. Lymphokines such as IL-2 or IL-12 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.


The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments in accordance with the invention.


V. EXAMPLES
Example 1
HLA Class I and Class II Binding Assays

The following example of peptide binding to HLA molecules demonstrates quantification of binding affinities of HLA class I and class II peptides. Binding assays can be performed with peptides that are either motif-bearing or not motif-bearing.


Epstein-Barr virus (EBV)-transformed homozygous cell lines, fibroblasts, CIR, or 721.22 transfectants were used as sources of HLA class I molecules. These cells were maintained in vitro by culture in RPMI 1640 medium supplemented with 2 mM L-glutamine (GIBCO, Grand Island, N.Y.), 50 μM 2-ME, 100 μg/ml of streptomycin, 100 U/ml of penicillin (Irvine Scientific) and 10% heat-inactivated FCS (Irvine Scientific, Santa Ana, Calif.). Cells were grown in 225-cm2 tissue culture flasks or, for large-scale cultures, in roller bottle apparatuses. The specific cell lines routinely used for purification of MHC class I and class II molecules are listed in Table XXIV.


Cell lysates were prepared and HLA molecules purified in accordance with disclosed protocols (Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., gJ. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells were lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. Lysates were cleared of debris and nuclei by centrifugation at 15,000×g for 30 min.


HLA molecules were purified from lysates by affinity chromatography. Lysates prepared as above were passed twice through two pre-columns of inactivated Sepharose CL4-B and protein A-Sepharose. Next, the lysate was passed over a column of Sepharose CL-4B beads coupled to an appropriate antibody. The antibodies used for the extraction of HLA from cell lysates are listed in Table XXV. The anti-HLA column was then washed with 10-column volumes of 10 mM Tris-HCL, pH 8.0, in 1% NP-40, PBS, 2-column volumes of PBS, and 2-column volumes of PBS containing 0.4% n-octylglucoside. Finally, MHC molecules were eluted with 50 mM diethylamine in 0.15M NaCl containing 0.4% n-octylglucoside, pH 11.5. A 1/25 volume of 2.0M Tris, pH 6.8, was added to the eluate to reduce the pH to ˜8.0. Eluates were then be concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, Mass.). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, Ill.) and confirmed by SDS-PAGE.


A detailed description of the protocol utilized to measure the binding of peptides to Class I and Class II MHC has been published (Sette et al., Mol. Immunol. 31:813, 1994; Sidney et al., in Current Protocols in Immunology, Margulies, Ed., John Wiley & Sons, New York, Section 18.3, 1998). Briefly, purified MHC molecules (5 to 500 nM) were incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides for 48 h in PBS containing 0.05% Nonidet P-40 (NP40) (or 20% w/v digitonin for H-2 IA assays) in the presence of a protease inhibitor cocktail. The final concentrations of protease inhibitors (each from CalBioChem, La Jolla, Calif.) were 1 mM PMSF, 1.3 nM 1.10 phenanthroline, 73 μM pepstatin A, 8 mM EDTA, 6 mM N-ethylmaleimide (for Class II assays), and 200 μM N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). All assays were performed at pH 7.0 with the exception of DRB1*0301, which was performed at pH 4.5, and DRB1*1601 (DR2w21·1) and DRB4*0101 (DRw53), which were performed at pH 5.0. pH was adjusted as described elsewhere (see Sidney et al., in Current Protocols in Immunology, Margulies, Ed., John Wiley & Sons, New York, Section 18.3, 1998).


Following incubation, MHC-peptide complexes were separated from free peptide by gel filtration on 7.8 mm×15 cm TSK200 columns (TosoHaas 16215, Montgomeryville, Pa.), eluted at 1.2 mls/min with PBS pH 6.5 containing 0.5% NP40 and 0.1% NaN3. Because the large size of the radiolabeled peptide used for the DRB1*1501 (DR2w2·1) assay makes separation of bound from unbound peaks more difficult under these conditions, all DRB1*1501 (DR2w2·1) assays were performed using a 7.8 mm×30 cm TSK2000 column eluted at 0.6 mls/min. The eluate from the TSK columns was passed through a Beckman 170 radioisotope detector, and radioactivity was plotted and integrated using a Hewlett-Packard 3396A integrator, and the fraction of peptide bound was determined.


Radiolabeled peptides were iodinated using the chloramine-T method. The specific radiolabeled probe peptide utilized in each assay, and its assay specific IC50 nM, is summarized in Table XXIV. Typically, in preliminary experiments, each MHC preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these HLA concentrations.


Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proven to be the most accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.


Because the antibody used for HLA-DR puriflcafion (LB3.1) is α-chain specific, β1 molecules are not separated from β3 (and/or β4 and β5) molecules. The β1 specificity of the binding assay is obvious in the cases of DRB1*0101 (DR1), DRB1*0802 (DR8w2), and DRB1*0803 (DR8w3), where no β3 is expressed. It has also been demonstrated for DRB1*0801 (DR3) and DRB3*0101 (DR52a), DRB1*0401 (DR4w4), DRB1*0404 (DR4w14), DRB1*0405 (DR4w15), DRB1*1101 (DR5), DRB1*1201 (DR5w12), DRB1*1302 (DR6w19) and DRB1*0701 (DR7). The problem of β chain specificity for DRB1*1501 (DR2w2·1), DRB5*0101 (DR2w2·2), DRB1*1601 (DR2w21·1), DRB5*0201 (DR2w21·3), and DRB4*0101 (DRw53) assays is circumvented by the use of fibroblasts. Development and validation of assays with regard to DRβ molecule specificity have been described previously (see, e.g., Southwood et al., J. Immunol. 160:3363-3373, 1998).


Binding assays as outlined above may be used to analyze supermotif and/or motif-bearing epitopes as, for example, described in Example 2.


Example 2
Identification of Conserved HLA Supermotif CTL Candidate Epitopes

Vaccine compositions of the invention may include multiple epitopes that comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification of supermotif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage was performed using the strategy described below. Epitopes were then selected to bear an HLA-A2, -A3, or -B7 supermotif or an HLA-A1 or -A24 motif.


Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes


Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs were performed as follows. All translated HBV isolate sequences were analyzed using a text string search software program, e.g., MotifSearch 1.4 (D. Brown, San Diego) to identify potential peptide sequences containing appropriate HLA binding motifs; alternative programs are readily produced in accordance with information in the art in view of the motif/supermotif disclosure herein. Furthermore, such calculations can be made mentally. Identified A2-, A3-, and DR-supermotif sequences were scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms take into account both extended and refined motifs (that is, to account for the impact of different amino acids at different positions), and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:





“ΔG”=a1i×a2i×a3i . . . ani


where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide. This assumption is justified by studies from our laboratories that demonstrated that peptides are bound to MHC and recognized by T cells in essentially an extended conformation (data omitted herein).


The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J; Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.


Selection of HLA-A2 Supertype Cross-Reactive Peptides

Complete sequences from 20 HBV isolates were aligned, then scanned, utilizing a customized computer program, to identify conserved 9- and 10-mer sequences containing the HLA-A2-supertype main anchor specificity.


A total of 150 conserved and motif-positive sequences were identified. These peptides were then evaluated for the presence of A*0201 preferred secondary anchor residues using an A*0201-specific polynomial algorithm. A total of 85 conserved, motif-positive sequences were selected and synthesized.


These 85 conserved, motif-containing 9- and 10-mer peptides were then tested for their capacity to bind purified HLA-A*0201 molecules in vitro. Thirty-four peptides were found to be good A*0201 binders (IC50≦500 nM); 15 were high binders (IC50≦50 nM) and 19 were intermediate binders (IC50 of 50-500 nM) (Table XXVI).


In the course of independent analyses, 25 conserved, HBV-derived, 8 or 11-mer sequences with appropriate A2-supertype main anchors were also synthesized and tested for A*0201 binding. One peptide, HBV env 259 11-mer (peptide 1147.14), bound A*0201 with an IC50 of 500 nM, or less, and has been included in Table XXVI. Also shown in Table XXVI is an analog peptide, representing a single substitution of the HBV pol 538 9-mer peptide, which binds A*0201 with an IC50 of 5.1 nM (see below).


Thirty of the 36 A*0201 binders were subsequently tested for the capacity to bind to additional A2-supertype alleles (A*0202, A*0203, A*0206, and A*6802). As shown in Table XXVI, 15/30 (50%) peptides were found to be A2-supertype cross-reactive binders, binding at least 3 of the 5 A2-supertype alleles tested. These 15 peptides were selected for further analysis.


Selection of HLA-A3 Supermotif-Bearing Epitopes

The sequences from the same 20 isolates were also examined for the presence of conserved peptides with the HLA-A3-supermotif primary anchors. A total of 80 conserved 9- or 10-mer motif-containing sequences were identified. Further analysis using the A03 and A11 algorithms identified 40 sequences which scored high in either or both algorithms. Thirty-six of the corresponding peptides were synthesized and tested for binding to HLA-A*03 and HLA-A*11, the two most prevalent A3-supertype alleles. Twenty-three peptides were identified which bound A3 and/or A11 with affinities or IC50 values of ≦500 nM (Table XXVII).


In the course of an independent series of studies 30 HBV-derived 8-mer, and 24 11-mer sequences, conserved in 75% or more of the isolates, were selected and tested for A*03 and A*11 binding. Four 8-mers and 9 11-mers were found to bind either or both alleles (Table XXVII). Finally, four 9-mer, and one 10-mer, conserved HBV-derived peptides not selected using the search criteria outlined above, but which have been shown to bind A*03 and/or A*11, have been identified, and are included in Table XXVII. Two of these peptides contain non-canonical anchors (peptides 20.0131, and 20.0130), and the other 3 are algorithm negative (peptides 1142.05, 1099.03, and 1090.15).


Thirty-eight of the 41 pepfides binding A*03 and/or A*11 were subsequently tested for binding crossreactivity to the other common A3-supertype alleles (A*3101, A*3301, and A*6801). It was found that 17 of these peptides were A3-supertype cross-reactive, binding at least 3 of the 5 A3-supertype alleles tested (Table XXVII).


Selection of HLA-B 7 Supermotif Bearing Epitopes

When the same 20 isolates were also analyzed for the presence of conserved 9- or 10-mer peptides with the HLA-B7-supermotif, 46 sequences were identified. Thirty-four of the corresponding peptides were synthesized and tested for binding to HLA-B*0702, the most common B7-supertype allele. Nine peptides bound B*0702 with an IC50 value of ≦500 nM (Table XXVIII). These 9 peptides were then tested for binding to other common B7-supertype alleles (B*3501, B*51, B*5301, and B*5401). Five of the 9 B*0702 binders were capable of binding to 3 or more of the 5 B7-supertype alleles tested.


In separate studies investigating the secondary anchor requirements of B7-supertype alleles, all available peptides with the B7-supermotif were tested for binding to all B7 supertype alleles. As a result, all 34 peptides described above were also tested for binding to other B7-supertype alleles. These experiments identified an additional 10 peptides which bound at least one B7-supertype allele with an IC50 value≦500 nM, including 2 peptides which bound 3 or more alleles. These 10 peptides are also shown in Table XXVIII.


Because of the low numbers of conserved B7-supertype degenerate HBV-derived 9- and 10-mer peptides, compared to the A2- and A3-supertypes, the 20 isolates were again examined to identify conserved, motif-containing 8- and 11-mers. This re-scan identified 51 peptides. Thirty-one of these were synthesized and tested for binding to each of the 5 most common B7-supertype alleles. Nineteen 8- and 11-mer peptides bound with high or intermediate affinity to at least one B7-supertype allele (IC50≦500 nM) (Table XXVIII). Two peptides were degenerate binders, binding 3 of the 5 alleles tested.


In summary, a total of 9 HBV-derived peptides, conserved in 75% or more of the isolates analyzed, have been identified which are degenerate B7-supertype binders (Table XXV1H).


Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes have been incorporated into the present analysis. A1 is, on average, present in 12%, and A24 is present in approximately 29%, of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Combined, these alleles would be represented with an average frequency of 39% in these same populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95.4%; by comparison, coverage by combing the A2-, A3-, and B7-supertypes is 86.2%.


Systematic analyses of HBV for A1 and A24 binders have yet to be completed. However, in the course of independent studies, 15 conserved HBV-derived peptides have been identified that bind A*0101 with IC50 less than 500 nM (Table XXIX); 7 of these bind with IC50 less than 100 nM. In a similar context, 14 conserved A*2402 binding HBV-derived peptides have also been identified, 6 of which bind A*2402 with IC50 less than 100 nM (Table XXIX).


Example 3
Confirmation of Immunogenicity
Evaluation of A*0201 Immunogenicity

The immunogenicity analysis of the 15 HBV-derived HLA-A2 supertype cross-reactive peptides identified above is summarized in Table XXX. Peptides were screened for immunogenicity in at least one of three systems. Peptides were screened for the induction of primary antigen-specific CTL in vitro using human PBMC (Wentworth et al., Molec. Immunol. 32:603, 1995); this data is indicated as “primary CTL” in Table XXX.


The protocol for in vitro induction of primary antigen-specific CTL from human PBMC has been described by Wentworth et al (Wentworth et al., Molec. Immunol. 32:603, 1995). PBMC from normal donors which had been enriched for CD8+ T cells were incubated with peptide loaded antigen-presenting cells (SAC-I activated PBMCs) in the presence of IL-7. After seven days cultures were restimulated using irradiated autologous adherent cells pulsed with peptide and then tested for cytotoxic activity seven days later.


In addition, HLA transgenic mice were used to evaluate peptide immunogenicity; this data is indicated as “transgenic CTL” in Table XXX. Previous studies have shown that CTL induced in A*0201/Kb transgenic mice exhibit specificity similar to CTL induced in humans (Vitiello et al., J. Exp. Med. 173:1007, 1991; Wentworth et al., Eur. J. Immunol. 26:97, 1996).


CTL induction in transgenic mice following peptide immunization has been described by Vitiello et al. (Vitiello et al., J. Exp. Med. 173:1007, 1991) and Alexander et al. (Alexander et al., J. Immunol. 159:4753, 1997). Briefly, synthetic peptides (50 μg/mouse) and the helper epitope HBV core 128 (140 μg/mouse) were emulsified in incomplete Freund's adjuvant (IFA) and injected subcutaneously at the base of the tail. Eleven days after injection, splenocytes were incubated in the presence of peptide-loaded syngenic LPS blasts. After six days cultures were assayed for cytotoxic activity using peptide-pulsed targets.


Peptides were also tested for the ability to stimulate recall CTL responses in acutely infected HBV patients (Bertoni et al., J. Clin. Invest. 100:503, 1997; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; Nayersina et al., J. Immunol. 150:4659, 1993); these data are indicated as “patient CTL” in Table XXX. Patient immunogenicity data is particularly informative as it indicates that a peptide is recognized during the course of a natural infection. These data demonstrate that a peptide is processed and presented in human cells that would represent the targets for CTL. Moreover, this data is especially relevant for vaccine design as the induction of CTL responses in patients has been correlated to the resolution of infection.


For the evaluation of recall CTL responses, screening was carried out as described by Bertoni et al. (Bertoni et al., J. Clin. Invest. 100:503, 1997). Briefly, PBMC from patients acutely infected with HBV were cultured in the presence of 10 μg/ml of synthetic peptide. After seven days, the cultures were restimulated with peptide. The cultures were assayed for cytotoxic activity on day 14 using target cells pulsed with peptide.


Of the 15 A2 supertype binding peptides, 11 were found to be immunogenic in at least one of the systems utilized. Five of the 11 peptides had previously been identified in the patients with acute HBV (Bertoni et al., J. Clin. Invest. 100:503, 1997). Five additional degenerate peptides (1069.06, 1090.77, 1147.14, 927.42 and 927.46) induced CTL responses in HLA-A*0201 transgenic mice. The 11 immunogenic supertype cross-reactive peptides are encoded by three HBV antigens; core, envelope and polymerase.


This set of 11 immunogenic A2-supermotif-bearing epitopes includes one analog peptide, 1090.77. The wild type peptide, 1090.14, from which this analog is derived is A2-supertype non-cross-reactive, but has been shown to be recognized in recall CTL responses from acute HBV patients, and to be immunogenic in HLA-A*0201 transgenic mice as well as primary human cultures (Table XXX). Further studies addressing the cross recognition of the wild type peptide 1090.14 and the 1090.77 analog are described in detail below.


In the course of independent analyses, 14 of the non-cross-reactive peptides shown in Table XXXb, including 1090.14, were found to be immunogenic in at least one system. Five peptides of these peptides were recognized in patients; 4 peptides induced CTL in transgenic mice.


In conclusion, 11 A2-supertype cross-reactive peptides have been identified that are capable of exhibiting immunogenicity in at least one of the three systems examined.


Evaluation of A*03/A11 immunogenicity


Seven of the 17 A3-supertype cross-reactive peptides have been evaluated for immunogenicity (Table XXXI). As described in the previous section, A3-supermotif-bearing peptides were screened using primary cultures, patient responses, or HLA-A11 transgenic mice (Alexander et al., J. Immunol. 159:4753, 1997). With the exception of peptide 1.0219, all of the conserved cross-reactive peptides listed in Table insert table XXXI were found to be immunogenic.


Additionally, a poorly conserved peptide (1150.51; 40% conserved) which exhibits cross-reactive supertype binding was found to be immunogenic in transgenic mice, and has been included in Table XXXI. Two other conserved, but non-cross-reactive, peptides have also been shown to be recognized in acutely infected HBV patients (Bertoni et al., J. Clin. Invest. 100:503, 1997). These epitiopes are shown in Table XXXI.


It is notable that for 7 of the 8 conserved immunogenic HBV-derived A3-supermotif-bearing epitopes, including all 6 of the cross-reactive peptides, positive data was obtained in patients. These epitopes are predominantly derived from the polymerase protein sequence, with only one epitope being derived from the core protein sequence. While a number of cross-reactive peptides have been identified in the X antigen (Table XXXI), to date these peptides have not been screened for immunogenicity.


In summary, 7 A3-supermotif-bearing, cross-reactive peptides have been identified that are recognized by CTL in acutely infected patients, or induce CTL in HLA-transgenic mice.


Evaluation of B7 Immunogenicity

The immunogenicity studies involving the HBV-derived HLA-B7-supermotif-bearing, cross-reactive peptides is summarized in Table XXXII. HLA-B7 peptides were screened exclusively in human systems measuring responses in either primary cultures or acutely infected HBV patients. Of the 7 degenerate peptides screened, 4 were shown to be immunogenic. One non-crossreactive peptide (XRN<3), 1147.04, was also shown to be recognized in acutely infected HBV patients (Bertoni et al., J. Clin. Invest. 100:503, 1997; see Table XXXII).


In summary, 5 conserved HBV-derived B7-supermotif-bearing epitopes that are recognized in acutely infected HBV patients have been identified. These epitopes afford coverage of 4 different HBV antigens (core, envelope, polymerase and X).


Example 4
Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Peptides by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in preparing highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged, or “fixed”, to confer upon a peptide certain characteristics, e.g., greater cross-reactivity within the group of HLA molecules that make-up the supertype, and/or greater binding affinity for some or all of those HLA molecules Examples of analog peptides that exhibit modulated binding affinity are provided.


Analoging at Primary Anchor Residues

It has been shown that class I peptide ligands can be modified, or “fixed” to increase their binding affinity and/or degeneracy (Sidney et al., J. Immunol. 157:3480, 1996). These fixed peptides may also demonstrate increased immunogenicity and crossreactive recognition by T cells specific for the wild type epitope (Parkhurst et al., J. Immunol. 157:2539, 1996; Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995). Specifically, the main anchors of A2 supertype peptides may be “fixed”, or analoged, to L or V (or M, if natural) at position 2, and V at the C-terminus. As indicated in Table XXVI, 9 of the 14 A2-supertype cross-reactive binding peptides are “fixable” by these criteria, as are 16 of the 21 non-cross-reactive binders. Ideal candidates for fixing would be peptides which bind at least 3 A2-supertype allele-specific molecules with IC50≦5000 nM.


An example of the efficacy of this strategy to generate more broadly cross-reactive epitopes is provided by the case of peptide 1090.14 (Table XXVI). Previously, this peptide was shown to be highly immunogenic in each of the systems examined. However, it only exhibits binding to a single A2-supertype allele-specific molecule, A*0201. The non-crossreactive binding capacity of this epitope limits the population coverage and consequently the value of including this peptide in a candidate vaccine. In an effort to increase binding affinity and cross-reactivity the C-terminus of peptide 1090.14 was altered from ‘alanine’ to the A2-supermotif preferred residue ‘valine’. This change resulted in a dramatic (40-fold) increase in binding capacity for A*0201 (from 200 nM to 5.1 nM), but also produced a peptide capable of binding 3 other A2-supertype allele-specific molecules. (see peptide 1090.77, Table XXVI).


Studies with HLA-A*0201 transgenic mice have shown that the CTL response from mice immunized with the 1090.77 peptide recognize target cells loaded with either the naturally occurring peptide 1090.14 or the valine-substituted analog (i.e., 1090.77). In fact, the lysis effected by 1090.77 induced CTL was indistinguishable regardless whether the analog or the wild-type sequence was used to load the target cells (B. Livingston, unpublished data).


The relevance of these observations for the design of vaccine constructs is indicated by studies in which chronic HBV patients were treated with the potent viral replication inhibitor, lamivudine. Extended therapy with lamivudine resulted in the selection of drug-resistant strains of HBV that have a substitution of valine for methione at position 2 in the 1090.14 epitope, suggesting that epitope-based vaccines used in combination with lamivudine may need to have the ability to induce CTL responses that recognize both wild type and mutant sequences.


To demonstrate that cross-recognition is possible between the native peptide (1090.14), the analog peptide, and the lamivudine induced mutant M2 peptide, CTL were generated using the 1090.77 analog peptide. These CTL cultures were then stimulated with either the wild type peptide (1090.14), or the lamivudine induced mutant M2 peptide. The ability of these CTL to then lyse target cells loaded with either the wild type, or the lamivudine induced mutant peptide was then assayed. Target cells presenting either peptide were similarly lysed by either CTL culture (Table XXVI).


These studies demonstrate how analoging a peptide can result in dramatically increased HLA-A2 supertype degeneracy while still allowing cross-recognition between wildtype and mutant epitopes. More specifically, these results indicate that a vaccine utilizing the analog peptide 1090.77 should stimulate a response that will recognize both wild-type and lamivudine-resistant strains of HBV.


Similarly, analogs of HLA-A3 supermotif-bearing epitopes may also be generated. For example, peptides could be analogued to possess a preferred V at position 2, and R or K at the C-terminus. Twelve of the A3-supertype degenerate peptides identified in Table XXVII are candidates for main anchor fixing, as are 19 of the 24 non-cross-reactive binders.


Analog peptides are initially tested for binding to A*03 and A*11, and those that demonstrate equivalent, or improved, binding capacity relative to the parent peptide would then be tested for A3-supertype cross-reactivity. Analogs demonstrating improved cross-reactivity are then further evaluated for immunogenicity, as necessary.


Typically, it is more difficult to identify B7 supermotif-bearing epitopes. As in the cases of A2- and A3-supertype epitopes, a peptide analoguing strategy can be utilized to generate additional B7 supermotif-bearing epitopes with increased cross-reactive binding. In general, B7 supermotif-bearing peptides should be fixed to possess P in position 2, and I at their C-terminus.


Analogs representing primary anchor single amino acid residues substituted with I residues at the C-terminus of two different B7-like peptides (HBV env 313 and HBV pol 541) were synthesized and tested for their B7-supertype binding capacity. It was found that the I substitution had an overall positive effect on binding affinity and/or cross-reactivity in both cases. In the case of HBV env 313 the I9 (I at C-terminal position 9) replacement was effective in increasing cross-reactivity from 4 to 5 alleles bound by virtue of an almost 400-fold increase B*5401 binding affinity. In the case of HBV pol 541, increased cross-reactivity was similarly achieved by a substantial increase in B*5401 binding. Also, significant gains in binding affinity for B*0702, B51, and B*5301 were observed with the HBV pol 541 I9 analog.


Analoging at Secondary Anchor Residues

Moreover, HLA superrnotifs are of value in engineering highly cross-reactive peptides by identifying particular residues at secondary anchor positions that are associated with such cross-reactive properties. Demonstrating this, the capacity of a second set of peptides representing discreet single amino acid substitutions at positions one and three of five different B7-supertype binding peptides were synthesized and tested for their B-7 supertype binding capacity. In 4/4 cases the effect of replacing the native residue at position 1 with the aromatic residue F (an “F1” substitution) resulted in an increase in cross-reactivity, compared to the parent peptide, and, in most instances, binding affinity was increased three-fold or better (Table XXVIII). More specifically, for HBV env 313, MAGE2 170, and HBV core 168 complete supertype cross-reactivity was achieved with the F1 substitution analogs. These gains were achieved by dramatically increasing B*5401 binding affinity. Also, gains in affinity were noted for other alleles in the cases of HBV core 168 (B*3501 and B*5301) and MAGE2 170 (B*3501, B51 and B*5301). Finally, in the case of MAGE3 196, the F1 replacement was effective in increasing cross-reactivity because of gains in B*0702 binding. An almost 70-fold increase in B51 binding capacity was also noted.


Two analogs were also made using the supermotif positive F substitution at position three (an “F3” substitution). In both instances increases in binding affinity and cross-reactivity were achieved. Specifically, in the case of HBV pol 541, the F3 substitution was effective in increasing cross-reactivity by virtue of its effect on B*5401 binding. In the case of MAGE3 196, complete supertype cross-reactivity was achieved by increasing B*0702 and B*3501 binding capacity. Also, in the case of MAGE3 196, it is notable that increases in binding capacity between 40- and 5000-fold were obtained for B*3501, B51, B*5301, and B*5401.


In conclusion, these data demonstrate that by the use of even single amino acid substitutions, it is possible to increase the binding affinity and/or cross-reactivity of peptide ligands for HLA supertype molecules.


Example 5
Identification of Conserved HBV-Derived Sequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif may also be identified as outlined below using methodology similar to that described in Examples 1-3.


Selection of HLA-DR-Supermotif-Bearing Epitopes

HLA-Class II molecules bind peptides typically between 12 and 20 residues in length. However, similar to HLA-Class I, the specificity and energy of interaction is usually contained within a short core region of about 9 residues. Most DR molecules share an overlapping specificity within this 9-mer core in which a hydrophobic residue in position 1 (P1) is the main anchor (O'Sullivan et al., J. Immunol. 147:2663, 1991; Southwood et al., J. Immunol. 160:3363, 1998). The presence of small or hydrophobic residues in position 6 (P6) is also important for most DR-peptide interactions. This overlapping P1-P6 specificity, within a 9-mer core region, has been defined as the DR-supermotif. Unlike Class I molecules, DR molecules are open at both ends of the binding groove, and can therefore accommodate longer peptides of varying length. Indeed, while most of the energy of peptide-DR interactions appears to be contributed by the core region, flanking residues appear to be important for high affinity interactions. Also, although not strictly necessary for MHC binding, flanking residues are clearly necessary in most instances for T cell recognition.


To identify HBV-derived DR cross-reactive HTL epitopes, the same 20 HBV polyproteins that were scanned for the identification of HLA Class I motif sequences were scanned for the presence of sequences with motifs for binding HLA-DR. Specifically, 15-mer sequences comprised of a DR-supermotif containing 9-mer core, and three residue N- and C-terminal flanking regions, were selected. It was also required that 100% of the 15-mer sequence be conserved in at least 85% (17/20) of the HBV strains scanned. Using these criteria, 36 non-redundant sequences were identified. Thirty-five of these peptides were subsequently synthesized.


Algorithms for predicting peptide binding to DR molecules have also been developed (Southwood et al., J. Immunol. 160:3363, 1998). These algorithms, specific for individual DR molecules, allow the scoring and ranking of 9-mer core regions. Using selection tables, it has been found that these algorithms efficiently select peptide sequences with a high probability of binding the appropriate DR molecule. Additionally, it has been found that running algorithms, specifically those for DR1, DR4w4, and DR7, sequentially can efficiently select DR cross-reactive peptides.


To see if these algorithms would identify additional peptides, the same HBV polyproteins used above were re-scanned for the presence of 15-mer peptides where 100% of the 9-mer core region was 385% (17/20 strains) conserved. Next, the 9-mer core region of each of these peptides was scored using the DR1, DR4w4, and DR7 algorithms. As a result, 8 additional sequences were identified and synthesized.


In summary, 44 15-mer peptides in which a 9-mer core region contained the DRsupermotif, or was selected using an algorithm predicting DR-binding sequences, were identified. Forty-three of these peptides were synthesized (Table XXXIII).


While performing the analyses of HBV-derived peptides described above, 9 peptides predicted on the basis of their DR1, DR4w4, and DR7 algorithm profiles to be DR-cross-reactive binding peptides, but which have 9-mer core regions that are only 80% conserved, were also identified. An additional peptide which contains a DR-supermotif core region that is 95% conserved, but is located only one residue removed from the N-terminus, was previously synthesized. These 10 peptides were also selected for further analysis, and are shown in Table XXXIII.


Finally, 2 peptides, CF-08 and 1186.25, which are redundant with a peptide selected above (27.0280), were considered for additional analysis. Peptide 1186.25 contains multiple DR-supermotif sequences. Peptide CF-08 is a 20-mer that nests both 27.0280 and 1186.25. These peptides are shown in Table XXXIII.


The 55 HBV-derived peptides identified above were tested for their capacity to bind common HLA-DR alleles. To maximize both population coverage, and the relationships between the binding repertoires of most DR alleles (see, e.g., Southwood et al., J. Immunol. 160:3363, 1998), peptides were screened for binding to sequential panels of DR assays. The composition of these screening panels, and the phenotypic frequency of associated antigens, are shown in Table XXXIV. All peptides were initially tested for binding to the alleles in the primary panel: DR1, DR4w4, and DR7. Only peptides binding at least 2 of these 3 alleles were then tested for binding in the secondary assays (DR2w2 β1, DR2w2 β2, DR6w19, and DR9). Finally, only peptides binding at least 2 of the 4 secondary panel alleles, and thus 4 of 7 alleles total, were screened for binding in the tertiary assays (DR4w15, DR5w11, and DR8w2).


Upon testing, it was found that 25 of the original 55 peptides (45%) bound two or more of the primary panel alleles. When these 25 peptides were subsequently tested in the secondary assays, 20 were found to bind at least 4 of the 7 DR alleles in the primary and secondary assay panels. Finally, 18 of the 20 peptides passing the secondary screening phase were tested for binding in the tertiary assays. As a result, 12 peptides were shown to bind at least 7 of 10 common HLA-DR alleles. The sequences of these 12 peptides, and their binding capacity for each assay in the primary through tertiary panels, are shown in Table XXXV. Also shown are peptides CF-08 and 857.02, which bound 5/5 and 5/6 of the alleles tested to date, respectively.


In summary, 14 peptides, derived from 12 independent regions of the HBV genome, have been identified that are capable of binding multiple HLA-DR alleles. This set of peptides includes at least 2 epitopes each from the Core (Nuc), Pol, and Env antigens.


Selection of Conserved DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is an important criterion in the selection of HTL epitopes. However, data generated previously indicated that DR3 only rarely cross-reacts with other DR alleles (Sidney et al., J. Immunol. 149:2634-2640, 1992; Geluk et al., J. Immunol. 152:5742-5748, 1994; Southwood et al., J. Immunol. 160:3363-3373, 1998). This is not entirely surprising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles.


To efficiently identify peptides that bind DR3, target proteins were analyzed for conserved sequences carrying one of the two DR3 specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Eighteen sequences were identified. Eight of these sequences were largely redundant with peptides shown in Table XXXVI, and 3 with peptides that had previously been synthesized for other studies. The 7 unique sequences were synthesized.


Seventeen of the eighteen peptides containing a DR3 motif have been tested for their DR3 binding capacity. Four peptides were found to bind DR3 with an affinity of 1000 nM or better (Table XXXVI).


Example 6
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.


In order to analyze population coverage, gene frequencies of HLA alleles were determined. Gene frequencies for each HLA allele were calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies were calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].


Where frequency data was not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies was assumed. To obtain total potential supertype population coverage no linkage disequilibrium was assumed, and only alleles confirmed to belong to each of the supertypes were included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations were made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may potentially include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602). Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups (see Table XXI). Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%.


Population coverage for HLA class II molecules can be developed analagously based on the present disclosure.


Summary of Candidate HLA class I and class II Epitopes


In summary, on the basis of the data presented above, 34 conserved CTL epitopes were selected as vaccine candidates (Table XXXVII). Of these 34 epitopes, 7 are derived from core, 18 from polymerase, and 9 from envelope. No epitopes from the X antigen were included in the package as this protein is expressed in low amounts and is, therefore, of less immunological interest.


The population coverage afforded by this panel of CTL epitopes is estimated to exceed 95% in each of 5 major ethnic populations. Using a Monte Carlo analysis (FIG. 1), it is predicted that approximately 90% of the individuals in a population comprised of Caucasians, North American Blacks, Japanese, Chinese and Hispanics would recognize five or more of the vaccine candidate epitopes.


While preferred CTL epitopes includes 34 discrete peptides, two peptides are entirely nested within longer peptides, thus effectively reducing the numbers of peptides that would have to be included in a vaccine candidate. Specifically, the A2-restricted peptide 927.15 is nested within the B7-restricted peptide 26.0570 and the B7-restricted peptide 988.05 is nested within the A2-restricted peptide 924.07. Similarly, the A24-restricted peptide 20.0136 and the A2-restricted peptide 1013.01 contain the same core region, differing only at the first amino acid. On a related note, the A2-restricted peptide 1090.14 and the B7-restricted peptide 1147.05 overlap by two amino acids, raising the possibility of delivering these two epitopes as one contiguous peptide sequence.


The set of recommended vaccine candidates includes 9 A2-restricted CTL epitopes; four polymerase-derived epitopes, four envelope-derived epitopes and a core epitope. Seven of these 9 peptides are recognized in recall CTL assays from acute patients. Of the 7 peptides recognized in patients, 2 are non-crossreactive binding peptides. The inclusion of these peptides as potential vaccine candidates stems from the observation that HLA-A*0201 is the predominantly expressed A2-supertype allele in all ethnicities examined. As such, inclusion of non-crossreactive A*0201 binding peptides increases the redundancy of antigen coverage and population coverage. The only two A2-restricted peptides that lack patient immunogenicity data are peptides 1090.77 and 1069.06. The 1090.77 peptide is an analog of a highly immunogenic peptide recognized in acute HBV patients. Although recall responses in patients have not been tested for the ability to recognize the analog peptide, immunogenicity studies conducted in HLA transgenic mice have shown that CTL induced with 1090.77 are capable of recognizing target cells loaded with the naturally occurring sequence. This data indicates that CTL raised to the 1090.77 peptide are cross-reactive and should recognize HBV-infected cells. The 1069.06 peptide was included as a potential vaccine epitope because its high binding affinity for A*6802 results in a greater population coverage. While peptide 1069.06 has not been tested for recognition by acute HBV patients, the peptide is immunogenic in HLA-A2 transgenic mice and primary human cultures.


Preferred CTL epitopes include 7 A3-supertype-restricted peptides; 6 derived from the polymerase antigen, and one from the core region. All of the A3-supertype vaccine candidate peptides are immunogenic in patients. Although peptide 1142.05 is a non-crossreactive A3-restricted peptide, it has been included because it has been shown to be recognized in patients and is capable of binding HLA-A1.


Nine B7-restricted peptides are preferred CTL epitopes. Of this group, 3 epitopes have been shown to be recognized in patients. While one of these peptides, 1147.04, is a non-crossreactive binder, it binds 2 of the major B7 supertype alleles with an IC50 or binding affinity value of less than 100 nM. Six B7-supertype epitopes were included as preferred epitopes based on supertype binding. Immunogenicity studies in humans (Bertoni et al., 1997; Doolan et al., 1997; Threlkeld et al., 1997) have demonstrated that highly cross-reactive peptides are almost always recognized as epitopes. Given these results, and in light of the limited immunogenicity data available, the use of B7-supertype binding affinity as a selection criterion was deemed appropriate.


Similarly, there is little immunogenicity data regarding A1- and A24-restricted peptides. One preferred CTL epitope, 1069.04, has been reported to be recognized in recall responses from acute HBV patients. As discussed in the preceding paragraph, a high percentage of the peptides with binding affinities<100 nM are found to be immunogenic. For this reason, all A1 and A24 peptides with binding affinities<100 nM were considered as preferred CTL epitopes. Using this selection criterion, 3 A1-restricted and 6 A24-restricted peptides are identified as candidate epitopes. Further analysis found that 3 core-derived peptides bound A24 with intermediate affinity. Since relatively few core epitopes were identified during the course of this study, the intermediate A24 binding core peptides were also included in the set of preferred epitopes to provide a greater degree of redundancy in antigen coverage.


The list of preferred HBV-derived HTL epitopes is summarized in Table XXXVII. The set of HTL epitopes includes 12 DR supermotif binding peptides and 4 DR3 binding peptides. The bulk of the HTL epitopes are derived from polymerase; 2 envelope and 2 core derived epitopes are also included in the set of preferred HTL epitopes. The total estimated population coverage represented by the panel of HTL epitopes is in excess of 91% in each of five major ethnic groups (Table XXXVIII)


Example 7
Recognition of Generation of Endogenous Processed Antigens after Priming

This example determines that CTL induced by native or analogued peptide epitopes identified and selected as described in Examples 1-5 recognize endogenously synthesized, i.e., native antigens.


Effector cells isolated from transgenic mice that are immunized with peptide epitopes as in Example 3 are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled 3A4-721.221-A11/Kb target cells, in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, e.g., cells that are stably transfected with HBV expression vectors.


The result will demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized HBV antigen.


Example 8
Activity Of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs in transgenic mice by use of an HBV CTL/HTL peptide conjugate. An analagous study may be found in Oseroffet al. Vaccine 16:823-833 (1998). The peptide composition can comprise multiple CTL and/or HTL epitopes. Such a peptide composition can comprise a lipidated HTL epitope conjugated to a preferred CTL epitope containing, for example, an A11 motif or an analog of that epitope.


Lipopeptides are prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide. A typical procedure is as follows: A dichloromethane solution of a four-fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid is added to the resin and the mixture is allowed to react for two hours. The resin is washed with dichloromethane and dried. The resin is then treated with trifluoroacetic acid in the presence of appropriate scavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. After evaporation of excess trifluoroacetic acid, the crude peptide is washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide is collected by filtration and dried.


Preparation of Peptides for Immunization: Peptide Compositions are Typically resuspended in DMSO at a concentration of 20 mg/ml. Before use, peptides are prepared at the required concentration by dilution in saline or the appropriate medium.


Immunization procedures: A11/Kb mice, which are transgenic for the human HLA A11 allele, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngeneic irradiated LPS-activated lymphoblasts coated with peptide.


Media:


a. RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mM Glutamine, 50 μg/ml Gentamicin and 5×10−5 M 2-mercaptoethanol serves as culture medium


b. RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2% (FCS) is used as cell washing medium.


Cell lines: The 3A4-721.221-A11/Kb cell line is used as target cells. This cell line is an EBV transformed cell line that was mutagenized and selected to be Class I negative which was transfected with an HLA-A11/Kb gene.


LPS-activated lymphoblasts: Splenocytes obtained from transgenic mice are resuspended at a concentration of 1-1.5×106/ml in culture medium supplemented with 25 μg/ml LPS and 7 μg/ml dextran sulfate in 75 cm2 tissue culture flasks. After 72 hours at 37° C., the lymphoblasts are collected for use by centrifugation.


Peptide coating of lymphoblasts: Peptide coating of the LPS activated lymphoblasts is achieved by incubating 30×106 irradiated (3000 rads) lymphoblasts with 100 μg of peptide in 1 ml of R10 medium for 1 hr at 37° C. Cells are then washed once and resuspended in culture medium at the desired concentration.


In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, the effector cells are harvested and assayed for cytotoxic activity.


Assay for cytotoxic activity: Target cells (1.0-1.5×106) are incubated at 37° C. in the presence of 200 μl of sodium 51Cr chromate. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the E:T of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: (1×106(5×104)−(1×106(5×105)=18LU/106.


The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using the CTL epitope as outlined in Example 3. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.


Example 9
Selection of CTL and HTL Epitopes for Inclusion in an HBV-Specific Vaccine

This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention.


The following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition, or for selecting epitopes to be included in a vaccine composition and/or to be encoded by a minigene. Each of the following principles are balanced in order to make the selection.


1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with HBV clearance. For HLA Class I this includes 3-4 epitopes that come from at least one antigen of HBV. In other words, it has been observed that in patients who spontaneously clear HBV, that they had generated an immune response to at least 3 epitopes on at least one HBV antigen. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one HBV antigen.


2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.


3.) Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, is employed to assess population coverage.


4.) When selecting epitopes for HBV antigens it is often preferable to select native epitopes. Therefore, of particular relevance for infectious disease vaccines, are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A peptide comprising “transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.


When providing nested epitopes, a sequence that has the greatest number of epitopes per provided sequence is provided. A limitation on this principle is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a longer peptide sequence, such as a sequence comprising nested epitopes, the sequence is screened in order to insure that it does not have pathological or other deleterious biological properties.


5.) When creating a minigene, as disclosed in greater detail in the Example 9, an objective is to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same as those employed when selecting a peptide comprising nested epitopes. Thus, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is an actual binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are to be avoided because the recipient may generate an immune response to that 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.


Peptide epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Table XXXVIIa and b. A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude of an immune response that clears an acute HBV infection.


Example 10
Construction of Minigene Multi-Epitope DNA Plasmids

This example provides an illustration of the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein. Expression plasmids have been constructed and evaluated as described, for example, in U.S. Ser. No. 60/085,751 filed May 15, 1998 and U.S. Ser. No. 09/078,904 filed May 13, 1998. An example of such a plasmid is shown in FIG. 2, which illustrates the orientation of HBV epitopes in minigene constructs. Such a plasmid may, for example, also include multiple CTL and HTL peptide epitopes.


A minigene expression plasmid may include multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes (FIG. 2). Preferred epitopes are identified, for example, in Tables XXVI-XXXIII, HLA class I supermotif or motif-bearing peptide epitopes derived from multiple HBV antigens, e.g., the core, polymerase, envelope and X proteins, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from multiple HBV antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.


This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.


The minigene DNA plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by a string of CTL and/or HTL epitopes selected in accordance with principles disclosed herein.


Overlapping oligonucleotides, for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (50 below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.


For the first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.


Example 11
The Plasmid Construct and the Degree to which it Induces Immunogenicity

The degree to which the plasmid construct prepared using the methodology outlined in Example 9 is able to induce immunogenicity is evaluated through in vivo injections into transgenic mice and in vitro culture of CTL and HTL, which are subsequently analysed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in U.S. Ser. No. 09/311,784 filed May 13, 1999 and Alexander et al., Immunity 1:751-761, 1994. To assess the capacity of the pMin minigene construct to induce CTLs in vivo, HLA-A11/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of plasmid cDNA. As a means of comparing the level of CTLs induced by DNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.


Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A3-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine. It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A3 supermotif peptide epitopes as does the polyepitopic peptide vaccine. Such an analysis is also performed using other HLA-A2 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A2 and HLA-B7 motif or supermotif epitopes.


To assess the capacity of a class II epitope encoding minigene to induce HTLs in vivo, I-Ab restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.


CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). the results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.


Example 12
Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention are used to prevent HBV infection in persons who are at risk for such an infection. For example, a polyepitopic peptide epitope composition containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is administered to individuals at risk for HBV infection. The composition is provided as a single lipidated polypeptide that encompasses multiple epitopes. The vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant. The dose of peptide for the initial immunization is from about 500 to about 50,000 μg for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient by techriiques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against HBV infection.


Alternatively, the polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.


Example 13
Polyepitopic Vaccine Compositions Derived from Native HBV Sequences

A native HBV polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is less than 250 amino acids in length, preferably less than 100 amino acids in length, and more preferably less than 75 or 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence. As noted herein, epitope motifs may be overlapping (i.e., frame shifted relative to one another) with frame shifted overlapping epitopes, e.g. two 9-mer epitopes can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.


The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from the source antigen. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.


The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent analogs) directs the immune response to peptide sequences that are present in native HBV antigens. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.


Related to this embodiment, computer programs can be derived which identify, in a target sequence, the greatest number of epitopes per sequence length.


Example 14
Polyepitopic Vaccine Compositions Directed to Multiple Diseases

The HBV peptide epitopes of the present invention are used in conjunction with peptide epitopes from target antigens related to one or more other diseases, to create a vaccine composition that is useful for the prevention or treatment of HBV as well as another disease. Examples of other diseases include, but are not limited to, HIV, HCV, and HPV.


For example, a polyepitopic peptide composition comprising multiple CTL and HTL epitopes that target greater than 98% of the population may be created for administration to individuals at risk for both HBV and HIV infection. The composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various disease-associated sources, or can be administered as a composition comprising one or more discrete epitopes.


Example 15
Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response for the presence of specific CTL populations corresponding to HBV. Such an analysis may be performed as described by Ogg et al., Science 279:2103-2106, 1998. In the following example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.


In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) may be used for a cross-sectional analysis of, for example, HBV Env-specific CTL frequencies from untreated HLA A*0201-positive indiviuals at different stages of infection using an HBV Env peptide containing an A2.1 extended motif. Tetrameric complexes are synethesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A2.1 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.


For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 ul of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain>99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the stage of infection with HBV or the status of exposure to HBV or to a vaccine that elicits a protective response.


Example 16
Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate T cell responses such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from infection or who are chronically infected with HBV or who have been vaccinated with an HBV vaccine.


For example, the class I restricted CTL response of persons at risk for HBV infection who have been vaccinated may be analyzed. The vaccine may be any HBV vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide reagents that, are highly conserved and, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members are then used for analysis of samples derived from individuals who bear that HLA type.


PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. Synthetic peptide is added at 10 μg/ml to each well and recombinant HBc Ag is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.


In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 ml of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with uninfected control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).


Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).


Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with synthetic peptide at 10 μM and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS. Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.


The results of such an analysis will indicate to what extent HLA-restricted CTL populations have been stimulated with the vaccine. Of course, this protocol can also be used to monitor prior HBV exposure.


The class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptide, whole antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.


The results of such an analysis will indicate to what extent HLA-restricted HTL populations have been stimulated with a vaccine or prior exposure to HBV.


Example 17
Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising HBV CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study (5, 50 and 500 μg) and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:


A total of about 27 subjects are enrolled and divided into 3 groups:


Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;


Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;


Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.


After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.


The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.


Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.


Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.


Thus, the vaccine is found to be both safe and efficacious.


Example 18
Phase II Trials in Patients Infected with HBV

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients (male and female) having chronic HBV infection. A main objective of the trials is to determine an effective dose and regimen for inducing CTLs in chronically infected HBV patients, to establish the safety of inducing a CTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of chronically infected CTL patients, as manifested by a transient flare in alanine aminotransferase (ALT), normalization of ALT, and reduction in HBV DNA. Such a study is designed, for example, as follows:


The studies are performed in multiple centers in the U.S. and Canada. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects are recorded.


There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and include both males and females. The patients represent diverse ethnic backgrounds. All of them are infected with HBV for over five years and are HIV, HCV and HDV negative, but have positive levels of HBe antigen and HBs antigen.


The magnitude and incidence of ALT flares and the levels of HBV DNA in the blood are monitored to assess the effects of administering the peptide compositions. The levels of HBV DNA in the blood are an indirect indication of the progress of treatment. The vaccine composition is found to be both safe and efficacious in the treatment of chronic HBV infection.


The examples herein are provided to illustrate the invention but not to limit its scope. For example, the human terminology for the Major Histocompatibility Complex, namely HLA, is used throughout this document. It is to be appreciated that these principles can be extended to other species as well. Moreover, peptide epitopes have been disclosed in the related application U.S. Ser. No. 08/820,360, which was previously incorporated by reference. Thus, 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.













TABLE I









POSITION



POSITION
POSITION
C Terminus



2
3 (Primary
(Primary



(Primary Anchor)
Anchor)
Anchor)



















SUPERMOTIFS





A1

TI
LVMS



FWY



A2

LIVM
ATQ



IV
MATL



A3

VSMA
TLI



RK



A24

YF
WIVLMT



FI
YWLM



B7

P



VILF
MWYA



B27

RHK



FYL
WMIVA



B44

E
D



FWYLIMVA



B58

ATS



FWY
LIVMA



B62

QL
IVMP



FWY
MIVLA



MOTIFS


A1

TSM



Y



A1


DE
AS


Y



A2.1

LM
VQIAT



V
LIMAT



A3

LMVISATF
CGD



KYR
HFA



A11

VTMLISAGN
CDF



K
RYH



A24

YFW
M



FLIW



A*3101

MVT
ALIS



R
K



A*3301

MVALF
IST



RK



A*6801

AVT
MSLI



RK



B*0702

P



LMF
WYAIV



B*3501

P



LMFWY
IVA



B51

P



LIVF
WYAM



B*5301

P



IMFWY
ALV



B*5401

P



ATIV
LMFWY






Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

















TABLE Ia









POSITION



POSITION
POSITION
C Terminus



2
3 (Primary
(Primary



(Primary Anchor)
Anchor)
Anchor)



















SUPERMOTIFS





A1

TI
LVMS



FWY



A2

VQAT



V
LIMAT



A3

VSMA
TLI



RK



A24

YF
WIVLMT



FI
YWLM



B7

P



VILF
MWYA



B27

RHK



FYL
WMIVA



B58

ATS



FWY
LIVMA



B62

QL
IVMP



FWY
MIVLA



MOTIFS


A1

TSM



Y



A1


DE
AS


Y



A2.1

VQAT*



V
LIMAT



A3.2

LMVISATF
CGD



KYR
HF



A11

VTMLISAGN
CDF



K
RH



A24

YFW



FLIW






*If 2 is V, or Q, the C-term is not L


Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.















TABLE II









POSITION

















1
2
3
4
5
6
7
8
C-terminus












SUPERMOTIFS


















A1



1° Anchor








1° Anchor






TILVMS






FWY


A2



1° Anchor








1° Anchor






LIVMATQ






LIVMAT


A3
preferred


1° Anchor

YFW (4/5)


YFW (3/5)
YFW (4/5)
P (4/5)

1° Anchor






VSMATLI






RK



deleterious
DE (3/5); P (5/5)

DE (4/5)


A24



1° Anchor








1° Anchor






YFWIVLMT






FIYWLM


B7
preferred
FWY (5/5)

1° Anchor

FWY (4/5)




FWY (3/5)

1° Anchor





LIVM (3/5)
P






VILFMWYA



deleterious
DE (3/5); P (5/5);



DE (3/5)
G (4/5)
QN (4/5)
DE (4/5)




G (4/5); A (3/5);




QN (3/5)


B27



1° Anchor








1° Anchor






RHK






FYLWMIVA


B44



1° Anchor








1° Anchor






ED






FWYLIMVA


B58



1° Anchor








1° Anchor






ATS






FWYLIVMA


B62



1° Anchor








1° Anchor






QLIVMP






FWYMIVLA








MOTIFS


















A1
preferred
GFYW

1° Anchor

DEA
YFW

P
DEQN
YFW

1° Anchor



9-mer


STM






Y



deleterious
DE

RHKLIVMP
A
G
A


A1
preferred
GRHK
ASTCLIVM

1° Anchor

GSTC

ASTC
LIVM
DE

1° Anchor



9-mer



DEAS





Y



deleterious
A
RHKDEPY

DE
PQN
RHK
PG
GP





FW













POSITION


















1
2
3
4
5







A1
peferred
YFW

1° Anchor

DEAQN
A
YFWQN



10-mer


STM




deleterious
GP

RHKGLIVM
DE
RHK



A1
preferred
YFW
STCLIVM

1° Anchor

A
YFW



10-mer



DEAS




deleterious
RHK
RHKDEPY


P






FW



A2.1
preferred
YFW

1° Anchor

YFW
STC
YFW



9-mer


LMIVQAT




deleterious
DEP

DERKH



A2.1
preferred
AYFW

1° Anchor

LVIM
G



10-mer


LMIVQAT




deleterious
DEP

DE
RKHA
P



A3
preferred
RHK

1° Anchor

YFW
PRHKYFW
A






LMVISAT






FCGD




deleterious
DEP

DE



A11
preferred
A

1° Anchor

YFW
YFW
A






VTLMISA






GNCDF




deleterious
DEP



A24
preferred
YFWRHK

1° Anchor


STC



9-mer


YFWM




deleterious
DEG

DE
G
QNP



A24
preferred


1° Anchor


P
YFWP



10-mer


YFWM




deleterious


GDE
QN
RHK



A3101
preferred
RHK

1° Anchor

YFW
P






MVTALIS




deleterious
DEP

DE

ADE



A3301
preferred


1° Anchor

YFW






MVALFIST




deleterious
GP

DE



A6801
preferred
YFWSTC

1° Anchor



YFWLIVM






AVTMSLI




deleterious
GP

DEG

RHK



B0702
preferred
RHKFWY

1° Anchor

RHK

RHK






P




deleterious
DEQNP

DEP
DE
DE



B3501
preferred
FWYLIVM

1° Anchor

FWY






P




deleterious
AGP



G



B51
preferred
LIVMFWY

1° Anchor

FWY
STC
FWY






P




deleterious
AGPDERHKSTC



DE



B5301
preferred
LIVMFWY

1° Anchor

FWY
STC
FWY






P




deleterious
AGPQN



B5401
preferred
FWY

1° Anchor

FWYLIVM

LIVM






P




deleterious
GPQNDE

GDESTC

RHKDE













POSITION

















6
7
8
9 or C-terminus
C-terminus







A1
peferred

PASTC
GDE
P

1° Anchor




10-mer





Y




deleterious
QNA
RHKYFW
RHK
A



A1
preferred

PG
G
YFW

1° Anchor




10-mer





Y




deleterious
G

PRHK
QN



A2.1
preferred

A
P

1° Anchor




9-mer




VLIMAT




deleterious
RKH
DERKH



A2.1
preferred
G

FYWL


1° Anchor




10-mer



VIM

VLIMAT




deleterious

RKH
DERKH
RKH



A3
preferred
YFW

P

1° Anchor









KYRHFA




deleterious



A11
preferred
YFW
YFW
P

1° Anchor









KRYH




deleterious

A
G



A24
preferred

YFW
YFW

1° Anchor




9-mer




FLIW




deleterious
DERHK
G
AQN



A24
preferred

P



1° Anchor




10-mer





FLIW




deleterious
DE
A
QN
DEA



A3101
preferred
YFW
YFW
AP

1° Anchor









RK




deleterious
DE
DE
DE



A3301
preferred

AYFW


1° Anchor









RK




deleterious



A6801
preferred

YFW
P

1° Anchor









RK




deleterious


A



B0702
preferred
RHK
RHK
PA

1° Anchor









LMFWYAIV




deleterious
GDE
QN
DE



B3501
preferred

FWY


1° Anchor









LMFWYIVA




deleterious
G



B51
preferred

G
FWY

1° Anchor









LIVFWYAM




deleterious
G
DEQN
GDE



B5301
preferred

LIVMFWY
FWY

1° Anchor









IMFWYALV




deleterious
G
RHKQN
DE



B5401
preferred

ALIVM
FWYAP

1° Anchor









ATIVLMFWY




deleterious
DE
QNDGE
DE







Italicized residues indicate less preferred or “tolerated” residues.



The information in Table II is specific for 9-mers unless otherwise specified.















TABLE III









POSITION
















MOTIFS
1° anchor 1
2
3
4
5
1° anchor 6
7
8
9




















DR4
preferred
FMYLIVW
M
T

I
VSTCPALIM
MH

MH



deleterious



W


R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious

C
CH
FD
CWD

GDE
D


DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious

C

G


GRD
N
G









DR Supermotif
MFLIVWY
VMSTACPLI


















DR3 MOTIFS
1° anchor 1
2
3
1° anchor 4
5
1° anchor 6







motif a



preferred
LIVMFY


D



motif b



preferred
LIVMFAY


DNQEST

KRH







Italicized residues indicate less preferred or “tolerated” residues.













TABLE IV







HLA Class I Standard Peptide Binding Affinity.















BINDING



STANDARD


AFFINITY


ALLELE
PEPTIDE
SEQUENCE
SEQ ID NO:
(nM)














A*0101
944.02
YLEPAIAKY
3475
25





A*0201
941.01
FLPSDYFPSV
3476
5.0





A*0202
941.01
FLPSDYFPSV
3476
4.3





A*0203
941.01
FLPSDYFPSV
3476
10





A*0206
941.01
FLPSDYFPSV
3476
3.7





A*0207
941.01
FLPSDYFPSV
3476
23





A*6802
1141.02
FTQAGYPAL
3477
40





A*0301
941.12
KVFPYALINK
3478
11





A*1101
940.06
AVDLYHFLK
3479
6.0





A*3101
941.12
KVFPYALINK
3478
18





A*3301
1083.02
STLPETYVVRR
3480
29





A*6801
941.12
KVFPYALINK
3479
8.0





A*2402
979.02
AYIDNYNKF
3481
12





B*0702
1075.23
APRTLVYLL
3482
5.5





B*3501
1021.05
FPFKYAAAF
3483
7.2





B51
1021.05
FPFKYAAAF
3483
5.5





B*5301
1021.05
FPFKYAAAF
3483
9.3





B*5401
1021.05
FPFKYAAAF
3483
10
















TABLE V







HLA Class II Standard Peptide Binding Affinity.


















Binding





Standard

SEQ ID
Affinity


Allele
Nomenclature
Peptide
Sequence
NO:
(nM)
















DRB1*0101
DR1
515.01
PKYVKQNTLKLAT
3484
5.0






DRB1*0301
DR3
829.02
YKTIAFDEEARR
3485
300





DRB1*0401
DR4w4
515.01
PKYVKQNTLKLAT
3484
45





DRB1*0404
DR4w14
717.01
YARFQSQTTLKQKT
3486
50





DRB1*0405
DR4w15
717.01
YARFQSQTTLKQKT
3486
38





DRB1*0701
DR7
553.01
QYIKANSKFIGITE
3487
25





DRB1*0802
DR8w2
553.01
QYIKANSKFIGITE
3487
49





DRB1*0803
DR8w3
553.01
QYIKANSKFIGITE
3487
1600





DRB1*0901
DR9
553.01
QYIKANSKFIGITE
3487
75





DRB1*1101
DR5w11
553.01
QYIKANSKFIGITE
3487
20





DRB1*1201
DR5w12
1200.05
EALIHQLKINPYVLS
3488
298





DRB1*1302
DR6w19
650.22
QYIKANAKFIGITE
3489
3.5





DRB1*1501
DR2w2β1
507.02
GRTQDENPVVHFFKNI
3490
9.1





VTPRTPPP





DRB3*0101
DR52a
511
NGQIGNDPNRDIL
3491
470





DRB4*0101
DRw53
717.01
YARFQSQTTLKQKT
3486
58





DRB5*0101
DR2w2β2
553.01
QYIKANSKFIGITE
3487
20





The “Nomenclature” column lists the allelic designations used in Tables XIX and XX.














TABLE VI







HLA-



super-
Allele-specific HLA-supertype members









type
Verifieda
Predictedb





A1
A*0101, A*2501, A*2601, A*2602, A*3201
A*0102, A*2604, A*3601, A*4301, A*8001


A2
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207,
A*0208, A*0210, A*0211, A*0212, A*0213



A*0209, A*0214, A*6802, A*6901


A3
A*0301, A*1101, A*3101, A*3301, A*6801
A*0302, A*1102, A*2603, A*3302, A*3303, A*3401, A*3402,




A*6601, A*6602, A*7401


A24
A*2301, A*2402, A*3001
A*2403, A*2404, A*3002, A*3003


B7
B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502,
B*1511, B*4201, B*5901



B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101,



B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501,



B*5502, B*5601, B*5602, B*6701, B*7801


B27
B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705,
B*2701, B*2707, B*2708, B*3802, B*3903, B*3904, B*3905,



B*2706, B*3801, B*3901, B*3902, B*7301
B*4801, B*4802, B*1510, B*1518, B*1503


B44
B*1801, B*1802, B*3701, B*4402, B*4403, B*4404, B*4001,
B*4101, B*4501, B*4701, B*4901, B*5001



B*4002, B*4006


B58
B*5701, B*5702, B*5801, B*5802, B*1516, B*1517


B62
B*1501, B*1502, B*1513, B*5201
B*1301, B*1302, B*1504, B*1505, B*1506, B*1507, B*1515,




B*1520, B*1521, B*1512, B*1514, B*1519






aVerified alleles includes alleles whose specificity has been determined by pool sequencing analysis, peptide binding assays, or by analysis of the sequences of CTL epitopes.




bPredicted alleles are alleles whose specificity is predicted on the basis of B and F pocket structure to overlap with the supertype specificity.














TABLE VII







HBV A01 SUPER MOTIF(With binding information)














Conservancy
Freq.
Protein
Position
Sequence
SEQ ID NO:
String
A*0101


















95
19
POL
521
AICSVVRRAF
1
XIXXXXXXXF







95
19
NUC
54
ALRQAILCW
2
XLXXXXXXW





80
16
ENV
108
AMQWNSTTF
3
XMXXXXXXF





100
20
POL
166
ASFCGSPY
4
XSXXXXXY





100
20
POL
166
ASFCGSPYSW
5
XSXXXXXXXW





90
18
NUC
19
ASKLCLGW
6
XSXXXXXW





85
17
NUC
19
ASKLCLGWLW
7
XSXXXXXXXW





80
16
POL
822
ASPLHVAW
8
XSXXXXXW





100
20
ENV
312
CIPIPSSW
9
XIXXXXXW





100
20
ENV
312
CIPIPSSWAF
10
XIXXXXXXXF





95
19
ENV
253
CLIFLLVLLDY
11
XLXXXXXXXXY





95
19
ENV
239
CLRRFIIF
12
XLXXXXXF





75
15
ENV
239
CLRRFIIFLF
13
XLXXXXXXXF





95
19
POL
523
CSVVRRAF
14
XSXXXXXF





100
20
ENV
310
CTCIPIPSSW
15
XTXXXXXXXW





90
18
NUC
31
DIDPYKEF
16
XIXXXXXF





85
17
NUC
29
DLLDTASALY
17
XLXXXXXXXY
11.1000





95
19
ENV
196
DSWWTSLNF
18
XSXXXXXXF





95
19
NUC
43
ELLSFLPSDF
19
XLXXXXXXXF





95
19
NUC
43
ELLSFLPSDFF
20
XLXXXXXXXXF





95
19
POL
374
ESRLVVDF
21
XSXXXXXF





95
19
POL
374
ESRLVVDFSQF
22
XSXXXXXXXXF





80
16
ENV
248
FILLLCLIF
23
XIXXXXXXF





80
16
ENV
246
FLFILLLCLIF
24
XLXXXXXXXXF





95
19
ENV
256
FLLVLLDY
25
XLXXXXXY





95
19
POL
658
FSPTYKAF
26
XSXXXXXF





90
18
X
63
FSSAGPCALRF
27
XSXXXXXXXXF





100
20
ENV
333
FSWLSLLVPF
28
XSXXXXXXXF





95
19
POL
656
FTFSPTYKAF
29
XTXXXXXXXF





95
19
ENV
346
FVGLSPTVW
30
XVXXXXXXW





95
19
POL
627
GLLGFAAPF
31
XLXXXXXXF





95
19
POL
509
GLSPFLLAQF
32
XLXXXXXXXF





85
17
NUC
29
GMDIDPYKEF
33
XMXXXXXXXF





95
19
NUC
123
GVWIRTPPAY
34
XVXXXXXXXY
0.0017





75
15
POL
569
HLNPNKTKRW
35
XLXXXXXXXW





80
16
POL
491
HLYSHPIILGF
36
XLXXXXXXXXF





85
17
POL
715
HTAELLAACF
37
XTXXXXXXXF





95
19
NUC
52
HTALRQAILCW
38
XTXXXXXXXXW





100
20
POL
149
HTLWKAGILY
39
XTXXXXXXXY
0.0300





100
20
ENV
249
ILLLCLIF
40
XLXXXXXF





80
16
POL
760
ILRGTSFVY
41
XLXXXXXXY
0.0017





90
18
ENV
188
ILTIPQSLDSW
42
XLXXXXXXXXW





90
18
POL
625
IVGLLGFAAPF
43
XVXXXXXXXXF





80
16
POL
503
KIPMGVGLSPF
44
XIXXXXXXXXF





85
17
NUC
21
KLCLGWLW
45
XLXXXXXW





75
15
POL
108
KLIMPARF
46
XLXXXXXF





75
15
POL
108
KLIMPARFY
47
XLXXXXXXY
0.0017





80
16
POL
610
KLPVNRPIDW
48
XLXXXXXXXW





85
17
POL
574
KTKRWGYSLNF
49
XTXXXXXXXXF





95
19
POL
55
KVGNFTGLY
50
XVXXXXXXY
0.0680





95
19
ENV
254
LIFLLVLLDY
51
XIXXXXXXXY
0.0084





100
20
POL
109
LIMPARFY
52
XIXXXXXY





85
17
NUC
30
LLDTASALY
53
XLXXXXXXY
25.0000





80
16
POL
752
LLGCAANW
54
XLXXXXXW





95
19
POL
628
LLGFAAPF
55
XLXXXXXF





100
20
ENV
378
LLPIFFCLW
56
XLXXXXXXW





100
20
ENV
378
LLPIFFCLWVY
57
XLXXXXXXXXY





95
19
NUC
44
LLSFLPSDF
58
XLXXXXXXF





95
19
NUC
44
LLSFLPSDFF
59
XLXXXXXXXF





90
18
POL
407
LLSSNLSW
60
XLXXXXXW





95
19
ENV
175
LLVLQAGF
61
XLXXXXXF





95
19
ENV
175
LLVLQAGFF
62
XLXXXXXXF





100
20
ENV
338
LLVPFVQW
63
XLXXXXXW





100
20
ENV
338
LLVPFVQWF
64
XLXXXXXXF





85
17
NUC
100
LLWFHISCLTF
65
XLXXXXXXXXF





95
19
NUC
45
LSFLPSDF
66
XSXXXXXF





95
19
NUC
45
LSFLPSDFF
67
XSXXXXXXF





95
19
POL
415
LSLDVSAAF
68
XSXXXXXXF





95
19
POL
415
LSLDVSAAFY
69
XSXXXXXXXY
4.2000





100
20
ENV
336
LSLLVPFVQW
70
XSXXXXXXXW





100
20
ENV
336
LSLLVPFVQWF
71
XSXXXXXXXXF





95
19
X
53
LSLRGLPVCAF
72
XSXXXXXXXXF





95
19
POL
510
LSPFLLAQF
73
XSXXXXXXF





75
15
ENV
349
LSPTVWLSVIW
74
XSXXXXXXXXW





85
17
POL
742
LSRKYTSF
75
XSXXXXXF





85
17
POL
742
LSRKYTSFPW
76
XSXXXXXXXW





75
15
ENV
16
LSVPNPLGF
77
XSXXXXXXF





75
15
NUC
137
LTFGRETVLEY
78
XTXXXXXXXXY





90
18
ENV
189
LTIPQSLDSW
79
XTXXXXXXXW





90
18
ENV
189
LTIPQSLDSWW
80
XTXXXXXXXXW





90
18
POL
404
LTNLLSSNLSW
81
XTXXXXXXXXW





95
19
ENV
176
LVLQAGFF
82
XVXXXXXF





100
20
ENV
339
LVPFVQWF
83
XVXXXXXF





100
20
POL
377
LVVDFSQF
84
XVXXXXXF





85
17
ENV
360
MMWYWGPSLY
85
XMXXXXXXXY
0.0810





75
15
X
103
MSTTDLEAY
86
XSXXXXXXY
0.8500





75
15
X
103
MSTTDLEAYF
87
XSXXXXXXXF





95
19
POL
42
NLGNLNVSIPW
88
XLXXXXXXXXW





90
18
POL
406
NLLSSNLSW
89
XLXXXXXXW





95
19
POL
45
NLNVSIPW
90
XLXXXXXW





75
15
ENV
15
NLSVPNPLGF
91
XLXXXXXXXF





90
18
POL
738
NSVVLSRKY
92
XSXXXXXXY
0.0005





100
20
ENV
380
PIFFCLWVY
93
XIXXXXXXY
0.0078





100
20
ENV
314
PIPSSWAF
94
XIXXXXXF





100
20
POL
124
PLDKGIKPY
95
XLXXXXXXY
0.0190





100
20
POL
124
PLDKGIKPYY
96
XLXXXXXXXY
0.1600





100
20
ENV
377
PLLPIFFCLW
97
XLXXXXXXXW





95
19
ENV
174
PLLVLQAGF
98
XLXXXXXXF





95
19
ENV
174
PLLVLQAGFF
99
XLXXXXXXXF





80
16
POL
505
PMGVGLSPF
100
XMXXXXXXF





85
17
POL
797
PTTGRTSLY
101
XTXXXXXXY
0.7700





75
15
ENV
351
PTVWLSVIW
102
XTXXXXXXW





85
17
POL
612
PVNRPIDW
103
XVXXXXXW





95
19
POL
685
QVFADATPTG
104
XVXXXXXXXXW





90
18
POL
624
RIVGLLGF
105
XIXXXXXF





75
15
POL
106
RLKLIMPARF
106
XLXXXXXXXF





75
15
POL
106
RLKLIMPARFY
107
XLXXXXXXXXY





95
19
POL
376
RLVVDFSQF
108
XLXXXXXXF





90
18
POL
353
RTPARVTGGVF
109
XTXXXXXXXXF





100
20
POL
49
SIPWTHKVGNF
110
XIXXXXXXXXF





95
19
ENV
194
SLDSWWTSLNF
111
XLXXXXXXXXF





95
19
POL
416
SLDVSAAF
112
XLXXXXXF





95
19
POL
416
SLDVSAAFY
113
XLXXXXXXY
17.2000





100
20
ENV
337
SLLVPFVQW
114
XLXXXXXXW





100
20
ENV
337
SLLVPFVQWF
115
XLXXXXXXXF





95
19
X
54
SLRGLPVCAF
116
XLXXXXXXXF





90
18
X
64
SSAGPCALRF
117
XSXXXXXXXF





75
15
X
104
STTDLEAY
118
XTXXXXXY





75
15
X
104
STTDLEAYF
119
XTXXXXXXF





75
15
ENV
17
SVPNPLGF
120
XVXXXXXF





90
18
POL
739
SVVLSRKY
121
XVXXXXXY





85
17
POL
739
SVVLSRKYTSF
122
XVXXXXXXXXF





90
18
ENV
190
TIPQSLDSW
123
XIXXXXXXW





90
18
ENV
190
TIPQSLDSWW
124
XIXXXXXXXW





100
20
POL
150
TLWKAGILY
125
XLXXXXXXY
0.0017





75
15
X
105
TTDLEAYF
126
XTXXXXXF





85
17
POL
798
TTGRTSLY
127
XTXXXXXY





80
16
NUC
16
TVQASKLCLGW
128
XVXXXXXXXXW





75
15
ENV
352
TVWLSVIW
129
XVXXXXXW





85
17
POL
741
VLSRKYTSF
130
XLXXXXXXF





85
17
POL
741
VLSRKYTSFPW
131
XLXXXXXXXXW





85
17
POL
740
VVLSRKYTSF
132
XVXXXXXXXF





80
16
POL
759
WILRGTSF
133
XIXXXXXF





80
16
POL
759
WILRGTSFVY
134
XIXXXXXXXY
0.0023





95
19
NUC
125
WIRTPPAY
135
XIXXXXXY





80
16
POL
751
WLLGCAANW
136
XLXXXXXXW





95
19
POL
414
WLSLDVSAAF
137
XLXXXXXXXF





95
19
POL
414
WLSLDVSAAFY
138
XLXXXXXXXXY





100
20
ENV
335
WLSLLVPF
139
XLXXXXXF





100
20
ENV
335
WLSLLVPFVQW
140
XLXXXXXXXXW





85
17
NUC
26
WLWGMDIDPY
141
XLXXXXXXXY
0.0810





95
19
ENV
237
WMCLRRFIIF
142
XMXXXXXXXF





85
17
ENV
359
WMMWYWGPS
143
XMXXXXXXXXY





100
20
POL
52
WTHKVGNF
144
XTXXXXXF





100
20
POL
122
YLPLDKGIKPY
145
XLXXXXXXXXY





90
18
NUC
118
YLVSFGVW
146
XLXXXXXW





80
16
POL
493
YSHPIILGF
147
XSXXXXXXF





85
17
POL
580
YSLNFMGY
148
XSXXXXXY
















TABLE VIII







HBV A02 SUPER MOTIF (With binding information)
























SEQ ID









Conservancy
Frequency
Protein
Position
Sequence
NO:
AA
A*0201
A*0202
A*0203
A*0206
A*6802






















85
17
POL
721
AACFARSRSGA
149
11











85
17
POL
431
AAMPHLLV
150
8





80
16
POL
756
AANWILRGT
151
9





95
19
POL
632
AAPFTQCGYPA
152
11





95
19
POL
521
AICSVVRRA
153
9
0.0001





90
18
NUC
58
AILCWGEL
154
8





90
18
NUC
58
AILCWGELM
155
9





95
19
POL
642
ALMPLYACI
156
9
0.5000
0.0340
3.3000
0.2500
0.0470





80
16
ENV
108
AMQWNSTT
157
8





75
15
X
102
AMSTTDLEA
158
9
0.0013





95
19
POL
516
AQFTSAICSV
159
10





95
19
POL
516
AQFTSAICSVV
160
11





95
19
POL
690
ATPTGWGL
161
8





80
16
POL
690
ATPTGWGLA
162
9





75
15
POL
690
ATPTGWGLAI
163
10





95
19
POL
397
AVPNLQSL
164
8





95
19
POL
397
AVPNLQSLT
165
9
0.0001





95
19
POL
397
AVPNLQSLTNL
166
11





80
16
POL
755
CAANWILRGT
167
10





95
19
X
61
CAFSSAGPCA
168
10
0.0001





95
19
X
61
CAFSSAGPCAL
169
11





90
18
X
69
CALRFTSA
170
8





100
20
ENV
312
CIPIPSSWA
171
9
0.0010





80
16
ENV
312
CIPIPSSWAFA
172
11





90
18
POL
533
CLAFSYMDDV
173
10
0.0008





90
18
POL
533
CLAFSYMDDVV
174
11





85
17
NUC
23
CLGWLWGM
175
8





85
17
NUC
23
CLGWLWGMDI
176
10
0.0093





100
20
ENV
253
CLIFLLVL
177
8
0.0002





100
20
ENV
253
CLIFLLVLL
178
9
0.0006





95
19
ENV
239
CLRRFIIFL
179
9
0.0002





75
15
ENV
239
CLRRFIIFLFI
180
11
0.0004





90
18
NUC
107
CLTFGRET
181
8





90
18
NUC
107
CLTFGRETV
182
9
0.0001





80
16
X
7
CQLDPARDV
183
9





80
16
X
7
CQLDPARDVL
184
10





85
17
POL
622
CQRIVGLL
185
8





85
17
POL
622
CQRIVGLLGFA
186
11





95
19
POL
684
CQVFADAT
187
8





95
19
POL
684
CQVFADATPT
188
10





100
20
ENV
310
CTCIPIPSSWA
189
11





95
19
POL
689
DATPTGWGL
190
9
0.0001





80
16
POL
689
DATPTGWGLA
191
10





75
15
POL
689
DATPTGWGLAI
192
11





90
18
NUC
31
DIDPYKEFGA
193
10





85
17
NUC
29
DLLDTASA
194
8





85
17
NUC
29
DLLDTASAL
195
9
0.0001





95
19
POL
40
DLNLGNLNV
196
9
0.0004





95
19
POL
40
DLNLGNLNVSI
197
11





80
16
NUC
32
DTASALYREA
198
10





80
16
NUC
32
DTASALYREAL
199
11





95
19
X
14
DVLCLRPV
200
8





95
19
X
14
DVLCLRPVGA
201
10
0.0001





90
18
POL
541
DVVLGAKSV
202
9
0.0003





100
20
POL
17
EAGPLEEEL
203
9
0.0001





80
16
X
122
ELGEEIRL
204
8





90
18
POL
718
ELLAACFA
205
8





75
15
NUC
142
ETVLEYLV
206
8





95
19
POL
687
FADATPTGWGL
207
11





85
17
POL
724
FARSRSGA
208
8





80
16
POL
821
FASPLHVA
209
8





95
19
POL
396
FAVPNLQSL
210
9





95
19
POL
396
FAVPNLQSLT
211
10
0.0003





80
16
ENV
243
FIIFLFIL
212
8
0.0006





80
16
ENV
243
FIIFLFILL
213
9
0.0002





80
16
ENV
243
FIIFLFILLL
214
10
0.0012





80
16
ENV
248
FILLLCLI
215
8
0.0003





80
16
ENV
248
FILLLCLIFL
216
10
0.0280





80
16
ENV
248
FILLLCLIFLL
217
11
0.0010





80
16
ENV
246
FLFILLLCL
218
9
0.0002





80
16
ENV
246
FLFILLLCLI
219
10
0.0013





75
15
ENV
171
FLGPLLVL
220
8





75
15
ENV
171
FLGPLLVLQA
221
10
0.0190





95
19
POL
513
FLLAQFTSA
222
9
0.2400





95
19
POL
513
FLLAQFTSAI
223
10
0.2100
0.0320
7.0000
0.1100
0.0880





95
19
POL
562
FLLSLGIHL
224
9
0.6500
0.0010
0.0100
0.1100
0.0035





80
16
ENV
183
FLLTRILT
225
8





80
16
ENV
183
FLLTRILTI
226
9
0.5100
0.0430
8.0000
0.2000
0.0010





95
19
ENV
256
FLLVLLDYQGM
227
11





100
20
POL
363
FLVDKNPHNT
228
10
0.0012





95
19
POL
656
FTFSPTYKA
229
9
0.0056
0.0150
0.0031
0.8000
7.3000





95
19
POL
656
FTFSPTYKAFL
230
11





95
19
POL
59
FTGLYSST
231
8





90
18
POL
59
FTGLYSSTV
232
9
0.0005





95
19
POL
635
FTQCGYPA
233
8





95
19
POL
635
FTQCGYPAL
234
9
0.0009





95
19
POL
635
FTQCGYPALM
235
10
0.0024





95
19
POL
518
FTSAICSV
236
8





95
19
POL
518
FTSAICSVV
237
9
0.0090





95
19
ENV
346
FVGLSPTV
238
8





95
19
ENV
346
FVGLSPTVWL
239
10
0.0008





90
18
X
132
FVLGGCRHKL
240
10
0.0030





90
18
X
132
FVLGGCRHKLV
241
11





95
19
ENV
342
FVQWFVGL
242
8





95
19
ENV
342
FVQWFVGLSPT
243
11





90
18
POL
766
FVYVPSAL
244
8





90
18
POL
766
FVYVPSALNPA
245
11





95
19
X
50
GAHLSLRGL
246
9
0.0001





90
18
X
50
GAHLSLRGLPV
247
11





85
17
POL
545
GAKSVQHL
248
8





85
17
POL
545
GAKSVQHLESL
249
11





75
15
POL
567
GIHLNPNKT
250
9





90
18
POL
155
GILYKRET
251
8





90
18
POL
155
GILYKRETT
252
9





85
17
POL
682
GLCQVFADA
253
9
0.0024





85
17
POL
682
GLCQVFADAT
254
10





95
19
POL
627
GLLGFAAPFT
255
10
0.0049





85
17
ENV
62
GLLGWSPQA
256
9
0.4000
0.0003
0.0350
0.2800
0.0005





95
19
X
57
GLPVCAFSSA
257
10
0.0008





95
19
POL
509
GLSPFLLA
258
8





95
19
POL
509
GLSPFLLAQFT
259
11





100
20
ENV
348
GLSPTVWL
260
8
0.0036





75
15
ENV
348
GLSPTVWLSV
261
10
0.2800





75
15
ENV
348
GLSPTVWLSVI
262
11
0.0036





90
18
ENV
265
GMLPVCPL
263
8





90
18
POL
735
GTDNSVVL
264
8





75
15
ENV
13
GTNLSVPNPL
265
10





80
16
POL
763
GTSFVYVPSA
266
10





80
16
POL
763
GTSFVYVPSAL
267
11





80
16
POL
507
GVGLSPFL
268
8





80
16
POL
507
GVGLSPFLL
269
9
0.0002





80
16
POL
507
GVGLSPFLLA
270
10





95
19
NUC
123
GVWIRTPPA
271
9
0.0030





90
18
NUC
104
HISCLTFGRET
272
11





80
16
POL
435
HLLVGSSGL
273
9
0.0031





90
18
X
52
HLSLRGLPV
274
9
0.0014





90
18
X
52
HLSLRGLPVCA
275
11





80
16
POL
491
HLYSHPII
276
8





80
16
POL
491
HLYSHPIIL
277
9
0.2200
0.0003
0.9300
0.1700
0.0530





85
17
POL
715
HTAELLAA
278
8





85
17
POL
715
HTAELLAACFA
279
11





100
20
NUC
52
HTALRQAI
280
8





95
19
NUC
52
HTALRQAIL
281
9
0.0001





100
20
POL
149
HTLWKAGI
282
8





100
20
POL
149
HTLWKAGIL
283
9
0.0001





80
16
ENV
244
IIFLFILL
284
8
0.0004





80
16
ENV
244
IIFLFILLL
285
9
0.0002





80
16
ENV
244
IIFLFILLLCL
286
11
0.0002





80
16
POL
497
IILGFRKI
287
8





80
16
POL
497
IILGFRKIPM
288
10





90
18
NUC
59
ILCWGELM
289
8





80
16
POL
498
ILGFRKIPM
290
9
0.0002





100
20
ENV
249
ILLLCLIFL
291
9
0.0015





100
20
ENV
249
ILLLCLIFLL
292
10
0.0190
0.0001
0.0002
0.1300
0.0015





100
20
ENV
249
ILLLCLIFLLV
293
11
0.0056





80
16
POL
760
ILRGTSFV
294
8





80
16
POL
760
ILRGTSFVYV
295
10
0.0160





100
20
NUC
139
ILSTLPET
296
8





100
20
NUC
139
ILSTLPETT
297
9
0.0001





100
20
NUC
139
ILSTLPETTV
298
10
0.0210
0.0085
0.0770
0.3100
0.0067





100
20
NUC
139
ILSTLPETTVV
299
11





95
19
ENV
188
ILTIPQSL
300
8





90
18
POL
156
ILYKRETT
301
8





90
18
POL
625
IVGLLGFA
302
8





90
18
POL
625
IVGLLGFAA
303
9
0.0009





90
18
POL
153
KAGILYKRET
304
10





90
18
POL
153
KAGILYKRETT
305
11





80
16
POL
503
KIPMGVGL
306
8





85
17
NUC
21
KLCLGWLWGM
307
10
0.0001





95
19
POL
489
KLHLYSHPI
308
9
0.0690
0.0340
2.7000
0.5900
0.0015





80
16
POL
489
KLHLYSHPII
309
10





80
16
POL
489
KLHLYSHPIIL
310
11





80
16
POL
610
KLPVNRPI
311
8





95
19
POL
653
KQAFTFSPT
312
9





95
19
POL
574
KTKRWGYSL
313
9
0.0001





85
17
POL
620
KVCQRIVGL
314
9
0.0003





85
17
POL
620
KVCQRIVGLL
315
10
0.0001





95
19
POL
55
KVGNFTGL
316
8





85
17
X
91
KVLHKRTL
317
8





85
17
X
91
KVLHKRTLGL
318
10
0.0004





90
18
POL
534
LAFSYMDDV
319
9
0.0002





90
18
POL
534
LAFSYMDDVV
320
10
0.0003





90
18
POL
534
LAFSYMDDVVL
321
11





95
19
POL
515
LAQFTSAI
322
8





95
19
POL
515
LAQFTSAICSV
323
11





100
20
ENV
254
LIFLLVLL
324
8
0.0025





95
19
POL
514
LLAQFTSA
325
8





95
19
POL
514
LLAQFTSAI
326
9
0.1000
0.2700
3.7000
0.2600
0.7900





100
20
ENV
251
LLCLIFLL
327
8
0.0004





100
20
ENV
251
LLCLIFLLV
328
9
0.0048





100
20
ENV
251
LLCLIFLLVL
329
10
0.0075





100
20
ENV
251
LLCLIFLLVLL
330
11
0.0013





85
17
NUC
30
LLDTASAL
331
8





95
19
ENV
260
LLDYQGML
332
8
0.0004





90
18
ENV
260
LLDYQGMLPV
333
10
0.0980
0.0001
0.0200
0.6700
0.0009





80
16
POL
752
LLGCAANWI
334
9
0.0011





80
16
POL
752
LLGCAANWIL
335
10
0.0140





95
19
POL
628
LLGFAAPFT
336
9
0.0008





85
17
ENV
63
LLGWSPQA
337
8





75
15
ENV
63
LLGWSPQAQGI
338
11





100
20
ENV
250
LLLCLIFL
339
8
0.0006





100
20
ENV
250
LLLCLIFLL
340
9
0.0065





100
20
ENV
250
LLLCLIFLLV
341
10
0.0036





100
20
ENV
250
LLLCLIFLLVL
342
11
0.0005





100
20
ENV
378
LLPIFFCL
343
8
0.0055





100
20
ENV
378
LLPIFFCLWV
344
10
0.0320
0.0008
0.0150
0.8000
0.0005





95
19
POL
563
LLSLGIHL
345
8





90
18
POL
407
LLSSNLSWL
346
9
0.0110
0.0780
3.9000
0.2700
0.0100





90
18
POL
407
LLSSNLSWLSL
347
11





80
16
ENV
184
LLTRILTI
348
8
0.0026





80
16
POL
436
LLVGSSGL
349
8





95
19
ENV
257
LLVLLDYQGM
350
10
0.0050





95
19
ENV
257
LLVLLDYQGML
351
11





90
18
ENV
175
LLVLQAGFFL
352
10
0.0310
0.0037
0.0045
0.1500
0.0110





90
18
ENV
175
LLVLQAGFFLL
353
11
0.0074





95
19
ENV
338
LLVPFVQWFV
354
10
0.6700
0.3800
1.7000
0.2900
0.1400





90
18
NUC
100
LLWFHISCL
355
9
0.0130
0.0002
0.0420
0.3100
0.0098





85
17
NUC
100
LLWFHISCLT
356
10





95
19
POL
643
LMPLYACI
357
8





95
19
ENV
178
LQAGFFLL
358
8





95
19
ENV
178
LQAGFFLLT
359
9





80
16
ENV
178
LQAGFFLLTRI
360
11





100
20
POL
401
LQSLTNLL
361
8





95
19
NUC
108
LTFGRETV
362
8





75
15
NUC
137
LTFGRETVL
363
9





90
18
POL
404
LTNLLSSNL
364
9





80
16
ENV
185
LTRILTIPQSL
365
11





85
17
POL
99
LTVNEKRRL
366
9





100
20
POL
364
LVDKNPHNT
367
9
0.0001





95
19
ENV
258
LVLLDVQGM
368
9
0.0001





95
19
ENV
258
LVLLDYQGML
369
10
0.0001





90
18
ENV
176
LVLQAGFFL
370
9
0.0096





90
18
ENV
176
LVLQAGFFLL
371
10
0.0022





90
18
ENV
176
LVLQAGFFLLT
372
11





95
19
ENV
339
LVPFVQWFV
373
9
0.0420
0.0150
0.0048
0.7900
2.8000





95
19
ENV
339
LVPFVQWFVGL
374
11





90
18
NUC
119
LVSFGVWI
375
8
0.0004





90
18
NUC
119
LVSFGVWIRT
376
10





85
17
ENV
360
MMWYWGPSL
377
9
0.6400





75
15
NUC
1
MQLFHLCL
378
8





100
20
NUC
136
NAPILSTL
379
8





100
20
NUC
136
NAPILSTLPET
380
11





95
19
POL
42
NLGNLNVSI
381
9
0.0047





90
18
POL
406
NLLSSNLSWL
382
10
0.0016





95
19
POL
45
NLNVSIPWT
383
9
0.0005





100
20
POL
400
NLQSLTNL
384
8





100
20
POL
400
NLQSLTNLL
385
9
0.0047





75
15
ENV
15
NLSVPNPL
386
8





90
18
POL
411
NLSWLSLDV
387
9
0.0650
0.0051
0.6400
0.1600
0.0990





90
18
POL
411
NLSWLSLDVSA
388
11





100
20
POL
47
NVSIPWTHKV
389
10
0.0001





100
20
POL
430
PAAMPHLL
390
8





85
17
POL
430
PAAMPHLLV
391
9





90
18
POL
775
PADDPSRGRL
392
10





90
18
ENV
131
PAGGSSSGT
393
9





90
18
ENV
131
PAGGSSSGTV
394
10





95
19
POL
641
PALMPLYA
395
8





95
19
POL
641
PALMPLYACI
396
10
0.0001





75
15
X
145
PAPCNFFT
397
8





75
15
X
145
PAPCNFFTSA
398
10





80
16
X
11
PARDVLCL
399
8





75
15
X
11
PARDVLCLRPV
400
11





90
18
POL
355
PARVTGGV
401
8





90
18
POL
355
PARVTGGVFL
402
10





90
18
POL
355
PARVTGGVFLV
403
11





95
19
NUC
130
PAYRPPNA
404
8





95
19
NUC
130
PAYRPPNAPI
405
10
0.0001





95
19
NUC
130
PAYRPPNAPIL
406
11





85
17
POL
616
PIDWKVCQRI
407
10
0.0001





85
17
POL
616
PIDWKVCQRIV
408
11





100
20
ENV
380
PIFFCLWV
409
8





100
20
ENV
380
PIFFCLWVYI
410
10
0.0004





85
17
POL
713
PIHTAELL
411
8





85
17
POL
713
PIHTAELLA
412
9





85
17
POL
713
PIHTAELLAA
413
10





80
16
POL
496
PIILGFRKI
414
9
0.0001





80
16
POL
496
PIILGFRKIPM
415
11





100
20
NUC
138
PILSTLPET
416
9
0.0001





100
20
NUC
138
PILSTLPETT
417
10
0.0001





100
20
NUC
138
PILSTLPETTV
418
11
0.0001





80
16
ENV
314
PIPSSWAFA
419
9





95
19
POL
20
PLEEELPRL
420
9
0.0003





90
18
POL
20
PLEEELPRLA
421
10
0.0001





95
19
ENV
10
PLGFFPDHQL
422
10
0.0002





100
20
POL
427
PLHPAAMPHL
423
10
0.0001





100
20
POL
427
PLHPAAMPHLL
424
11





100
20
ENV
377
PLLPIFFCL
425
9
0.0650
0.0001
0.0018
0.1100
0.0047





100
20
ENV
377
PLLPIFFCLWV
426
11





90
18
ENV
174
PLLVLQAGFFL
427
11
0.0008





80
16
POL
711
PLPIHTAEL
428
9
0.0004





80
16
POL
711
PLPIHTAELL
429
10
0.0001





80
16
POL
711
PLPIHTAELLA
430
11





75
15
POL
2
PLSYQHFRKL
431
10
0.0001





75
15
POL
2
PLSYQHFRKLL
432
11





85
17
POL
98
PLTVNEKRRL
433
10
0.0001





80
16
POL
505
PMGVGLSPFL
434
10
0.0001





80
16
POL
505
PMGVGLSPFLL
435
11





95
19
ENV
106
PQAMQWNST
436
9





80
16
ENV
106
PQAMQWNSTT
437
10





90
18
ENV
192
PQSLDSWWT
438
g





90
18
ENV
192
PQSLDSWWTSL
439
11





75
15
POL
692
PTGWGLAI
440
8





80
16
ENV
219
PTSNHSPT
441
8





85
17
POL
797
PTTGRTSL
442
8





85
17
POL
797
PTTGRTSLYA
443
10





80
16
NUC
15
PTVQASKL
444
8





80
16
NUC
15
PTVQASKLCL
445
10





75
15
ENV
351
PTVWLSVI
446
8





75
15
ENV
351
PTVWLSVIWM
447
10





95
19
X
59
PVCAFSSA
448
8





85
17
POL
612
PVNRPIDWKV
449
10
0.0002





95
19
POL
654
QAFTFSPT
450
8





95
19
POL
654
QAFTFSPTYKA
451
11





95
19
ENV
179
QAGFFLLT
452
8





80
16
ENV
179
QAGFFLLTRI
453
10





80
16
ENV
179
QAGFFLLTRIL
454
11





90
18
NUC
57
QAILCWGEL
455
9





90
18
NUC
57
QAILCWGELM
456
10





95
19
ENV
107
QAMQWNST
457
8





80
16
ENV
107
QAMQWNSTT
458
9





80
16
NUC
18
QASKLCLGWL
459
10





80
16
X
8
QLDPARDV
460
8
0.0001





80
16
X
8
QLDPARDVL
461
9
0.0001





80
16
X
8
QLDPARDVLCL
462
11
0.0001





90
18
NUC
99
QLLWFHISCL
463
10
0.0060





85
17
NUC
99
QLLWFHISCLT
464
11





95
19
POL
685
QVFADATPT
465
9
0.0001





95
19
POL
528
RAFPHCLA
466
8





80
16
ENV
187
RILTIPQSL
467
9
0.0010





90
18
POL
624
RIVGLLGFA
468
9





90
18
POL
624
RIVGLLGFAA
469
10





75
15
POL
106
RLKLIMPA
470
8





90
18
NUC
56
RQAILCWGEL
471
10





90
18
NUC
56
RQAILCWGELM
472
11





90
18
NUC
98
RQLLWFHI
473
8





90
18
NUC
98
RQLLWFHISCL
474
11





85
17
ENV
88
RQSGRQPT
475
8





90
18
POL
353
RTPARVTGGV
476
10





95
19
NUC
127
RTPPAYRPPNA
477
11





95
19
POL
36
RVAEDLNL
478
8





90
18
POL
36
RVAEDLNLGNL
479
11





80
16
POL
818
RVHFASPL
480
8





75
15
POL
818
RVHFASPLHV
481
10
0.0001





75
15
POL
818
RVHFASPLHVA
482
11





100
20
POL
357
RVTGGVFL
483
8





100
20
POL
357
RVTGGVFLV
484
9
0.0041





90
18
X
65
SAGPCALRFT
485
10





95
19
POL
520
SAICSVVRRA
486
10
0.0001





90
18
NUC
35
SALYREAL
487
8





100
20
POL
49
SIPWTHKV
488
8





95
19
ENV
194
SLDSWWTSL
489
9





75
15
POL
565
SLGIHLNPNKT
490
11





95
19
ENV
337
SLLVPFVQWFV
491
11





75
15
POL
581
SLNFMGYV
492
8





75
15
POL
581
SLNFMGYVI
493
9
0.0038





95
19
X
54
SLRGLPVCA
494
9
0.0007





90
18
POL
403
SLTNLLSSNL
495
10
0.0014





75
15
ENV
216
SQSPTSNHSPT
496
11





75
15
ENV
280
STGPCKTCT
497
9





100
20
NUC
141
STLPETTV
498
8





100
20
NUC
141
STLPETTVV
499
9
0.0019





80
16
ENV
85
STNRQSGRQPT
500
11





85
17
POL
548
SVQHLESL
501
8





80
16
ENV
330
SVRFSWLSL
502
9
0.0001





80
16
ENV
330
SVRFSWLSLL
503
10
0.0004





80
16
ENV
330
SVRFSWLSLLV
504
11





90
18
POL
739
SVVLSRKYT
505
9





95
19
POL
524
SVVRRAFPHCL
506
11





85
17
POL
716
TAELLAACFA
507
10





95
19
NUC
53
TALRQAIL
508
8





80
16
NUC
33
TASALYREA
509
9





80
16
NUC
33
TASALYREAL
510
10





90
18
ENV
190
TIPQSLDSWWT
511
11





100
20
NUC
142
TLPETTVV
512
8





100
20
POL
150
TLWKAGIL
513
8





95
19
POL
636
TQCGYPAL
514
8





95
19
POL
636
TQCGYPALM
515
9





95
19
POL
636
TQCGYPALMPL
516
11





85
17
POL
798
TTGRTSLYA
517
9





75
15
ENV
278
TTSTGPCKT
518
9





75
15
ENV
278
TTSTGPCKTCT
519
11





85
17
POL
100
TVNEKRRL
520
8





80
16
NUC
16
TVQASKLCL
521
9
0.0002





75
15
ENV
352
TVWLSVIWM
522
9
0.0002





95
19
POL
37
VAEDLNLGNL
523
10
0.0001





95
19
X
15
VLCLRPVGA
524
9
0.0014





85
17
POL
543
VLGAKSVQHL
525
10
0.0001





90
18
X
133
VLGGCRHKL
526
9
0.0009





90
18
X
133
VLGGCRHKLV
527
10
0.0001





85
17
X
92
VLHKRTLGL
528
9
0.0012





95
19
ENV
259
VLLDYQGM
529
8





95
19
ENV
259
VLLDYQGML
530
9
0.0440
0.0001
0.0210
0.9000
0.0002





90
18
ENV
259
VLLDYQGMLPV
531
11
0.5800
0.2200
4.9000
0.3400
0.0170





95
19
ENV
177
VLQAGFFL
532
8
0.0019





95
19
ENV
177
VLQAGFFLL
533
9
0.0660





95
19
ENV
177
VLQAGFFLLT
534
10
0.0011





80
16
NUC
17
VQASKLCL
535
8





80
16
NUC
17
VQASKLCLGWL
536
11





95
19
ENV
343
VQWFVGLSPT
537
10





95
19
ENV
343
VQWFVGLSPTV
538
11





100
20
POL
358
VTGGVFLV
539
8





90
18
POL
542
VVLGAKSV
540
8





80
16
POL
542
VVLGAKSVQHL
541
11





90
18
POL
740
VVLSRKYT
542
8





95
19
POL
525
VVRRAFPHCL
543
10
0.0003





95
19
POL
525
VVRRAFPHCLA
544
11





80
16
POL
759
WILRGTSFV
545
9
0.0270





80
16
POL
759
WILRGTSFVYV
546
11





80
16
POL
751
WLLGCAANWI
547
10
0.0053





80
16
POL
751
WLLGCAANWIL
548
11





100
20
POL
414
WLSLDVSA
549
8





95
19
POL
414
WLSLDVSAA
550
9
0.0059





100
20
ENV
335
WLSLLVPFV
551
9
1.1000
0.0380
7.2000
0.3600
0.0310





95
19
ENV
237
WMCLRRFI
552
8





95
19
ENV
237
WMCLRRFII
553
9
0.0005





95
19
ENV
237
WMCLRRFIIFL
554
11
0.0019





85
17
ENV
359
WMMWYWGPSL
555
10
0.0009





100
20
POL
52
WTHKVGNFT
556
9
0.0001





95
19
POL
52
WTHKVGNFTGL
557
11





100
20
POL
147
YLHTLWKA
558
8





100
20
POL
147
YLHTLWKAGI
559
10
0.0160
0.0005
0.5600
0.1000
0.0320





100
20
POL
147
YLHTLWKAGIL
560
11





100
20
POL
122
YLPLDKGI
561
8





90
18
NUC
118
YLVSFGVWI
562
9
0.3800





90
18
NUC
118
YLVSFGVWIRT
563
11





90
18
POL
538
YMDDVVLGA
564
9
0.0250
0.0001
0.0024
0.1000
0.0002





90
18
ENV
263
YQGMLPVCPL
565
10





75
15
POL
5
YQHFRKLL
566
8





75
15
POL
5
YQHFRKLLL
567
9





75
15
POL
5
YQHFRKLLLL
568
10





85
17
POL
746
YTSFPWLL
569
8





75
15
POL
746
YTSFPWLLGCA
570
11





90
18
POL
768
YVPSALNPA
571
9
0.0039
















TABLE IX







HBV A03 SUPER MOTIF (With binding information)





















Conserv-





C-






SEQ ID



ancy
Frequency
Protein
Position
Sequence
P2
term
AA
A*0301
A*1101
A*3101
A*3301
A*6801
NO:
























85
17
POL
721
AACFARSR
A
R
8
0.0004
0.0003
0.0056
0.0035
0.0014
572






95
19
POL
521
AICSVVRR
I
R
8
−0.0002
0.0003
0.0014
−0.0009
0.0006
573





90
18
POL
772
ALNPADDPSR
L
R
10
0.0003
0.0001



574





85
17
X
70
ALRFTSAR
L
R
8
0.0047
0.0009
0.0450
0.0230
0.0004
575





80
16
POL
822
ASPLHVAWR
S
R
9





576





75
15
ENV
84
ASTNRQSGR
S
R
9
0.0009
0.0002
0.0088
0.0008
0.0001
577





80
16
POL
755
CAANWILR
A
R
8





578





85
17
X
69
CALRFTSAR
A
R
9
0.0034
0.0230
1.5000
8.0000
0.7300
579





90
18
X
17
CLRPVGAESR
L
R
10
0.0011
0.0001



580





100
20
NUC
48
CSPHHTALR
S
R
9
0.0029
0.0001
0.0520
0.0250
0.0440
581





85
17
NUC
29
DLLDTASALYR
L
R
11
0.0042
−0.0003
−0.0012
3.7000
0.0410
582





85
17
NUC
32
DTASALYR
T
R
8
0.0004
−0.0002
−0.0009
0.0018
0.0009
583





95
19
POL
17
EAGPLEEELPR
A
R
11
−0.0009
−0.0003
−0.0012
0.0015
0.0110
584





90
18
POL
718
ELLAACFAR
L
R
9
0.0002
0.0004



585





85
17
POL
718
ELLAACFARSR
L
R
11
0.0062
0.0016
0.0200
0.2000
0.1600
586





95
19
NUC
174
ETTVVRRR
T
R
8
0.0003
−0.0002
−0.0009
0.1400
0.0027
587





80
16
NUC
174
ETTVVRRRGR
T
R
10
0.0003
0.0001



588





80
16
POL
821
FASPLHVAWR
A
R
10





589





90
18
X
63
FSSAGPCALR
S
R
10





590





95
19
POL
656
FTFSPTYK
T
K
8
0.0100
0.0100
0.0023
0.2100
0.0590
591





95
19
POL
518
FTSAICSVVR
T
R
10
0.0003
0.0003



592





95
19
POL
518
FTSAICSVVRR
T
R
11
0.0065
0.0092
0.0170
0.0350
1.5000
593





90
18
X
132
FVLGGCRHK
V
K
9
0.0430
0.0090



594





75
15
POL
567
GIHLNPNK
I
K
8





595





75
15
POL
567
GIHLNPNKTK
I
K
10
0.0025
0.0011
0.0009
0.0009
0.0003
596





75
15
POL
567
GIHLNPNKTKR
I
R
11





597





85
17
NUC
29
GMDIDPYK
M
K
8
0.0006
0.0004
−0.0009
−0.0009
0.0001
598





90
18
POL
735
GTDNSVVLSR
T
R
10
0.0010
0.0420
0.0030
0.0019
0.0008
599





90
18
POL
735
GTDNSVVLSRK
T
K
11
0.0140
0.5600
−0.0002
−0.0006
0.0001
600





95
19
NUC
123
GVWIRTPPAYR
V
R
11
0.1900
0.1700
6.8000
0.7300
0.6600
601





90
18
NUC
104
HISCLTFGR
I
R
9
0.0160
0.0065



602





75
15
POL
569
HLNPNKTK
L
K
8





603





75
15
POL
569
HLNPNKTKR
L
R
9
0.0025
0.0001



604





100
20
POL
149
HTLWKAGILYK
T
K
11
0.5400
0.4400
0.0370
0.0720
0.1900
605





90
18
NUC
105
ISCLTFGR
S
R
8
0.0004
0.0002
0.0017
−0.0009
0.0017
606





100
20
POL
153
KAGILYKR
A
R
8
0.0002
−0.0002
0.0015
−0.0009
0.0001
607





80
16
POL
610
KLPVNRPIDWK
L
K
11





608





75
15
X
130
KVFVLGGCR
V
R
9
0.0420
0.0820
0.6000
0.0710
0.0030
609





85
17
POL
720
LAACFARSR
A
R
9
0.0058
0.0065



610





90
18
POL
719
LLAACFAR
L
R
8
0.0024
0.0003
0.0015
0.0029
0.0064
611





85
17
POL
719
LLAACFARSR
L
R
10





612





85
17
NUC
30
LLDTASALYR
L
R
10
0.0050
0.0002



613





80
16
POL
752
LLGCAANWILR
L
R
11





614





75
15
POL
564
LSLGIHLNPNK
S
K
11





615





95
19
NUC
169
LSTLPETTVVR
S
R
11
−0.0009
0.0008
−0.0012
−0.0023
0.0078
616





75
15
POL
3
LSYQHFRK
S
K
8





617





85
17
POL
99
LTVNEKRR
T
R
8
−0.0002
−0.0002
−0.0009
−0.0009
0.0001
618





90
18
NUC
119
LVSFGVWIR
V
R
9
0.0028
0.0120



619





100
20
POL
377
LVVDFSQFSR
V
R
10
0.0016
0.3600
0.0260
0.2300
0.4900
620





75
15
X
103
MSTTDLEAYFK
S
K
11





621





90
18
NUC
75
NLEDPASR
L
R
8
−0.0002
−0.0002
−0.0009
−0.0009
0.0001
622





95
19
POL
45
NLNVSIPWTHK
L
K
11
−0.0009
0.0005
−0.0012
−0.0023
0.0019
623





90
18
POL
738
NSVVLSRK
S
K
8
0.0006
0.0010
−0.0009
−0.0009
0.0007
624





100
20
POL
47
NVSIPWTHK
V
K
9
0.0820
0.0570
0.0002
0.0100
0.0320
625





90
18
POL
775
PADDPSRGR
A
R
9
0.0008
0.0002
0.0004
0.0015
0.0002
626





80
16
X
11
PARDVLCLR
A
R
9
0.0002
0.0002
0.0100
0.0180
0.0002
627





75
15
ENV
83
PASTNRQSGR
A
R
10





628





90
18
POL
616
PIDWKVCQR
I
R
9
0.0002
0.0005



629





80
16
POL
496
PIILGFRK
I
K
8





630





95
19
POL
20
PLEEELPR
L
R
8
0.0002
−0.0002
−0.0009
−0.0009
0.0001
631





100
20
POL
2
PLSYQHFR
L
R
8
−0.0002
−0.0002
−0.0009
−0.0009
0.0001
632





75
15
POL
2
PLSYQHFRK
L
K
9
0.0011
0.0031
0.0006
0.0008
0.0002
633





85
17
POL
98
PLTVNEKR
L
R
8
0.0002
−0.0002
−0.0009
−0.0009
0.0001
634





85
17
POL
98
PLTVNEKRR
L
R
9
0.0008
0.0005
0.0004
0.0027
0.0002
635





90
18
X
20
PVGAESRGR
V
R
9
0.0002
0.0005
0.0004
0.0043
0.0002
636





85
17
POL
612
PVNRPIDWK
V
K
9
0.0310
0.1400
0.0002
0.0006
0.0009
637





95
19
POL
654
QAFTFSPTYK
A
K
10
0.0450
0.5400
0.0010
0.0057
1.2000
638





80
16
ENV
179
QAGFFLLTR
A
R
9





639





75
15
NUC
169
QSPRRRRSQSR
S
R
11





640





80
16
POL
189
QSSGILSR
S
R
8





641





75
15
POL
106
RLKLIMPAR
L
R
9
0.0950
0.0002
3.1000
0.0490
0.0002
642





75
15
X
128
RLKVFVLGGCR
L
R
11





643





95
19
POL
376
RLVVDFSQFSR
L
R
11
0.2800
3.8000
2.6000
1.2000
6.1000
644





95
19
NUC
183
RSPRRRTPSPR
S
R
11
−0.0007
−0.0003
0.0190
−0.0023
0.0003
645





75
15
NUC
167
RSQSPRRR
S
R
8





646





75
15
NUC
167
RSQSPRRRR
S
R
9





647





95
19
NUC
188
RTPSPRRR
T
R
8
−0.0002
−0.0002
0.0033
0.0014
0.0002
648





95
19
NUC
188
RTPSPRRRR
T
R
9
0.0054
0.0005
0.2000
0.0016
0.0003
649





100
20
POL
357
RVTGGVFLVDK
V
K
11
0.0190
0.0290
−0.0002
−0.0003
0.0001
650





90
18
X
65
SAGPCALR
A
R
8
−0.0002
0.0020
0.0029
0.0024
0.0360
651





95
19
POL
520
SAICSVVR
A
R
8
−0.0002
0.0071
0.0280
0.0081
0.0690
652





95
19
POL
520
SAICSVVRR
A
R
9
0.0058
0.2100
0.1500
0.0650
0.3800
653





90
18
POL
771
SALNPADDPSR
A
R
11
−0.0004
−0.0003
−0.0012
−0.0023
0.0003
654





75
15
POL
565
SLGIHLNPNK
L
K
10





655





90
18
X
64
SSAGPCALR
S
R
9
0.0080
0.1400
0.3300
0.1600
0.7500
656





95
19
NUC
170
STLPETTVVR
T
R
10
0.0007
0.0600
0.0080
0.0240
0.0250
657





95
19
NUC
170
STLPETTVVRR
T
R
11
0.0150
1.4000
0.1000
0.1600
0.3100
658





80
16
ENV
85
STNRQSGR
T
R
8





659





75
15
X
104
STTDLEAYFK
T
K
10
0.0066
2.7000



660





85
17
POL
716
TAELLAACFAR
A
R
11
0.0006
0.0023
0.0066
0.1600
0.0590
661





95
19
NUC
171
TLPETTVVR
L
R
9
0.0008
0.0002
0.0009
0.0024
0.0180
662





95
19
NUC
171
TLPETTVVRR
L
R
10
0.0007
0.0230
0.0006
0.0120
0.0440
663





95
19
NUC
171
TLPETTVVRRR
L
R
11
0.0005
0.0160
0.0061
0.0710
0.6400
664





100
20
POL
150
TLWKAGILYK
L
K
10
5.3000
0.3600
0.0051
0.0010
0.0130
665





100
20
POL
150
TLWKAGILYKR
L
R
11
0.0082
0.0095
0.1000
0.1100
0.0640
666





95
19
POL
519
TSAICSVVR
S
R
9
0.0005
0.0008
0.0600
0.0200
0.0820
667





95
19
POL
519
TSAICSVVRR
S
R
10
0.0018
0.0006
0.0030
0.0066
0.0048
668





75
15
X
105
TTDLEAYFK
T
K
9
0.0006
0.9200
0.0006
0.0012
0.0170
669





75
15
ENV
278
TTSTGPCK
T
K
8





670





80
16
NUC
175
TTVVRRRGR
T
R
9
0.0008
0.0005
0.2500
0.1400
0.0095
671





80
16
NUC
176
TVVRRRGR
V
R
8
0.0003
0.0001



672





80
16
NUC
176
TVVRRRGRSPR
V
R
11





673





90
18
X
133
VLGGCRHK
L
K
8
0.0150
0.0002
−0.0005
−0.0009
0.0001
674





80
16
ENV
177
VLQAGFFLLTR
L
R
11





675





90
18
NUC
120
VSFGVWIR
S
R
8
0.0040
0.0290
0.0750
0.0270
0.0360
676





100
20
POL
48
VSIPWTHK
S
K
8
0.0130
0.0170
0.0031
0.0013
0.0004
677





100
20
POL
358
VTGGVFLVDK
T
K
10
0.0390
0.0920
0.0002
0.0006
0.0022
678





100
20
POL
378
VVDFSQFSR
V
R
9
0.0015
0.0750
0.0013
0.0170
0.0330
679





80
16
NUC
177
VVRRRGRSPR
V
R
10
0.0027
0.0001



680





80
16
NUC
177
VVRRRGRSPRR
V
R
11





681





95
19
NUC
125
VVIRTPPAYR
I
R
9
0.0008
0.0005



682





90
18
POL
314
WLQFRNSK
L
K
8
−0.0002
0.0005
0.0020
0.0052
0.0001
683





85
17
NUC
26
WLWGMDIDPYK
L
K
11
0.0030
0.0013
−0.0003
0.0039
0.0490
684





100
20
POL
122
YLPLDKGIK
L
K
9
0.0001
0.0001
0.0006
0.0006
0.0002
685





90
18
NUC
118
YLVSFGVWIR
L
R
10
0.0005
0.0002



686





90
18
POL
538
YMDDVVLGAK
M
K
10
0.0330
0.0043
0.0002
0.0006
0.0001
687





80
16
POL
493
YSHPIILGFR
S
R
10





688





80
16
POL
493
YSHPIILGFRK
S
K
11





689
















TABLE X







HBV A24 SUPER MOTIF (With binding information)














Conservancy
Freq
Protein
Position
Sequence
String
A*2401
SEQ ID NO:


















95
19
POL
529
AFPHCLAF
XFXXXXXF

690






95
19
POL
529
AFPHCLAFSY
XFXXXXXXXY

691





95
19
POL
529
AFPHCLAFSYM
XFXXXXXXXXM

692





95
19
X
62
AFSSAGPCAL
XFXXXXXXXL
0.0012
693





90
18
POL
535
AFSYMDDVVL
XFXXXXXXXL
0.0009
694





95
19
POL
655
AFTFSPTY
XFXXXXXY

695





95
19
POL
655
AFTFSPTYKAF
XFXXXXXXXXF

696





95
19
POL
521
AICSVVRRAF
XIXXXXXXXF

697





90
18
NUC
58
AILCWGEL
XIXXXXXL

698





90
18
NUC
58
AILCWGELM
XIXXXXXXM

699





95
19
POL
642
ALMPLYACI
XLXXXXXXI

700





95
19
NUC
54
ALRQAILCW
XLXXXXXXW

701





80
16
ENV
108
AMQWNSTTF
XMXXXXXXF

702





95
19
POL
690
ATPTGWGL
XTXXXXXL

703





75
15
POL
690
ATPTGWGLAI
XTXXXXXXXI

704





95
19
POL
397
AVPNLQSL
XVXXXXXL

705





95
19
POL
397
AVPNLQSLTNL
XVXXXXXXXXL

706





100
20
NUC
131
AYRPPNAPI
XYXXXXXXI
0.0260
707





100
20
NUC
131
AYRPPNAPIL
XYXXXXXXXL
0.0220
708





75
15
POL
607
CFRKLPVNRPI
XFXXXXXXXX1

709





100
20
ENV
312
CIPIPSSW
XIXXXXXW

710





100
20
ENV
312
CIPIPSSWAF
XIXXXXXXXF

711





85
17
NUC
23
CLGWLWGM
XLXXXXXM

712





85
17
NUC
23
CLGWLWGMDI
XLXXXXXXXI

713





100
20
ENV
253
CLIFLLVL
XLXXXXXL

714





100
20
ENV
253
CLIFLLVLL
XLXXXXXXL

715





95
19
ENV
253
CLIFLLVLLDY
XLXXXXXXXXY

716





95
19
ENV
239
CLRRFIIF
XLXXXXXF

717





95
19
ENV
239
CLRRFIIFL
XLXXXXXXL

718





75
15
ENV
239
CLRRFIIFLF
XLXXXXXXXF

719





75
15
ENV
239
CLRRFIIFLFI
XLXXXXXXXXI

720





100
20
ENV
310
CTCIPIPSSW
XTXXXXXXXW

721





90
18
NUC
31
DIDPYKEF
XIXXXXXF

722





85
17
NUC
29
DLLDTASAL
XLXXXXXXL

723





85
17
NUC
29
DLLDTASALY
XLXXXXXXXY

724





95
19
POL
40
DLNLGNLNVSI
XLXXXXXXXXI

725





80
16
NUC
32
DTASALYREAL
XTXXXXXXXXL

726





85
17
POL
618
DWKVCQRI
XWXXXXXI

727





85
17
POL
618
DWKVCQRIVGL
XWXXXXXXXXL

728





90
18
ENV
262
DYQGMLPVCPL
XYXXXXXXXXL
0.0002
729





80
16
X
122
ELGEEIRL
XLXXXXXL

730





95
19
NUC
43
ELLSFLPSDF
XLXXXXXXXF

731





95
19
NUC
43
ELLSFLPSDFF
XLXXXXXXXXF

732





90
18
NUC
117
EYLVSFGVW
XYXXXXXXW

733





90
18
NUC
117
EYLVSFGVWI
XYXXXXXXXI
0.0340
734





100
20
ENV
382
FFCLWVYI
XFXXXXXI

735





80
16
ENV
182
FFLLTRIL
XFXXXXXL

736





80
16
ENV
182
FFLLTRILTI
XFXXXXXXXI

737





85
17
ENV
13
FFPDHQLDPAF
XFXXXXXXXXF

738





80
16
ENV
243
FIIFLFIL
XIXXXXXL

739





80
16
ENV
243
FIIFLFILL
XIXXXXXXL

740





80
16
ENV
243
FIIFLFILLL
XIXXXXXXXL

741





80
16
ENV
248
FILLLCLI
XIXXXXXI

742





80
16
ENV
248
FILLLCLIF
XIXXXXXXF

743





80
16
ENV
248
FILLLCLIFL
XIXXXXXXXL

744





80
16
ENV
248
FILLLCLIFLL
XIXXXXXXXXL

745





80
16
ENV
246
FLFILLLCL
XLXXXXXXL

746





80
16
ENV
246
FLFILLLCLI
XLXXXXXXXI

747





80
16
ENV
246
FLFILLLCLIF
XLXXXXXXXXF

748





75
15
ENV
171
FLGPLLVL
XLXXXXXL

749





95
19
POL
513
FLLAQFTSAI
XLXXXXXXXI

750





95
19
POL
562
FLLSLGIHL
XLXXXXXXL

751





80
16
ENV
183
FLLTRILTI
XLXXXXXXI

752





95
19
ENV
256
FLLVLLDY
XLXXXXXY

753





95
19
ENV
256
FLLVLLDYQGM
XLXXXXXXXXM

754





95
19
POL
656
FTFSPTYKAF
XTXXXXXXXF

755





95
19
POL
656
FTFSPTYKAFL
XTXXXXXXXXL

756





95
19
POL
635
FTQCGYPAL
XTXXXXXXL

757





95
19
POL
635
FTQCGYPALM
XTXXXXXXXM

758





95
19
ENV
346
FVGLSPTVW
XVXXXXXXW

759





95
19
ENV
346
FVGLSPTVWL
XVXXXXXXXL

760





90
18
X
132
FVLGGCRHKL
XVXXXXXXXL

761





95
19
ENV
342
FVQWFVGL
XVXXXXXL

762





90
18
POL
766
FVYVPSAL
XVXXXXXL

763





95
19
POL
630
GFAAPFTQCGY
XFXXXXXXXXY

764





80
16
ENV
181
GFFLLTRI
XFXXXXXI

765





80
16
ENV
181
GFFLLTRIL
XFXXXXXXL

766





80
16
ENV
181
GFFLLTRILTI
XFXXXXXXXXI

767





95
19
ENV
12
GFFPDHQL
XFXXXXXL

768





75
15
ENV
170
GFLGPLLVL
XFXXXXXXL

769





80
16
POL
500
GFRKIPMGVGL
XFXXXXXXXXL

770





95
19
POL
627
GLLGFAAPF
XLXXXXXXF

771





95
19
POL
509
GLSPFLLLAQF
XLXXXXXXXF

772





100
20
ENV
348
GLSPTVWL
XLXXXXXL

773





75
15
ENV
348
GLSPTVWLSVI
XLXXXXXXXXI

774





85
17
NUC
29
GMDIDPYKEF
XMXXXXXXXF

775





90
18
ENV
265
GMLPVCPL
XMXXXXXL

776





90
18
POL
735
GTDNSVVL
XTXXXXXL

777





75
15
ENV
13
GTNLSVPNPL
XTXXXXXXXL

778





80
16
POL
763
GTSFVYVPSAL
XTXXXXXXXXL

779





80
16
POL
507
GVGLSPFL
XVXXXXXL

780





80
16
POL
507
GVGLSPFLL
XVXXXXXXL

781





95
19
NUC
123
GVWIRTPPAY
XVXXXXXXXY

782





85
17
NUC
25
GWLWGMDI
XWXXXXXI

783





85
17
NUC
25
GWLWGMDIDPY
XWXXXXXXXXY

784





85
17
ENV
65
GWSPQAQGI
XWXXXXXXI
0.0024
785





85
17
ENV
65
GWSPQAQGIL
XWXXXXXXXL
0.0003
786





95
19
POL
639
GYPALMPL
XYXXXXXL

787





95
19
POL
639
GYPALMPLY
XYXXXXXXY
0.0490
788





95
19
ENV
234
GYRWMCLRRF
XYXXXXXXXF
0.0110
789





95
19
ENV
234
GYRWMCLRRFI
XYXXXXXXXXI

790





85
17
POL
579
GYSLNFMGY
XYXXXXXXY
0.0002
791





75
15
POL
579
GYSLNFMGYVI
XYXXXXXXXXI

792





80
16
POL
820
HFASPLHVAW
XFXXXXXXXW

793





75
15
POL
7
HFRKLLLL
XFXXXXXL

794





80
16
POL
435
HLLVGSSGL
XLXXXXXXL

795





75
15
POL
569
HLNPNKTKRW
XLXXXXXXXW

796





80
16
POL
491
HLYSHPII
XLXXXXXI

797





80
16
POL
491
HLYSHPIIL
XLXXXXXXL

798





80
16
POL
491
HLYSHPIILGF
XLXXXXXXXXF

799





85
17
POL
715
HTAELLAACF
XTXXXXXXXF

800





100
20
NUC
52
HTALRQAI
XTXXXXXI

801





95
19
NUC
52
HTALRQAIL
XTXXXXXXL

802





95
19
NUC
52
HTALRQAILCW
XTXXXXXXXXW

803





100
20
POL
149
HTLWKAGI
XTXXXXXI

804





100
20
POL
149
HTLWKAGIL
XTXXXXXXL

805





100
20
POL
149
HTLWKAGILY
XTXXXXXXXY

806





100
20
POL
146
HYLHTLWKAGI
XYXXXXXXXXI

807





100
20
ENV
381
IFFCLWVY
XFXXXXXY

808





100
20
ENV
381
IFFCLWVYI
XFXXXXXXI
0.0087
809





80
16
ENV
245
IFLFILLL
XFXXXXXL

810





80
16
ENV
245
IFLFILLLCL
XFXXXXXXXL

811





80
16
ENV
245
IFLFILLLCLI
XFXXXXXXXXI

812





95
19
ENV
255
IFLLVLLDY
XFXXXXXXY

813





80
16
ENV
244
IIFLFILL
XIXXXXXL

814





80
16
ENV
244
IIFLFILLL
XIXXXXXXL

815





80
16
ENV
244
IIFLFILLLCL
XIXXXXXXXXL

816





80
16
POL
497
IILGFRKI
XIXXXXXI

817





80
16
POL
497
IILGFRKIPM
XIXXXXXXXM

818





90
18
NUC
59
ILCWGELM
XLXXXXXM

819





80
16
POL
498
ILGFRKIPM
XLXXXXXXM

820





100
20
ENV
249
ILLLCLIF
XLXXXXXF

821





100
20
ENV
249
ILLLCLIFL
XLXXXXXXL

822





100
20
ENV
249
ILLLCLIFLL
XLXXXXXXXL

823





80
16
POL
760
ILRGTSFVY
XLXXXXXXY

824





95
19
ENV
188
ILTIPQSL
XLXXXXXL

825





90
18
ENV
188
ILTIPQSLDSW
XLXXXXXXXXW

826





90
18
POL
625
IVGLLGFAAPF
XVXXXXXXXXF

827





85
17
ENV
358
IWMMWYWGPS
XWXXXXXXXXL
0.0004
828





95
19
POL
395
KFAVPNLQSL
XFXXXXXXXL
0.0020
829





80
16
POL
503
KIPMGVGL
XIXXXXXL

830





80
16
POL
503
KIPMGVGLSPF
XIXXXXXXXXF

831





85
17
NUC
21
KLCLGWLW
XLXXXXXW

832





85
17
NUC
21
KLCLGWLWGM
XLXXXXXXXM

833





95
19
POL
489
KLHLYSHPI
XLXXXXXXI

834





80
16
POL
489
KLHLYSHPII
XLXXXXXXXI

835





80
16
POL
489
KLHLYSHPIIL
XLXXXXXXXXL

836





75
15
POL
108
KLIMPARF
XLXXXXXF

837





75
15
POL
108
KLIMPARFY
XLXXXXXXY

838





80
16
POL
610
KLPVNRPI
XLXXXXXI

839





80
16
POL
610
KLPVNRPIDW
XLXXXXXXXW

840





95
19
POL
574
KTKRWGYSL
XTXXXXXXL

841





85
17
POL
574
KTKRWGYSLNF
XTXXXXXXXXF

842





85
17
POL
620
KVCQRIVGL
XVXXXXXXL

843





85
17
POL
620
KVCQRIVGLL
XVXXXXXXXL

844





95
19
POL
55
KVGNFTGL
XVXXXXXL

845





95
19
POL
55
KVGNFTGLY
XVXXXXXXY

846





85
17
X
91
KVLHKRTL
XVXXXXXL

847





85
17
X
91
KVLHKRTLGL
XVXXXXXXXL

848





100
20
POL
121
KYLPLDKGI
XYXXXXXXI
0.0028
849





85
17
POL
745
KYTSFPWL
XYXXXXXL

850





85
17
POL
745
KYTSFPWLL
XYXXXXXXL
3.6000
851





80
16
ENV
247
LFILLLCL
XFXXXXXL

852





80
16
ENV
247
LFILLLCLI
XFXXXXXXI

853





80
16
ENV
247
LFILLLCLIF
XFXXXXXXXF

854





80
16
ENV
247
LFILLLCLIFL
XFXXXXXXXXL

855





100
20
ENV
254
LIFLLVLL
XIXXXXXL

856





95
19
ENV
254
LIFLLVLLDY
XIXXXXXXXY

857





100
20
POL
109
LIMPARFY
XIXXXXXY

858





95
19
POL
514
LLAQFTSAI
XLXXXXXXI

859





100
20
ENV
251
LLCLIFLL
XLXXXXXL

860





100
20
ENV
251
LLCLIFLLVL
XLXXXXXXXL

861





100
20
ENV
251
LLCLIFLLVLL
XLXXXXXXXXL

862





85
17
NUC
30
LLDTASAL
XLXXXXXL

863





85
17
NUC
30
LLDTASALY
XLXXXXXXY

864





95
19
ENV
260
LLDYQGML
XLXXXXXL

865





80
16
POL
752
LLGCAANW
XLXXXXXW

866





80
16
POL
752
LLGCAANWI
XLXXXXXXI

867





80
16
POL
752
LLGCAANWIL
XLXXXXXXXL

868





95
19
POL
628
LLGFAAPF
XLXXXXXF

869





75
15
ENV
63
LLGWSPQAQGI
XLXXXXXXXXI

870





100
20
ENV
250
LLLCLIFL
XLXXXXXL

871





100
20
ENV
250
LLLCLIFLL
XLXXXXXXL

872





100
20
ENV
250
LLLCLIFLLVL
XLXXXXXXXXL

873





100
20
ENV
378
LLPIFFCL
XLXXXXXL

874





100
20
ENV
378
LLPIFFCLW
XLXXXXXXW

875





100
20
ENV
378
LLPIFFCLWVY
XLXXXXXXXXY

876





95
19
NUC
44
LLSFLPSDF
XLXXXXXXF

877





95
19
NUC
44
LLSFLPSDFF
XLXXXXXXXF

878





95
19
POL
563
LLSLGIHL
XLXXXXXL

879





90
18
POL
407
LLSSNLSW
XLXXXXXW

880





90
18
POL
407
LLSSNLSWL
XLXXXXXXL

881





90
18
POL
407
LLSSNLSWLSL
XLXXXXXXXXL

882





80
16
ENV
184
LLTRILTI
XLXXXXXI

883





80
16
POL
436
LLVGSSGL
XLXXXXXL

884





95
19
ENV
257
LLVLLDYQGM
XLXXXXXXXM

885





95
19
ENV
257
LLVLLDYQGML
XLXXXXXXXXL

886





95
19
ENV
175
LLVLQAGF
XLXXXXXF

887





95
19
ENV
175
LLVLQAGFF
XLXXXXXXF

888





90
18
ENV
175
LLVLQAGFFL
XLXXXXXXXL

889





90
18
ENV
175
LLVLQAGFFLL
XLXXXXXXXXL

890





100
20
ENV
338
LLVPFVQW
XLXXXXXW

891





100
20
ENV
338
LLVPFVQWF
XLXXXXXXF

892





90
18
NUC
100
LLWFHISCL
XLXXXXXXL

893





85
17
NUC
100
LLWFHISCLTF
XLXXXXXXXXF

894





95
19
POL
643
LMPLYACI
XMXXXXXI

895





75
15
NUC
137
LTFGRETVL
XTXXXXXXL

896





75
15
NUC
137
LTFGRETVLEY
XTXXXXXXXXY

897





90
18
ENV
189
LTIPQSLDSW
XTXXXXXXXW

898





90
18
ENV
189
LTIPQSLDSWW
XTXXXXXXXXW

899





90
18
POL
404
LTNLLSSNL
XTXXXXXXL

900





90
18
POL
404
LTNLLSSNLSW
XTXXXXXXXXW

901





80
16
ENV
185
LTRILTIPQSL
XTXXXXXXXXL

902





85
17
POL
99
LTVNEKRRL
XTXXXXXXL

903





95
19
ENV
258
LVLLDYQGM
XVXXXXXXM

904





95
19
ENV
258
LVLLDYQGML
XVXXXXXXXL

905





95
19
ENV
176
LVLQAGFF
XVXXXXXF

906





90
18
ENV
176
LVLQAGFFL
XVXXXXXXL

907





90
18
ENV
176
LVLQAGFFLL
XVXXXXXXXL

908





100
20
ENV
339
LVPFVQWF
XVXXXXXF

909





95
19
ENV
339
LVPFVQWFVGL
XVXXXXXXXXL

910





90
18
NUC
119
LVSFGVWI
XVXXXXXI

911





100
20
POL
377
LVVDFSQF
XVXXXXXF

912





90
18
NUC
101
LWFHISCL
XWXXXXXL

913





85
17
NUC
101
LWFHISCLTF
XWXXXXXXXF

914





85
17
NUC
27
LWGMDIDPY
XWXXXXXXY

915





100
20
POL
151
LWKAGILY
XWXXXXXY

916





80
16
POL
492
LYSHPIIL
XYXXXXXL

917





80
16
POL
492
LYSHPIILGF
XYXXXXXXXF
1.1000
918





85
17
ENV
360
MMWYWGPSL
XMXXXXXXL
0.0012
919





85
17
ENV
360
MMWYWGPSLY
XMXXXXXXXY
0.0001
920





85
17
ENV
361
MWYWGPSL
XWXXXXXL

921





85
17
ENV
361
MWYWGPSLY
XWXXXXXXY
0.0027
922





95
19
POL
561
NFLLSLGI
XFXXXXXI

923





95
19
POL
561
NFLLSLGIHL
XFXXXXXXXL
0.0099
924





95
19
POL
42
NLGNLNVSI
XLXXXXXXI

925





95
19
POL
42
NLGNLNVSIPW
XLXXXXXXXXW

926





90
18
POL
406
NLLSSNLSW
XLXXXXXXW

927





90
18
POL
406
NLLSSNLSWL
XLXXXXXXXL

928





95
19
POL
45
NLNVSIPW
XLXXXXXW

929





100
20
POL
400
NLQSLTNL
XLXXXXXL

930





100
20
POL
400
NLQSLTNLL
XLXXXXXXL

931





75
15
ENV
15
NLSVPNPL
XLXXXXXL

932





75
15
ENV
15
NLSVPNPLGF
XLXXXXXXXF

933





80
16
POL
758
NWILRGTSF
XWXXXXXXF

934





80
16
POL
758
NWILRGTSFVY
XWXXXXXXXXY

935





95
19
POL
512
PFLLAQFTSAI
XFXXXXXXXXI

936





95
19
POL
634
PFTQCGYPAL
XFXXXXXXXL
0.0002
937





95
19
POL
634
PFTQCGYPALM
XFXXXXXXXXM

938





95
19
ENV
341
PFVQWFVGL
XFXXXXXXL
0.0003
939





85
17
POL
616
PIDWKVCQRI
XIXXXXXXXI

940





100
20
ENV
380
PIFFCLWVY
XIXXXXXXY

941





100
20
ENV
380
PIFFFCLWVYI
XIXXXXXXXI

942





85
17
POL
713
PIHTAELL
XIXXXXXL

943





80
16
POL
496
PIILGFRKI
XIXXXXXXI

944





80
16
POL
496
PIILGFRKIPM
XIXXXXXXXXM

945





100
20
ENV
314
PIPSSWAF
XIXXXXXF

946





100
20
POL
124
PLDKGIKPY
XLXXXXXXY

947





100
20
POL
124
PLDKGIKPYY
XLXXXXXXXY

948





95
19
POL
20
PLEEELPRL
XLXXXXXXL

949





95
19
ENV
10
PLGFFPDHQL
XLXXXXXXXL

950





100
20
POL
427
PLHPAAMPHL
XLXXXXXXXL

951





100
20
POL
427
PLHPAAMPHLL
XLXXXXXXXXL

952





100
20
ENV
377
PLLPIFFCL
XLXXXXXXL

953





100
20
ENV
377
PLLPIFFCLW
XLXXXXXXXW

954





95
19
ENV
174
PLLVLQAGF
XLXXXXXXF

955





95
19
ENV
174
PLLVLQAGFF
XLXXXXXXXF

956





90
18
ENV
174
PLLVLQAGFFL
XLXXXXXXXXL

957





80
16
POL
711
PLPIHTAEL
XLXXXXXXL

958





80
16
POL
711
PLPIHTAELL
XLXXXXXXXL

959





75
15
POL
2
PLSYQHFRKL
XLXXXXXXXL

960





75
15
POL
2
PLSYQHFRKLL
XLXXXXXXXXL

961





85
17
POL
98
PLTVNEKRRL
XLXXXXXXXL

962





80
16
POL
505
PMGVGLSPF
XMXXXXXXF

963





80
16
POL
505
PMGVGLSPFL
XMXXXXXXXL

964





80
16
POL
505
PMGVGLSPFLL
XMXXXXXXXXL

965





75
15
POL
692
PTGWGLAI
XTXXXXXI

966





85
17
POL
797
PTTGRTSL
XTXXXXXL

967





85
17
POL
797
PTTGRTSLY
XTXXXXXXY

968





80
16
NUC
15
PTVQASKL
XTXXXXXL

969





80
16
NUC
15
PTVQASKLCL
XTXXXXXXXL

970





75
15
ENV
351
PTVWLSVI
XTXXXXXI

971





75
15
ENV
351
PTVWLSVIW
XTXXXXXXW

972





75
15
ENV
351
PTVWLSVIWM
XTXXXXXXXM

973





85
17
POL
612
PVNRPIDW
XVXXXXXW

974





80
16
POL
750
PWLLGCAANW
XWXXXXXXXW

975





80
16
POL
750
PWLLGCAANWI
XWXXXXXXXXI

976





100
20
POL
51
PWTHKVGNF
XWXXXXXXF
0.0290
977





80
16
X
8
QLDPARDVL
XLXXXXXXL

978





80
16
X
8
QLDPARDVLCL
XLXXXXXXXXL

979





90
18
NUC
99
QLLWFHISCL
XLXXXXXXXL

980





95
19
POL
685
QVFADATPTGW
XVXXXXXXXXW

981





95
19
ENV
344
QWFVGLSPTVW
XWXXXXXXXX

982





75
15
ENV
242
RFIIFLFI
XFXXXXXI

983





75
15
ENV
242
RFIIFLFIL
XFXXXXXXL

984





75
15
ENV
242
RFIIFLFILL
XFXXXXXXXL

985





75
15
ENV
242
RFIIFLFILLL
XFXXXXXXXXL

986





100
20
ENV
332
RFSWLSLL
XFXXXXXL

987





100
20
ENV
332
RFSWLSLLVPF
XFXXXXXXXXF

988





80
16
ENV
187
RILTIPQSL
XIXXXXXXL

989





90
18
POL
624
RIVGLLGF
XIXXXXXF

990





75
15
POL
106
RLKLIMPARF
XLXXXXXXXF

991





75
15
POL
106
RLKLIMPARFY
XLXXXXXXXXY

992





95
19
POL
376
RLVVDFSQF
XLXXXXXXF

993





90
18
POL
353
RTPARVTGGVF
XTXXXXXXXXF

994





95
19
POL
36
RVAEDLNL
XVXXXXXL

995





90
18
POL
36
RVAEDLNLGNL
XVXXXXXXXXL

996





80
16
POL
818
RVHFASPL
XVXXXXXL

997





100
20
POL
357
RVTGGVFL
XVXXXXXL

998





85
17
POL
577
RWGYSLNF
XWXXXXXF

999





85
17
POL
577
RWGYSLNFM
XWXXXXXXM

1000





85
17
POL
577
RWGYSLNFMGY
XWXXXXXXXXY

1001





95
19
ENV
236
RWMCLRRF
XWXXXXXF

1002





95
19
ENV
236
RWMCLRRFI
XWXXXXXXI
0.0710
1003





95
19
ENV
236
RWMCLRRFII
XWXXXXXXXI
1.1000
1004





95
19
ENV
236
RWMCLRRFIIF
XWXXXXXXXXF

1005





100
20
POL
167
SFCGSPYSW
XFXXXXXXW
0.0710
1006





95
19
NUC
46
SFLPSDFF
XFXXXXXF

1007





80
16
POL
765
SFVYVPSAL
XFXXXXXXL

1008





100
20
POL
49
SIPWTHKVGNF
XIXXXXXXXXF

1009





95
19
ENV
194
SLDSWWTSL
XLXXXXXXL

1010





95
19
ENV
194
SLDSWWTSLNF
XLXXXXXXXXF

1011





95
19
POL
416
SLDVSAAF
XLXXXXXF

1012





95
19
POL
416
SLDVSAAFY
XLXXXXXXY

1013





100
20
ENV
337
SLLVPFVQW
XLXXXXXXW

1014





100
20
ENV
337
SLLVPFVQWF
XLXXXXXXXF

1015





75
15
POL
581
SLNFMGYVI
XLXXXXXXI

1016





95
19
X
54
SLRGLPVCAF
XLXXXXXXXF

1017





90
18
POL
403
SLTNLLSSNL
XLXXXXXXXL

1018





75
15
X
104
STTDLEAY
XTXXXXXY

1019





75
15
X
104
STTDLEAYF
XTXXXXXXF

1020





75
15
ENV
17
SVPNPLGF
XVXXXXXF

1021





85
17
POL
548
SVQHLESL
XVXXXXXL

1022





80
16
ENV
330
SVRFSWLSL
XVXXXXXXL

1023





80
16
ENV
330
SVPFSWLSLL
XVXXXXXXXL

1024





90
18
POL
739
SVVLSRKY
XVXXXXXY

1025





85
17
POL
739
SVVLSRKYTSF
XVXXXXXXXXF

1026





95
19
POL
524
SVVRRAFPHCL
XVXXXXXXXXL

1027





95
19
POL
413
SWLSLDVSAAF
XWXXXXXXXXF

1028





100
20
ENV
334
SWLSLLVPF
XWXXXXXXF
0.3900
1029





95
19
POL
392
SWPKFAVPNL
XWXXXXXXXL
5.6000
1030





100
20
ENV
197
SWWTSLNF
XWXXXXXF

1031





95
19
ENV
197
SWWTSLNFL
XWXXXXXXL
0.3800
1032





90
18
POL
537
SYMDDVVL
XYXXXXXL

1033





75
15
POL
4
SYQHFRKL
XYXXXXXL

1034





75
15
POL
4
SYQHFRKLL
XYXXXXXXL
0.0051
1035





75
15
POL
4
SYQHFRKLLL
XYXXXXXXXL
0.0660
1036





75
15
POL
4
SYQHFRKLLLL
XYXXXXXXXXL

1037





75
15
NUC
138
TFGRETVL
XFXXXXXL

1038





75
15
NUC
138
TFGRETVLEY
XFXXXXXXY

1039





75
15
NUC
138
TFGRETVLEYL
XFXXXXXXXXL

1040





95
19
POL
657
TFSPTYKAF
XFXXXXXXF
0.0060
1041





95
19
POL
657
TFSPTYKAF
XFXXXXXXL
0.0043
1042





90
18
ENV
190
TIPQSLDSW
XIXXXXXXW

1043





90
18
ENV
190
TIPQSLDSWW
XIXXXXXXXW

1044





100
20
POL
150
TLWKAGIL
XLXXXXXL

1045





100
20
POL
150
TLWKAGILY
XLXXXXXXY

1046





75
15
X
105
TTDLEAYF
XTXXXXXF

1047





85
17
POL
798
TTGRTSLY
XTXXXXXY

1048





85
17
POL
100
TVNEKRRL
XVXXXXXL

1049





80
16
NUC
16
TVQASKLCL
XVXXXXXXL

1050





80
16
NUC
16
TVQASKLCLGW
XVXXXXXXXXW

1051





75
15
ENV
352
TVWLSVIW
XVXXXXXW

1052





75
15
ENV
352
TVWLSVIWM
XVXXXXXXM

1053





95
19
POL
686
VFADATPTGW
XFXXXXXXXW
0.0180
1054





75
15
X
131
VFVLGGCRHKL
XFXXXXXXXXL

1055





85
17
POL
543
VLGAKSVQHL
XLXXXXXXXL

1056





90
18
X
133
VLGGCRHKL
XLXXXXXXL

1057





85
17
X
92
VLHKRTLGL
XLXXXXXXL

1058





95
19
ENV
259
VLLDYQGM
XLXXXXXM

1059





95
19
ENV
259
VLLDYQGML
XLXXXXXXL

1060





95
19
ENV
177
VLQAGFFL
XLXXXXXL

1061





95
19
ENV
177
VLQAGFFLL
XLXXXXXXL

1062





85
17
POL
741
VLSRKYTSF
XLXXXXXXF

1063





85
17
POL
741
VLSRKYTSFPW
XLXXXXXXXXW

1064





80
16
POL
542
VVLGAKSVQHL
XVXXXXXXXXL

1065





85
17
POL
740
VVLSRKYTSF
XVXXXXXXXF

1066





95
19
POL
525
VVRRAFPHCL
XVXXXXXXXL

1067





95
19
NUC
124
VWIRTPPAY
XWXXXXXXY

1068





75
15
ENV
353
VWLSVIWM
XWXXXXXM

1069





90
18
NUC
102
WFHISCLTF
XFXXXXXXF
0.0300
1070





95
19
ENV
345
WFVGLSPTVW
XFXXXXXXXW
0.0120
1071





95
19
ENV
345
WFVGLSPTVWL
XFXXXXXXXXL

1072





80
16
POL
759
WILRGTSF
XIXXXXXF

1073





80
16
POL
759
WILRGTSFVY
XIXXXXXXXY

1074





95
19
NUC
125
WIRTPPAY
XIXXXXXY

1075





80
16
POL
751
WLLGCAANW
XLXXXXXXW

1076





80
16
POL
751
WLLGCAANWI
XLXXXXXXXI

1077





80
16
POL
751
WLLGCAANWIL
XLXXXXXXXXL

1078





95
19
POL
414
WLSLDVSAAF
XLXXXXXXXF

1079





95
19
POL
414
WLSLDVSAAFY
XLXXXXXXXXY

1080





100
20
ENV
335
WLSLLVPF
XLXXXXXF

1081





100
20
ENV
335
WLSLLVPFVQW
XLXXXXXXXXW

1082





85
17
NUC
26
WLWGMDIDPY
XLXXXXXXXY

1083





95
19
ENV
237
WMCLRRFI
XMXXXXXI

1084





95
19
ENV
237
WMCLRRFII
XMXXXXXXI
0.0230
1085





95
19
ENV
237
WMCLRRFIIF
XMXXXXXXXF
0.0013
1086





95
19
ENV
237
WMCLRRFIIFL
XMXXXXXXXXL

1087





85
17
ENV
359
WMMWYWGPSL
XMXXXXXXXL
0.0005
1088





85
17
ENV
359
WMMWYWGPSL
XMXXXXXXXXY

1089





100
20
POL
52
WTHKVGNF
XTXXXXXF

1090





95
19
POL
52
WTHKVGNFTGL
XTXXXXXXXXL

1091





95
19
ENV
198
WWTSLNFL
XWXXXXXL

1092





85
17
ENV
362
WYWGPSLY
XYXXXXXY
0.0001
1093





100
20
POL
147
YLHTLWKAGI
XLXXXXXXXI

1094





100
20
POL
147
YLHTLWKAGIL
XLXXXXXXXXL

1095





100
20
POL
122
YLPLDKGI
XLXXXXXI

1096





100
20
POL
122
YLPLDKGIKPY
XLXXXXXXXXY

1097





90
18
NUC
118
YLVSFGVW
XLXXXXXW

1098





90
18
NUC
118
YLVSFGVWI
XLXXXXXXI

1099





85
17
POL
746
YTSFPWLL
XTXXXXXL

1100
















TABLE XI







HBV B07 SUPER MOTIF (With binding information)





















Conserv-
Fre-




C-










ancy
quency
Protein
Position
Sequence
P2
term
AA
B*0702
B*3501
B*5101
B*5301
B*5401
SEQ ID NO
























75
15
X
146
APCNFFTSA
P
A
9





1101






95
19
POL
633
APFTQCGY
P
Y
8
0.0001
0.0012
0.0019
0.0002
0.0002
1102





95
19
POL
633
APFTQCGYPA
P
A
10
0.0029
0.0001

0.0002
1.4000
1103





95
19
POL
633
APFTQCGYPAL
P
L
11
0.2300
0.0010
0.0004
−0.0003
0.0093
1104





100
20
ENV
232
CPGYRWMCL
P
L
9





1105





80
16
NUC
14
CPTVQASKL
P
L
9





1106





80
16
NUC
14
CPTVQASKLCL
P
L
11





1107





80
16
X
10
DPARDVLCL
P
L
9





1108





80
16
ENV
122
DPRVRGLY
P
Y
8





1109





90
18
POL
778
DPSRGRLGL
P
L
9
0.0120
0.0001
0.0001
0.0001
0.0001
1110





90
18
NUC
33
DPYKEFGA
P
A
8
0.0001
0.0001
0.0019
0.0002
0.0019
1111





75
15
ENV
130
FPAGGSSSGTV
P
V
11





1112





90
18
ENV
14
FPDHQLDPA
P
A
9





1113





85
17
ENV
14
FPDHQLDPAF
P
F
10
0.0002
0.0016
0.0003
0.0011
0.0021
1114





95
19
POL
530
FPHCLAFSY
P
Y
9
0.0001
0.5250
0.0665
0.5400
0.0199
1115





95
19
POL
530
FPHCLAFSYM
P
M
10
0.0990
0.2200
0.0900
0.0790
0.0480
1116





75
15
POL
749
FPWLLGCA
P
A
8





1117





75
15
POL
749
FPWLLGCAA
P
A
9





1118





75
15
POL
749
FPWLLGCAANW
P
W
11





1119





90
18
X
67
GPCALRFTSA
P
A
10
0.0900
0.0001
0.0001
0.0002
0.0035
1120





95
19
POL
19
GPLEEELPRL
P
L
10
0.0001
0.0001
0.0002
0.0001
0.0002
1121





90
18
POL
19
GPLEEELPRLA
P
A
11
−0.0002
0.0001
0.0001
−0.0003
0.0001
1122





95
19
ENV
173
GPLLVLQA
P
A
8
0.0003
0.0001
0.0110
0.0002
0.0065
1123





95
19
ENV
173
GPLLVLQAGF
P
F
10
0.0001
0.0001
0.0002
0.0001
0.0002
1124





95
19
ENV
173
GPLLVLQAGFF
P
F
11
0.0011
0.0001
0.0001
0.0008
0.0009
1125





95
17
POL
97
GPLTVNEKRRL
P
L
11
0.0031
0.0001
0.0001
−0.0003
0.0001
1126





100
20
POL
429
HPAAMPHL
P
L
8
0.0650
0.0004
0.3100
0.0037
0.0160
1127





100
20
POL
429
HPAAMPHLL
P
L
9
0.0980
0.0270
0.0110
0.0500
0.0120
1128





85
17
POL
429
HPAAMPHLLV
P
V
10
0.0160
0.0020
0.0078
0.0140
0.0170
1129





80
16
POL
495
HPIILGFRKI
P
I
10





1130





100
20
ENV
313
IPIPSSWA
P
A
8
0.0004
0.0004
0.0019
0.0002
0.0600
1131





100
20
ENV
313
IPIPSSWAF
P
F
9
0.1300
2.7679
2.3500
0.7450
0.0034
1132





80
16
ENV
313
IPIPSSWAFA
P
A
10
0.0013
0.0024

0.0014
0.4500
1133





80
16
POL
504
IPMGVGLSPF
P
F
10





1134





80
16
POL
504
IPMGVGLSPFL
P
L
11





1135





90
18
ENV
191
IPQSLDSW
P
W
8





1136





90
18
ENV
191
IPQSLDSWW
P
W
9





1137





80
16
ENV
315
IPSSWAFA
P
A
8





1138





100
20
POL
50
IPWTHKVGNF
P
F
10
0.0013
0.0001
0.0007
0.0001
0.0002
1139





100
20
ENV
379
LPIFFCLW
P
W
8
0.0001
0.0001
0.0360
0.1400
0.0035
1140





100
20
ENV
379
LPIFFCLWV
P
V
9





1141





100
20
ENV
379
LPIFFCLWVY
P
Y
10
0.0002
0.0079
0.0002
0.0006
0.0002
1142





100
20
ENV
379
LPIFFCLWVYI
P
I
11
0.0002
0.0001
0.0043
0.0139
0.0021
1143





85
17
POL
712
LPIHTAEL
P
L
8





1144





85
17
POL
712
LPIHTAELL
P
L
9
0.0040
0.0630
0.0052
0.3100
0.0005
1145





85
17
POL
712
LPIHTAELLA
P
A
10
0.0018
0.0011

0.0016
0.3300
1146





85
17
POL
712
LPIHTAELLAA
P
A
11
0.0090
0.0027
−0.0003
0.0120
2.7500
1147





80
16
X
89
LPKVLHKRTL
P
L
10





1148





100
20
POL
123
LPLDKGIKPY
P
Y
10
0.0001
0.0290
0.0002
0.0003
0.0002
1149





100
20
POL
123
LPLDKGIKPYY
P
Y
11
−0.0002
0.0009
0.0001
0.0007
0.0001
1150





95
19
X
58
LPVCAFSSA
P
A
9
0.0480
0.0710
0.0110
0.0009
19.0000
1151





80
16
POL
611
LPVNRPIDW
P
W
9





1152





80
16
POL
611
LPVNRPIDWKV
P
V
11





1153





80
16
POL
433
MPHLLVGSSGL
P
L
11





1154





100
20
POL
1
MPLSYQHF
P
F
8
0.0001
0.0097
0.0120
0.0370
0.0190
1155





75
15
POL
1
MPLSYQHFRKL
P
L
11





1156





90
18
POL
774
NPADDPSRGRL
P
L
11
0.0120
0.0001
0.0001
−0.0003
0.0001
1157





95
19
ENV
9
NPLGFFPDHQL
P
L
11
0.0012
0.0021
0.0001
0.0028
0.0001
1158





75
15
POL
571
NPNKTKRW
P
W
8





1159





75
15
POL
571
NPNKTKRWGY
P
Y
10





1160





95
19
NUC
129
PPAYRPPNA
P
A
9
0.0001
0.0001
0.0001
0.0002
0.0003
1161





95
19
NUC
129
PPAYRPPNAPI
P
I
11
0.0003
0.0001
0.0001
−0.0003
0.0001
1162





85
17
ENV
58
PPHGGLLGW
P
W
9
0.0001
0.0002
0.0001
0.0003
0.0002
1163





100
20
NUC
134
PPNAPILSTL
P
L
10
0.0001
0.0001
0.0035
0.0001
0.0002
1164





80
16
POL
615
RPIDWKVCQRI
P
I
11





1165





100
20
NUC
133
RPPNAPIL
P
L
8
0.0076
0.0001
0.0280
0.0002
0.0002
1166





100
20
NUC
133
RPPNAPILSTL
P
L
11
0.1300
0.0001
0.0018
−0.0003
0.0001
1167





100
20
NUC
44
SPEHCSPHHTA
P
A
11
−0.0002
0.0001
0.0001
−0.0003
0.0011
1168





95
19
POL
511
SPFLLAQF
P
F
8
0.5500
0.0009
0.0180
0.0009
0.0093
1169





95
19
POL
511
SPFLLAQFTSA
P
A
11
0.0820
0.0001
0.0001
−0.0003
12.0500
1170





100
20
NUC
49
SPHHTALRQA
P
A
10
0.0012
0.0001

0.0002
0.0035
1171





100
20
NUC
49
SPHHTALRQAI
P
I
11
0.5800
0.0001
0.0004
0.0005
0.0002
1172





85
17
ENV
67
SPQAQGIL
P
L
8





1173





85
17
POL
808
SPSVPSHL
P
L
8





1174





75
15
ENV
350
SPTVWLSV
P
V
8





1175





75
15
ENV
350
SPTVWLSVI
P
I
9





1176





75
15
ENV
350
SPTVWLSVIW
P
W
10





1177





75
15
ENV
350
SPTVWLSVIWM
P
M
11





1178





95
19
POL
659
SPTYKAFL
P
L
8
0.3900
0.0001
0.0019
0.0002
0.0002
1179





90
18
POL
354
TPARVTGGV
P
V
9
0.0078
0.0001
0.0013
0.0001
0.0015
1180





90
18
POL
354
TPARVTGGVF
P
F
10
0.3200
0.1000
0.0001
0.0099
0.0006
1181





90
18
POL
354
TPARVTGGVFL
P
L
11
0.0950
0.0001
0.0001
0.0005
0.0005
1182





95
19
NUC
128
TPPAYRPPNA
P
A
10
0.0001
0.0001

0.0002
0.0100
1183





75
15
ENV
57
TPPHGGLL
P
L
8





1184





75
15
ENV
57
TPPHGGLLGW
P
W
10





1185





80
16
POL
691
TPTGWGLA
P
A
8





1186





75
15
POL
691
TPTGWGLAI
P
I
9





1187





95
19
ENV
340
VPFVQWFV
P
V
8
0.0010
0.0001
19.0000
0.0002
0.1100
1188





95
19
ENV
340
VPFVQWFVGL
P
L
10
0.0011
0.0001
0.0100
0.0001
0.0025
1189





95
19
POL
398
VPNLQSLTNL
P
L
10
0.0006
0.0001
0.0004
0.0001
0.0002
1190





95
19
POL
398
VPNLQSLTNLL
P
L
11
0.0004
0.0001
0.0001
−0.0003
0.0002
1191





90
18
POL
769
VPSALNPA
P
A
8
0.0011
0.0001
0.0070
0.0002
1.0000
1192





95
19
POL
393
WPKFAVPNL
P
L
9
0.0054
0.0002
0.0015
0.0001
0.0015
1193





95
19
POL
640
YPALMPLY
P
Y
8
0.0004
0.2600
0.4100
0.0450
0.0056
1194





95
19
POL
640
YPALMPLYA
P
A
9
0.0180
0.0480
0.0340
0.0140
16.0000
1195





95
19
POL
640
YPALMPLYACI
P
I
11
0.0040
0.0001
0.0470
0.0320
0.0700
1196
















TABLE XII







HBV B27 Super Motif (No binding data available)
















Position in
No. of
Sequence
Conservancy




Protein
Sequence
HBV
Amino Acids
Frequency
(%)
SeqID Num























1197






AYW
AHLSLRGL
51
8
19
95
1198





AYW
ARVTGGVF
356
8
18
90
1199





AYW
DHGAHLSL
48
8
19
95
1200





AYW
DHQLDPAF
16
8
18
90
1201





AYW
DKGIKPYY
126
8
20
100
1202





AYW
FHISCLTF
103
8
18
90
1203





AYW
FRKIPMGV
501
8
16
80
1204





AYR
GRETVLEY
140
8
15
75
1205





AYW
HHTALRQA
51
8
20
100
1206





AYW
IHTAELLA
714
8
17
85
1207





AYW
LHKRTLGL
93
8
18
90
1208





AYW
LHLYSHPI
490
8
19
95
1209





AYW
LRGLPVCA
55
8
19
95
1210





AYW
LRGTSFVY
761
8
16
80
1211





AYW
LRQAILCW
55
8
19
95
1212





AYW
LRRFIIFL
240
8
19
95
1213





AYW
NKTKRWGY
573
8
15
75
1214





AYW
NRPIDWKV
614
8
18
90
1215





AYW
NRRVAEDL
34
8
17
85
1216





AYW
PHCLAFSY
531
8
19
95
1217





AYW
PHGGLLGW
59
8
17
85
1218





AYW
PKFAVPNL
394
8
19
95
1219





AYR
QHFRKLLL
6
8
15
75
1220





AYW
RHYLHTLW
145
8
20
100
1221





AYW
RKYTSFPW
744
8
17
85
1222





AYW
RRAFPHCL
527
8
19
95
1223





AYW
RRFIIFLF
241
8
15
75
1224





AYW
SHPIILGF
494
8
16
80
1225





AYW
SKLCLGWL
20
8
18
90
1226





AYW
SRNLYVSL
472
8
16
80
1227





AYW
TKRWGYSL
575
8
19
95
1228





AYW
TRHYLHTL
144
8
20
100
1229





AYW
VRFSWLSL
331
8
16
80
1230





AYW
WKVCQRIV
619
8
17
85
1231





AYW
YRPPNAPI
132
8
20
100
1232





AYW
ARVTGGVFL
356
9
18
90
1233





AYW
EHCSPHHTA
46
9
20
100
1234





AYR
GRETVLEYL
140
9
15
75
1235





AYW
HHTALRQAI
51
9
20
100
1236





AYW
HKVGNFTGL
54
9
19
95
1237





AYW
IHTAELLAA
714
9
17
85
1238





AYW
KRWGYSLNF
576
9
17
85
1239





AYW
LHLYSHPII
490
9
16
80
1240





AYW
LHPAAMPHL
428
9
20
100
1241





AYW
LHTLWKAGI
148
9
20
100
1242





AYR
LKLIMPARF
107
9
15
75
1243





AYW
LRGLPVCAF
55
9
19
95
1244





AYW
LRGTSFVYV
761
9
16
80
1245





AYW
LRRFIIFLF
240
9
15
75
1246





AYW
PHCLAFSYM
531
9
19
95
1247





AYW
PHHTALRQA
50
9
20
100
1248





AYW
PKVLHKRTL
90
9
17
85
1249





AYR
QHFRKLLLL
6
9
15
75
1250





AYW
QRIVGLLGF
623
9
18
90
1251





AYW
RKIPMGVGL
502
9
16
80
1252





AYW
RKLPVNRPI
609
9
16
80
1253





AYW
RKYTSFPWL
744
9
17
85
1254





AYW
RRAFPHCLA
527
9
19
95
1255





AYW
RRFIIFLFI
241
9
15
75
1256





AYR
RRLKLIMPA
105
9
15
75
1257





AYW
RRVAEDLNL
35
9
18
90
1258





AYW
SKLCLGWLW
20
9
17
85
1259





AYW
SRKYTSFPW
743
9
17
85
1260





AYW
TRHYLHTLW
144
9
20
100
1261





AYW
VHFASPLHV
819
9
16
80
1262





AYW
VRFSWLSLL
331
9
16
80
1263





AYW
VRRAFPHCL
526
9
19
95
1264





AYW
YRPPNAPIL
132
9
20
100
1265





AYW
YRWMCLRRF
235
9
19
95
1266





AYW
AHLSLRGLPV
51
10
18
90
1267





AYW
AKSVQHLESL
546
10
17
85
1268





AYW
ARDVLCLRPV
12
10
15
75
1269





AYW
ARVTGGVFLV
356
10
18
90
1270





AYW
EHCSPHHTAL
46
10
20
100
1271





AYW
FRKIPMGVGL
501
10
16
80
1272





AYW
FRKLPVNRPI
608
10
16
80
1273





AYR
GRETVLEYLV
140
10
15
75
1274





AYW
HHTALRQAIL
51
10
19
95
1275





AYW
HKVGNFTGLY
54
10
19
95
1276





AYW
KRWGYSLNFM
576
10
17
85
1277





AYW
LHLYSHPIIL
490
10
16
80
1278





AYW
LHPAAMPHLL
428
10
20
100
1279





AYW
LHTLWKAGIL
148
10
20
100
1280





AYR
LKLIMPARFY
107
10
15
75
1281





AYW
LRRFIIFLFI
240
10
15
75
1282





AYW
NKTKRWGYSL
573
10
15
75
1283





AYW
NRRVAEDLNL
34
10
17
85
1284





AYW
PHHTALRQAI
50
10
20
100
1285





AYW
PHLLVGSSGL
434
10
16
80
1286





AYW
QRIVGLLGFA
623
10
18
90
1287





AYW
RHYLHTLWKA
145
10
20
100
1288





AYW
RKYTSFPWLL
744
10
17
85
1289





AYW
RRAFPHCLAF
527
10
19
95
1290





AYW
RRFIIFLFIL
241
10
15
75
1291





AYW
SRKYTSFPWL
743
10
17
85
1292





AYW
SRLVVDFSQF
375
10
19
95
1293





AYW
THKVGNFTGL
53
10
19
95
1294





AYW
TKRWGYSLNF
575
10
17
85
1295





AYW
TKYLPLDKGI
120
10
20
100
1296





AYW
TRILTIPQSL
186
10
16
80
1297





AYW
VHFASPLHVA
819
10
16
80
1298





AYW
VRFSWLSLLV
331
10
16
80
1299





AYW
VRRAFPHCLA
526
10
19
95
1300





AYW
WKVCQRIVGL
619
10
17
85
1301





AYW
YRWMCLRRFI
235
10
19
95
1302





AYW
DHGAHLSLRGL
48
11
19
95
1303





AYW
IHLNPNKTKRW
568
11
15
75
1304





AYW
IHTAELLAACF
714
11
17
85
1305





AYW
LHPAAMPHLLV
428
11
17
85
1306





AYW
LHTLWKAGILY
148
11
20
100
1307





AYW
LRQAILCWGEL
55
11
18
90
1308





AYW
LRRFIIFLFIL
240
11
15
75
1309





AYW
PHHTALRQAIL
50
11
19
95
1310





AYW
PKFAVPNLQSL
394
11
19
95
1311





AYW
PKVLHKRTLGL
90
11
17
85
1312





AYW
PRTPARVTGGV
352
11
18
90
1313





AYW
QRIVGLLGFAA
623
11
18
90
1314





AYW
RKLPVNRPIDW
609
11
16
80
1315





AYW
RRFIIFLFILL
241
11
15
75
1316





AYR
RRLKLIMPARF
105
11
15
75
1317





AYW
SHPIILGFRKI
494
11
16
80
1318





AYW
SKLCLGWLWGM
20
11
17
85
1319





AYW
SRKYTSFPWLL
743
11
17
85
1320





AYW
THKVGNFTGLY
53
11
19
95
1321





AYW
TKRWGYSLNFM
575
11
17
85
1322





AYW
TRHYLHTLWKA
144
11
20
100
1323





AYW
VHFASPLHVAW
819
11
16
80
1324





AYW
VRRAFPHCLAF
526
11
19
95
1325





AYW
WKVCQRIVGLL
619
11
17
85
1326





AYW
YRWMCLRRFII
235
11
19
95
1327
















TABLE XIII







HBV B58 Super Motif

















No. of
Sequence
Conservancy




Protein
Sequence
Position
Amino Acids
Frequency
(%)
SEQ ID NO:

















POL
AAMPHLLV
431
8
17
85
1328






NUC
ASALYREA
34
8
17
85
1329





POL
ASFCGSPY
166
8
20
100
1330





NUC
ASKLCLGW
19
8
18
90
1331





POL
ASPLHVAW
822
8
16
80
1332





ENV
ASVRFSWL
329
8
16
80
1333





POL
ATPTGWGL
690
8
19
95
1334





X
CALRFTSA
69
8
18
90
1335





NUC
CSPHHTAL
48
8
20
100
1336





POL
CSVVRRAF
523
8
19
95
1337





POL
ESRLVVDF
374
8
19
95
1338





NUC
ETVLEYLV
142
8
15
75
1339





POL
FARSRSGA
724
8
17
85
1340





POL
FASPLHVA
821
8
16
80
1341





POL
FSPTYKAF
658
8
19
95
1342





X
FSSAGPCA
63
8
19
95
1343





ENV
FSWLSLLV
333
8
20
100
1344





POL
FSYMDDVV
536
8
18
90
1345





POL
FTQCGYPA
635
8
19
95
1346





POL
FTSAICSV
518
8
19
95
1347





POL
GAKSVQHL
545
8
17
85
1348





POL
GTDNSVVL
735
8
18
90
1349





POL
HTAELLAA
715
8
17
85
1350





NUC
HTALRQAI
52
8
20
100
1351





POL
HTLWKAGI
149
8
20
100
1352





POL
LAQFTSAI
515
8
19
95
1353





NUC
LSFLPSDF
45
8
19
95
1354





POL
LSLDVSAA
415
8
19
95
1355





ENV
LSLLVPFV
336
8
20
100
1356





X
LSLRGLPV
53
8
19
95
1357





POL
LSRKYTSF
742
8
17
85
1358





POL
LSSNLSWL
408
8
18
90
1359





POL
LSWLSLDV
412
8
20
100
1360





NUC
LTFGRETV
108
8
19
95
1361





X
MSTTDLEA
103
8
16
80
1362





NUC
NAPILSTL
136
8
20
100
1363





POL
PAAMPHLL
430
8
20
100
1364





POL
PALMPLYA
641
8
19
95
1365





X
PARDVLCL
11
8
16
80
1366





POL
PARVTGGV
355
8
18
90
1367





NUC
PAYRPPNA
130
8
19
95
1368





POL
PSRGRLGL
779
8
18
90
1369





POL
PTGWGLAI
692
8
15
75
1370





POL
PTTGRTSL
797
8
17
85
1371





NUC
PTVQASKL
15
8
16
80
1372





ENV
PTVWLSVI
351
8
15
75
1373





POL
RAFPHCLA
528
8
19
95
1374





X
RTLGLSAM
96
8
24
120
1375





NUC
SALYREAL
35
8
18
90
1376





X
SSAGPCAL
64
8
19
95
1377





ENV
SSGTVNPV
136
8
15
75
1378





ENV
SSKPRQGM
5
8
18
90
1379





NUC
STLPETTV
141
8
20
100
1380





X
STTDLEAY
104
8
15
75
1381





NUC
TALRQAIL
53
8
19
95
1382





POL
TSAICSVV
519
8
19
95
1383





ENV
TSGFLGPL
168
8
16
80
1384





X
TTDLEAYF
105
8
15
75
1385





POL
TTGRTSLY
798
8
17
85
1386





POL
VSWPKFAV
391
8
19
95
1387





NUC
VSYVNVNM
115
8
20
100
1388





POL
VTGGVFLV
358
8
20
100
1389





ENV
WSPQAQGI
66
8
17
85
1390





POL
WTHKVGNF
52
8
20
100
1391





POL
YSLNFMGY
580
8
17
85
1392





POL
YTSFPWLL
746
8
17
85
1393





POL
AAPFTQCGY
632
9
19
95
1394





NUC
ASALYREAL
34
9
17
85
1395





NUC
ASKLCLGWL
19
9
18
90
1396





POL
ATPTGWGLA
690
9
16
80
1397





POL
CSRNLYVSL
471
9
16
80
1398





POL
DATPTGWGL
689
9
19
95
1399





ENV
DSWWTSLNF
196
9
19
95
1400





POL
EAGPLEEEL
17
9
20
100
1401





POL
FADATPTGW
687
9
19
95
1402





POL
FASPLHVAW
821
9
16
80
1403





POL
FAVPNLQSL
396
9
19
95
1404





POL
FSPTYKAFL
658
9
19
95
1405





X
FSSAGPCAL
63
9
19
95
1406





POL
FSYMDDVVL
536
9
18
90
1407





POL
FTFSPTYKA
656
9
19
95
1408





POL
FTGLYSSTV
59
9
18
90
1409





POL
FTQCGYPAL
635
9
19
95
1410





POL
FTSAICSVV
518
9
19
95
1411





X
GAHLSLRGL
50
9
19
95
1412





NUC
HTALRQAIL
52
9
19
95
1413





POL
HTLWKAGIL
149
9
20
100
1414





POL
KSVQHLESL
547
9
17
85
1415





POL
KTKRWGYSL
574
9
19
95
1416





POL
LAFSYMDDV
534
9
18
90
1417





NUC
LSFLPSDFF
45
9
19
95
1418





POL
LSLDVSAAF
415
9
19
95
1419





POL
LSPFLLAQF
510
9
19
95
1420





ENV
LSPTVWLSV
349
9
15
75
1421





NUC
LSTLPETTV
140
9
20
100
1422





ENV
LSVPNPLGF
16
9
15
75
1423





POL
LSYQHFRKL
3
9
15
75
1424





NUC
LTFGRETVL
137
9
15
75
1425





POL
LTNLLSSNL
404
9
18
90
1426





POL
LTVNEKRRL
99
9
17
85
1427





X
MSTTDLEAY
103
9
15
75
1428





POL
NSVVLSRKY
738
9
18
90
1429





POL
PAAMPHLLV
430
9
17
85
1430





POL
PARVTGGVF
355
9
18
90
1431





POL
PTTGRTSLY
797
9
17
85
1432





ENV
PTVWLSVIW
351
9
15
75
1433





POL
QAFTFSPTY
654
9
19
95
1434





NUC
QAILCWGEL
57
9
18
90
1435





NUC
QASKLCLGW
18
9
16
80
1436





POL
RAFPHCLAF
528
9
19
95
1437





ENV
RTGDPAPNM
167
9
16
80
1438





X
SAGPCALRF
65
9
18
90
1439





POL
SASFCGSPY
165
9
20
100
1440





POL
SSNLSWLSL
409
9
18
90
1441





ENV
SSSGTVNPV
135
9
15
75
1442





NUC
STLPETTVV
141
9
20
100
1443





X
STTDLEAYF
104
9
15
75
1444





POL
TAELLAACF
716
9
17
85
1445





NUC
TASALYREA
33
9
16
80
1446





POL
TSFVYVPSA
764
9
16
80
1447





ENV
TSGFLGPLL
168
9
15
75
1448





POL
TTGRTSLYA
798
9
17
85
1449





POL
VSIPWTHKV
48
9
20
100
1450





ENV
WSPQAQGIL
66
9
17
85
1451





ENV
WSSKPRQGM
4
9
18
90
1452





POL
YSHPIILGF
493
9
16
80
1453





POL
YSLNFMGYV
580
9
15
75
1454





POL
ASFCGSPYSW
166
10
20
100
1455





NUC
ASKLCLGWLW
19
10
17
85
1456





ENV
ASVRFSWLSL
329
10
16
80
1457





POL
ATPTGWGLAI
690
10
15
75
1458





X
CAFSSAGPCA
61
10
19
95
1459





ENV
CTCIPIPSSW
310
10
20
100
1460





ENV
CTIPAQGTSM
298
10
16
80
1461





POL
DATPTGWGLA
689
10
16
80
1462





ENV
DSWWTSLNFL
196
10
18
90
1463





NUC
DTASALYREA
32
10
16
80
1464





POL
FAAPFTQCGY
631
10
19
95
1465





ENV
FSWLSLLVPF
333
10
20
100
1466





POL
FTFSPTYKAF
656
10
19
95
1467





POL
FTQCGYPALM
635
10
38
190
1468





ENV
GSSSGTVNPV
134
10
15
75
1469





ENV
GTNLSVPNPL
13
10
15
75
1470





POL
GTSFVYVPSA
763
10
16
80
1471





POL
HTAELLAACF
715
10
17
85
1472





POL
HTLWKAGILY
149
10
20
100
1473





POL
LAFSYMDDVV
534
10
18
90
1474





POL
LSLDVSAAFY
415
10
19
95
1475





ENV
LSLLVPFVQW
336
10
20
100
1476





X
LSLRGLPVCA
53
10
19
95
1477





ENV
LSPTVWLSVI
349
10
15
75
1478





POL
LSRKYTSFPW
742
10
17
85
1479





POL
LSSNLSWLSL
408
10
18
90
1480





NUC
LSTLPETTVV
140
10
20
100
1481





POL
LSWLSLDVSA
412
10
20
100
1482





POL
LSYQHFRKLL
3
10
15
75
1483





ENV
LTIPQSLDSW
189
10
18
90
1484





X
MSTTDLEAYF
103
10
15
75
1485





POL
PADDPSRGRL
775
10
18
90
1486





ENV
PAGGSSSGTV
131
10
18
90
1487





POL
PALMPLYACI
641
10
19
95
1488





X
PAPCNFFTSA
145
10
15
75
1489





POL
PARVTGGVFL
355
10
18
90
1490





NUC
PAYRPPNAPI
130
10
19
95
1491





POL
PTTGRTSLYA
797
10
17
85
1492





NUC
PTVQASKLCL
15
10
16
80
1493





ENV
PTVWLSVIWM
351
10
30
150
1494





ENV
QAGFFLLTRI
179
10
16
80
1495





NUC
QAILCWGELM
57
10
36
180
1496





ENV
QAMQWNSTTF
107
10
16
80
1497





NUC
QASKLCLGWL
18
10
16
80
1498





ENV
QSLDSWWTSL
193
10
18
90
1499





POL
RTPARVTGGV
353
10
18
90
1500





POL
SAICSVVRRA
520
10
19
95
1501





X
SSAGPCALRF
64
10
18
90
1502





POL
TAELLAACFA
716
10
17
85
1503





NUC
TALRQAILCW
53
10
19
95
1504





NUC
TASALYREAL
33
10
16
80
1505





POL
TSFPWLLGCA
747
10
15
75
1506





POL
TSFVYVPSAL
764
10
16
80
1507





ENV
TSGFLGPLLV
168
10
15
75
1508





POL
VAEDLNLGNL
37
10
19
95
1509





POL
YSLNFMGYVI
580
10
15
75
1510





POL
AACFARSRSGA
721
11
17
85
1511





POL
AAPFTQCGYPA
632
11
19
95
1512





ENV
ASVRFSWLSLL
329
11
16
80
1513





X
CAFSSAGPCAL
61
11
19
95
1514





X
CALRFTSARRM
69
11
26
130
1515





NUC
CSPHHTALRQA
48
11
20
100
1516





ENV
CTCIPIPSSWA
310
11
20
100
1517





POL
DATPTGWGLAI
689
11
15
75
1518





NUC
DTASALYREAL
32
11
16
80
1519





POL
ESRLVVDFSQF
374
11
19
95
1520





POL
FADATPTGWGL
687
11
19
95
1521





X
FSSAGPCALRF
63
11
18
90
1522





ENV
FSWLSLLVPFV
333
11
20
100
1523





POL
FSYMDDVVLGA
536
11
18
90
1524





POL
FTFSPTYKAFL
656
11
19
95
1525





X
GAHLSLRGLPV
50
11
18
90
1526





POL
GAKSVQHLESL
545
11
17
85
1527





POL
GTSFVYVPSAL
763
11
16
80
1528





POL
HTAELLAACFA
715
11
17
85
1529





NUC
HTALRQAILCW
52
11
19
95
1530





NUC
ISCLTFGRETV
105
11
18
90
1531





POL
KTKRWGYSLNF
574
11
17
85
1532





POL
LAFSYMDDVVL
534
11
18
90
1533





POL
LAQFTSAICSV
515
11
19
95
1534





ENV
LSLLVPFVQWF
336
11
20
100
1535





X
LSLRGLPVCAF
53
11
19
95
1536





ENV
LSPTVWLSVIW
349
11
15
75
1537





POL
LSRKYTSFPWL
742
11
17
85
1538





POL
LSWLSLDVSAA
412
11
19
95
1539





POL
LSYQHFRKLLL
3
11
15
75
1540





NUC
LTFGRETVLEY
137
11
15
75
1541





ENV
LTIPQSLDSWW
189
11
18
90
1542





POL
LTNLLSSNLSW
404
11
18
90
1543





ENV
LTRILTIPQSL
185
11
16
80
1544





X
PARDVLCLRPV
11
11
15
75
1545





POL
PARVTGGVFLV
355
11
18
90
1546





NUC
PAYRPPNAPIL
130
11
19
95
1547





ENV
PTVWLSVIWMM
351
11
28
140
1548





POL
QAFTFSPTYKA
654
11
19
95
1549





ENV
QAGFFLLTRIL
179
11
16
80
1550





NUC
QASKLCLGWLW
18
11
15
75
1551





POL
QSLTNLLSSNL
402
11
18
90
1552





POL
RAFPHCLAFSY
528
11
19
95
1553





POL
RTPARVTGGVF
353
11
18
90
1554





NUC
RTPPAYRPPNA
127
11
19
95
1555





POL
SAICSVVRRAF
520
11
19
95
1556





POL
SASFCGSPYSW
165
11
20
100
1557





POL
SSNLSWLSLDV
409
11
18
90
1558





POL
TSAICSVVRRA
519
11
19
95
1559





POL
TSFPWLLGCAA
747
11
15
75
1560





ENV
TSGFLGPLLVL
168
11
15
75
1561





POL
VSWPKFAVPNL
391
11
19
95
1562





POL
WTHKVGNFTGL
52
11
19
95
1563





POL
YTSFPWLLGCA
746
11
15
75
1564
















TABLE XIV







HBV B62 Super Motif

















No. of
Sequence
Conservancy




Protein
Sequence
Position
Amino Acids
Frequency
(%)
SEQ ID NO:

















NUC
AILCWGEL
58
8
18
90
1565






POL
APFTQCGY
633
8
19
95
1566





POL
AVPNLQSL
397
8
19
95
1567





ENV
CIPIPSSW
312
8
20
100
1568





NUC
CLGWLWGM
23
8
17
85
1569





ENV
CLIFLLVL
253
8
20
100
1570





ENV
CLRRFIIF
239
8
19
95
1571





POL
CQRIVGLL
622
8
17
85
1572





NUC
DIDPYKEF
31
8
18
90
1573





NUC
DLLDTASA
29
8
17
85
1574





ENV
DPRVRGLY
122
8
16
80
1575





NUC
DPYKEFGA
33
8
18
90
1576





X
DVLCLRPV
14
8
19
95
1577





X
ELGEEIRL
122
8
16
80
1578





POL
ELLAACFA
718
8
18
90
1579





ENV
FIIFLFIL
243
8
16
80
1580





ENV
FILLLCLI
248
8
16
80
1581





ENV
FLGPLLVL
171
8
15
75
1582





ENV
FLLVLLDY
256
8
19
95
1583





POL
FPWLLGCA
749
8
15
75
1584





ENV
FVGLSPTV
346
8
19
95
1585





ENV
FVQWFVGL
342
8
19
95
1586





POL
FVYVPSAL
766
8
18
90
1587





POL
GLSPFLLA
509
8
19
95
1588





ENV
GLSPTVWL
348
8
20
100
1589





ENV
GMLPVCPL
265
8
18
90
1590





ENV
GPLLVLQA
173
8
19
95
1591





POL
GVGLSPFL
507
8
16
80
1592





POL
HLYSHPII
491
8
16
80
1593





POL
HPAAMPHL
429
8
20
100
1594





ENV
IIFLFILL
244
8
16
80
1595





POL
IILGFRKI
497
8
16
80
1596





NUC
ILCWGELM
59
8
18
90
1597





ENV
ILLLCLIF
249
8
20
100
1598





POL
ILRGTSFV
760
8
16
80
1599





ENV
ILTIPQSL
188
8
19
95
1600





ENV
IPIPSSWA
313
8
20
100
1601





ENV
IPQSLDSW
191
8
18
90
1602





ENV
IPSSWAFA
315
8
16
80
1603





POL
IVGLLGFA
625
8
18
90
1604





POL
KIPMGVGL
503
8
16
80
1605





NUC
KLCLGWLW
21
8
17
85
1606





POL
KLIMPARF
108
8
15
75
1607





POL
KLPVNRPI
610
8
16
80
1608





POL
KVGNFTGL
55
8
19
95
1609





X
KVLHKRTL
91
8
17
85
1610





ENV
LIFLLVLL
254
8
20
100
1611





POL
LIMPARFY
109
8
20
100
1612





POL
LLAQFTSA
514
8
19
95
1613





ENV
LLCLIFLL
251
8
20
100
1614





NUC
LLDTASAL
30
8
17
85
1615





ENV
LLDYQGML
260
8
19
95
1616





POL
LLGCAANW
752
8
16
80
1617





POL
LLGFAAPF
628
8
19
95
1618





ENV
LLGWSPQA
63
8
17
85
1619





ENV
LLLCLIFL
250
8
20
100
1620





ENV
LLPIFFCL
378
8
20
100
1621





POL
LLSLGIHL
563
8
19
95
1622





POL
LLSSNLSW
407
8
18
90
1623





ENV
LLTRILTI
184
8
16
80
1624





POL
LLVGSSGL
436
8
16
80
1625





ENV
LLVLQAGF
175
8
19
95
1626





ENV
LLVPFVQW
338
8
20
100
1627





POL
LMPLYACI
643
8
19
95
1628





ENV
LPIFFCLW
379
8
20
100
1629





POL
LPIHTAEL
712
8
17
85
1630





ENV
LQAGFFLL
178
8
19
95
1631





POL
LQSLTNLL
401
8
20
100
1632





ENV
LVLQAGFF
176
8
19
95
1633





ENV
LVPFVQWF
339
8
20
100
1634





NUC
LVSFGVWI
119
8
18
90
1635





POL
LVVDFSQF
377
8
20
100
1636





POL
MPLSYQHF
1
8
20
100
1637





NUC
MQLFHLCL
1
8
15
75
1638





ENV
MQWNSTTF
109
8
16
80
1639





POL
NLNVSIPW
45
8
19
95
1640





POL
NLQSLTNL
400
8
20
100
1641





ENV
NLSVPNPL
15
8
15
75
1642





POL
NPNKTKRW
571
8
15
75
1643





ENV
PIFFCLWV
380
8
20
100
1644





POL
PIHTAELL
713
8
17
85
1645





ENV
PIPSSWAF
314
8
20
100
1646





ENV
PQSLDSWW
192
8
18
90
1647





X
PVCAFSSA
59
8
19
95
1648





POL
PVNRPIDW
612
8
17
85
1649





X
QLDPARDV
8
8
16
80
1650





POL
RIVGLLGF
624
8
18
90
1651





POL
RLKLIMPA
106
8
15
75
1652





NUC
RPPNAPIL
133
8
20
100
1653





NUC
RQLLWFHI
98
8
18
90
1654





POL
RVAEDLNL
36
8
19
95
1655





POL
RVHFASPL
818
8
16
80
1656





POL
RVTGGVFL
357
8
20
100
1657





POL
SIPWTHKV
49
8
20
100
1658





POL
SLDVSAAF
416
8
19
95
1659





POL
SLNFMGYV
581
8
15
75
1660





POL
SPFLLAQF
511
8
19
95
1661





ENV
SPQAQGIL
67
8
17
85
1662





POL
SPSVPSHL
808
8
17
85
1663





ENV
SPTVWLSV
350
8
15
75
1664





POL
SPTYKAFL
659
8
19
95
1665





ENV
SVPNPLGF
17
8
15
75
1666





POL
SVQHLESL
548
8
17
85
1667





POL
SVVLSRKY
739
8
18
90
1668





NUC
TLPETTVV
142
8
20
100
1669





POL
TLWKAGIL
150
8
20
100
1670





ENV
TPPHGGLL
57
8
15
75
1671





POL
TPTGWGLA
691
8
16
80
1672





POL
TQCGYPAL
636
8
19
95
1673





POL
TVNEKRRL
100
8
17
85
1674





ENV
TVWLSVIW
352
8
15
75
1675





ENV
VLLDYQGM
259
8
19
95
1676





ENV
VLQAGFFL
177
8
19
95
1677





ENV
VPFVQWFV
340
8
19
95
1678





POL
VPSALNPA
769
8
18
90
1679





NUC
VQASKLCL
17
8
16
80
1680





POL
VVLGAKSV
542
8
18
90
1681





POL
WILRGTSF
759
8
16
80
1682





NUC
WIRTPPAY
125
8
19
95
1683





POL
WLSLDVSA
414
8
20
100
1684





ENV
WLSLLVPF
335
8
20
100
1685





ENV
WMCLRRFI
237
8
19
95
1686





POL
YLHTLWKA
147
8
20
100
1687





POL
YLPLDKGI
122
8
20
100
1688





NUC
YLVSFGVW
118
8
18
90
1689





POL
YPALMPLY
640
8
19
95
1690





POL
YQHFRKLL
5
8
15
75
1691





POL
AICSVVRRA
521
9
19
95
1692





NUC
AILCWGELM
58
9
18
90
1693





POL
ALMPLYACI
642
9
19
95
1694





NUC
ALRQAILCW
54
9
19
95
1695





ENV
AMQWNSTTF
108
9
16
80
1696





X
AMSTTDLEA
102
9
15
75
1697





X
APCNFFTSA
146
9
15
75
1698





ENV
CIPIPSSWA
312
9
20
100
1699





ENV
CLIFLLVLL
253
9
20
100
1700





ENV
CLRRFIIFL
239
9
19
95
1701





NUC
CLTFGRETV
107
9
18
90
1702





ENV
CPGYRWMCL
232
9
20
100
1703





NUC
CPTVQASKL
14
9
16
80
1704





X
CQLDPARDV
7
9
16
80
1705





NUC
DLLDTASAL
29
9
17
85
1706





POL
DLNLGNLNV
40
9
19
95
1707





X
DPARDVLCL
10
9
16
80
1708





POL
DPSRGRLGL
778
9
18
90
1709





POL
DVVLGAKSV
541
9
18
90
1710





ENV
FIIFLFILL
243
9
16
80
1711





ENV
FILLLCLIF
248
9
16
80
1712





ENV
FLFILLLCL
246
9
16
80
1713





POL
FLLAQFTSA
513
9
19
95
1714





POL
FLLSLGIHL
562
9
19
95
1715





ENV
FLLTRILTI
183
9
16
80
1716





ENV
FPDHQLDPA
14
9
18
90
1717





POL
FPHCLAFSY
530
9
19
95
1718





POL
FPWLLGCAA
749
9
15
75
1719





ENV
FVGLSPTVW
346
9
19
95
1720





POL
GLCQVFADA
682
9
17
85
1721





POL
GLLGFAAPF
627
9
19
95
1722





ENV
GLLGWSPQA
62
9
17
85
1723





POL
GVGLSPFLL
507
9
16
80
1724





NUC
GVWIRTPPA
123
9
19
95
1725





POL
HLLVGSSGL
435
9
16
80
1726





X
HLSLRGLPV
52
9
18
90
1727





POL
HLYSHPIIL
491
9
16
80
1728





POL
HPAAMPHLL
429
9
20
100
1729





ENV
IIFLFILLL
244
9
16
80
1730





POL
ILGFRKIPM
498
9
16
80
1731





ENV
ILLLCLIFL
249
9
20
100
1732





POL
ILRGTSFVY
760
9
16
80
1733





ENV
IPIPSSWAF
313
9
20
100
1734





ENV
IPQSLDSWW
191
9
18
90
1735





POL
IVGLLGFAA
625
9
18
90
1736





POL
KLHLYSHPI
489
9
19
95
1737





POL
KLIMPARFY
108
9
15
75
1738





POL
KVCQRIVGL
620
9
17
85
1739





POL
KVGNFTGLY
55
9
19
95
1740





POL
LLAQFTSAI
514
9
19
95
1741





ENV
LLCLIFLLV
251
9
20
100
1742





NUC
LLDTASALY
30
9
17
85
1743





POL
LLGCAANWI
752
9
16
80
1744





ENV
LLLCLIFLL
250
9
20
100
1745





ENV
LLPIFFCLW
378
9
20
100
1746





NUC
LLSFLPSDF
44
9
19
95
1747





POL
LLSSNLSWL
407
9
18
90
1748





ENV
LLVLQAGFF
175
9
19
95
1749





ENV
LLVPFVQWF
338
9
20
100
1750





NUC
LLWFHISCL
100
9
18
90
1751





ENV
LPIFFCLWV
379
9
20
100
1752





POL
LPIHTAELL
712
9
17
85
1753





X
LPVCAFSSA
58
9
19
95
1754





POL
LPVNRPIDW
611
9
16
80
1755





ENV
LVLLDYQGM
258
9
19
95
1756





ENV
LVLQAGFFL
176
9
18
90
1757





ENV
LVPFVQWFV
339
9
19
95
1758





ENV
MMWYWGPSL
360
9
17
85
1759





POL
NLGNLNVSI
42
9
19
95
1760





POL
NLLSSNLSW
406
9
18
90
1761





POL
NLQSLTNLL
400
9
20
100
1762





POL
NLSWLSLDV
411
9
18
90
1763





ENV
PIFFCLWVY
380
9
20
100
1764





POL
PIHTAELLA
713
9
17
85
1765





POL
PIILGFRKI
496
9
16
80
1766





ENV
PIPSSWAFA
314
9
16
80
1767





POL
PLDKGIKPY
124
9
20
100
1768





POL
PLEEELPRL
20
9
19
95
1769





ENV
PLLPIFFCL
377
9
20
100
1770





ENV
PLLVLQAGF
174
9
19
95
1771





POL
PLPIHTAEL
711
9
16
80
1772





POL
PMGVGLSPF
505
9
16
80
1773





NUC
PPAYRPPNA
129
9
19
95
1774





ENV
PPHGGLLGW
58
9
17
85
1775





X
QLDPARDVL
8
9
16
80
1776





ENV
RILTIPQSL
187
9
16
80
1777





POL
RIVGLLGFA
624
9
18
90
1778





POL
RLVVDFSQF
376
9
19
95
1779





POL
RVTGGVFLV
357
9
20
100
1780





ENV
SLDSWWTSL
194
9
19
95
1781





POL
SLDVSAAFY
416
9
19
95
1782





ENV
SLLVPFVQW
337
9
20
100
1783





POL
SLNFMGYVI
581
9
15
75
1784





X
SLRGLPVCA
54
9
19
95
1785





ENV
SPTVWLSVI
350
9
15
75
1786





ENV
SVRFSWLSL
330
9
16
80
1787





ENV
TIPQSLDSW
190
9
18
90
1788





POL
TLWKAGILY
150
9
20
100
1789





POL
TPARVTGGV
354
9
18
90
1790





POL
TPTGWGLAI
691
9
15
75
1791





POL
TQCGYPALM
636
9
19
95
1792





NUC
TVQASKLCL
16
9
16
80
1793





ENV
TVWLSVIWM
352
9
15
75
1794





X
VLCLRPVGA
15
9
19
95
1795





X
VLGGCRHKL
133
9
18
90
1796





X
VLHKRTLGL
92
9
17
85
1797





ENV
VLLDYQGML
259
9
19
95
1798





ENV
VLQAGFFLL
177
9
19
95
1799





POL
VLSRKYTSF
741
9
17
85
1800





POL
WILRGTSFV
759
9
16
80
1801





POL
WLLGCAANW
751
9
16
80
1802





POL
WLSLDVSAA
414
9
19
95
1803





ENV
WLSLLVPFV
335
9
20
100
1804





ENV
WMCLRRFII
237
9
19
95
1805





POL
WPKFAVPNL
393
9
19
95
1806





NUC
YLVSFGVWI
118
9
18
90
1807





POL
YMDDVVLGA
538
9
18
90
1808





POL
YPALMPLYA
640
9
19
95
1809





POL
YQHFRKLLL
5
9
15
75
1810





POL
YVPSALNPA
768
9
18
90
1811





POL
AICSVVRRAF
521
10
19
95
1812





POL
APFTQCGYPA
633
10
19
95
1813





POL
AQFTSAICSV
516
10
19
95
1814





ENV
CIPIPSSWAF
312
10
20
100
1815





POL
CLAFSYMDDV
533
10
18
90
1816





NUC
CLGWLWGMDI
23
10
17
85
1817





ENV
CLRRFIIFLF
239
10
15
75
1818





X
CQLDPARDVL
7
10
16
80
1819





POL
CQRIVGLLGF
622
10
17
85
1820





NUC
DIDPYKEFGA
31
10
18
90
1821





NUC
DLLDTASALY
29
10
17
85
1822





X
DVLCLRPVGA
14
10
19
95
1823





NUC
ELLSFLPSDF
43
10
19
95
1824





ENV
FIIFLFILLL
243
10
16
80
1825





ENV
FILLLCLIFL
248
10
16
80
1826





ENV
FLFILLLCLI
246
10
16
80
1827





ENV
FLGPLLVLQA
171
10
15
75
1828





POL
FLLAQFTSAI
513
10
19
95
1829





ENV
FPDHQLDPAF
14
10
17
85
1830





POL
FPHCLAFSYM
530
10
19
95
1831





ENV
FVGLSPTVWL
346
10
19
95
1832





X
FVLGGCRHKL
132
10
18
90
1833





X
GLPVCAFSSA
57
10
19
95
1834





POL
GLSPFLLAQF
509
10
19
95
1835





ENV
GLSPTVWLSV
348
10
15
75
1836





NUC
GMDIDPYKEF
29
10
17
85
1837





X
GPCALRFTSA
67
10
18
90
1838





POL
GPLEEELPRL
19
10
19
95
1839





ENV
GPLLVLQAGF
173
10
19
95
1840





POL
GVGLSPFLLA
507
10
16
80
1841





NUC
GVWIRTPPAY
123
10
19
95
1842





POL
HLNPNKTKRW
569
10
15
75
1843





POL
HPAAMPHLLV
429
10
17
85
1844





POL
HPIILGFRKI
495
10
16
80
1845





POL
IILGFRKIPM
497
10
16
80
1846





ENV
ILLLCLIFLL
249
10
20
100
1847





POL
ILRGTSFVYV
760
10
16
80
1848





NUC
ILSTLPETTV
139
10
20
100
1849





ENV
IPIPSSWAFA
313
10
16
80
1850





POL
IPMGVGLSPF
504
10
16
80
1851





POL
IPWTHKVGNF
50
10
20
100
1852





NUC
KLCLGWLWGM
21
10
17
85
1853





POL
KLHLYSHPII
489
10
16
80
1854





POL
KLPVNRPIDW
610
10
16
80
1855





POL
KQAFTFSPTY
653
10
19
95
1856





POL
KVCQRIVGLL
620
10
17
85
1857





X
KVLHKRTLGL
91
10
17
85
1858





ENV
LIFLLVLLDY
254
10
19
95
1859





ENV
LLCLIFLLVL
251
10
20
100
1860





ENV
LLDYQGMLPV
260
10
18
90
1861





POL
LLGCAANWIL
752
10
16
80
1862





ENV
LLCLIFLLV
250
10
20
100
1863





ENV
LLPIFFCLWV
378
10
20
100
1864





NUC
LLSFLPSDFF
44
10
19
95
1865





ENV
LLVLLDYQGM
257
10
19
95
1866





ENV
LLVQAGFFL
175
10
18
90
1867





ENV
LLVPFVQWFV
338
10
19
95
1868





ENV
LPIFFCLWVY
379
10
20
100
1869





POL
LPIHTAELLA
712
10
17
85
1870





X
LPKVLHKRTL
89
10
16
80
1871





POL
LPLDKGIKPY
123
10
20
100
1872





ENV
LVLLDYQGML
258
10
19
95
1873





ENV
LVLQAGFFLL
176
10
18
90
1874





ENV
MMWYWGPSLY
360
10
17
85
1875





POL
NLLSSNLSWL
406
10
18
90
1876





ENV
NLSVPNPLGF
15
10
15
75
1877





POL
NPNKTKRWGY
571
10
15
75
1878





POL
NVSIPWTHKV
47
10
20
100
1879





POL
PIDWKVCQRI
616
10
17
85
1880





ENV
PIFFCLWVYI
380
10
20
100
1881





POL
PIHTAELLAA
713
10
17
85
1882





POL
PLDKGIKPYY
124
10
20
100
1883





POL
PLEEELPRLA
20
10
18
90
1884





ENV
PLGFFDHQL
10
10
19
95
1885





POL
PLHPAAMPHL
427
10
20
100
1886





ENV
PLLPIFFCLW
377
10
20
100
1887





ENV
PLLVLQAGFF
174
10
19
95
1888





POL
PLPIHTAELL
711
10
16
80
1889





POL
PLSYQHFRKL
2
10
15
75
1890





POL
PLTVNEKRRL
98
10
17
85
1891





POL
PMGVGLSPFL
505
10
16
80
1892





NUC
PPNAPILSTL
134
10
20
100
1893





POL
PVNRPIDWKV
612
10
17
85
1894





NUC
QLLWFHISCL
99
10
18
90
1895





POL
RIVGLLGFAA
624
10
18
90
1896





POL
RLKLIMPARF
106
10
15
75
1897





NUC
RQAILCWGEL
56
10
18
90
1898





POL
RVHFASPLHV
818
10
15
75
1899





ENV
SLLVPFVQWF
337
10
20
100
1900





X
SLRGLPVCAF
54
10
19
95
1901





POL
SLTNLLSSNL
403
10
18
90
1902





NUC
SPHHTALRQA
49
10
20
100
1903





ENV
SPTVWLSVIW
350
10
15
75
1904





ENV
SVRFSWLSLL
330
10
16
80
1905





ENV
TIPQSLDSWW
190
10
18
90
1906





POL
TPARVTGGVF
354
10
18
90
1907





NUC
TPPAYRPPNA
128
10
19
95
1908





ENV
TPPHGGLLGW
57
10
15
75
1909





POL
VLGKSVQHL
543
10
17
85
1910





X
VLGGCRHKLV
133
10
18
90
1911





ENV
VPFVQWFVGL
340
10
19
95
1912





POL
VPNLQSLTNL
398
10
19
95
1913





NUC
VQASKLCLGW
17
10
16
80
1914





POL
VVLSRKYTSF
740
10
17
85
1915





POL
VVRRAFPHCL
525
10
19
95
1916





POL
WILRGTSFVY
759
10
16
80
1917





POL
WLLGCAANWI
751
10
16
80
1918





POL
WLSLDVSAAF
414
10
19
95
1919





NUC
WLWGMDIDPY
26
10
17
85
1920





ENV
WMCLRRFIIF
237
10
19
95
1921





ENV
WMMWYWGPSL
359
10
17
85
1922





POL
YLHTLWKAGI
147
10
20
100
1923





ENV
YQGMLPVCPL
263
10
18
90
1924





POL
YQHFRKLLLL
5
10
15
75
1925





POL
APFTQCGYPAL
633
11
19
95
1926





POL
AQFTSAICSVV
516
11
19
95
1927





POL
AVPNLQSLTNL
397
11
19
95
1928





ENV
CIPIPSSWAFA
312
11
16
80
1929





POL
CLAFSYMDDVV
533
11
18
90
1930





ENV
CLIFLLVLLDY
253
11
19
95
1931





ENV
CLRRFIIFLFI
239
11
15
75
1932





NUC
CPTVQASKLCL
14
11
16
80
1933





POL
CQRIVGLLGFA
622
11
17
85
1934





POL
DLNLGNLNVSI
40
11
19
95
1935





NUC
ELLSFLPSDFF
43
11
19
95
1936





ENV
FILLLCLIFLL
248
11
16
80
1937





ENV
FLFILLLCLIF
246
11
16
80
1938





ENV
FLLVLLDYQCM
256
11
19
95
1939





ENV
FPAGGSSSGTV
130
11
15
75
1940





POL
FPWLLGCAANW
749
11
15
75
1941





X
FVLGGCRHKLV
132
11
18
90
1942





POL
FVYVPSALNPA
766
11
18
90
1943





ENV
GLSPTVWLSVI
348
11
15
75
1944





POL
GPLEEELPRLA
19
11
18
90
1945





ENV
GPLLVLQAGFF
173
11
19
95
1946





POL
GPLTVNXEKRRL
97
11
17
85
1947





X
HLSLRGLPVCA
52
11
18
90
1948





POL
HLYSHPIILGF
491
11
16
80
1949





ENV
IIFLFILLLCL
244
11
16
80
1950





ENV
ILLLCLIFLLV
249
11
20
100
1951





NUC
ILSTLPETTVV
139
11
20
100
1952





ENV
ILTIPQSLDSW
188
11
18
90
1953





POL
IPMGVGLSPFL
504
11
16
80
1954





POL
IVGLLGFAAPF
625
11
18
90
1955





POL
KIPMGVGLSPF
503
11
16
80
1956





POL
KLHLYSHPIIL
489
11
16
80
1957





ENV
LLCLIFLLVLL
251
11
20
100
1958





ENV
LLGWSPQAQGI
63
11
15
75
1959





ENV
LLLCLIFLLVL
250
11
20
100
1960





ENV
LLPIFFCLWVY
378
11
20
100
1961





POL
LLSSNLSWLSL
407
11
18
90
1962





ENV
LLVLLDYQGML
257
11
19
95
1963





ENV
LLVLQAGFFLL
175
11
18
90
1964





NUC
LLWFHISCLTF
100
11
17
85
1965





ENV
LPIFFCLWVYI
379
11
20
100
1968





POL
LPIHTAELLAA
712
11
17
85
1967





POL
LPLDKGIKPYY
123
11
20
100
1968





POL
LPVNRPIDWKV
611
11
16
80
1969





ENV
LQAGFFLLTRI
178
11
16
80
1970





ENV
LVPFVQWFVGL
339
11
19
95
1971





POL
MPHLLVGSSGL
433
11
16
80
1972





POL
MPLSYQHFRKL
1
11
15
75
1973





POL
NLGNLNVSIPW
42
11
19
95
1974





POL
NLSWLSLDVSA
411
11
18
90
1975





POL
NPADDPSRGRL
774
11
18
90
1976





ENV
NPLGFFPDHQL
9
11
19
95
1977





POL
PIDWKVCQRIV
616
11
17
85
1978





POL
PIILGFRKIPM
496
11
16
80
1979





NUC
PILSTLPETTV
138
11
20
100
1980





POL
PLHPAAMPHLL
427
11
20
100
1981





ENV
PLLPIFFCLWV
377
11
20
100
1982





ENV
PLLVLQAGFFL
174
11
18
90
1983





POL
PLPIHTAELLA
711
11
16
80
1984





POL
PLSYQHFRKLL
2
11
15
75
1985





POL
PMGVGLSPFLL
505
11
16
80
1986





NUC
PPAYRPPNAPI
129
11
19
95
1987





ENV
PQAMQWNSTTF
106
11
16
80
1988





ENV
PQSLDSWWTSL
192
11
18
90
1989





X
QLDPARDVLCL
8
11
16
80
1990





POL
QVFADATPTGW
685
11
19
95
1991





POL
RLKMPARFY
106
11
15
75
1992





POL
RPIDWKVCQRI
615
11
16
80
1993





NUC
RPPNAPILSTL
133
11
20
100
1994





NUC
RQAILCWGELM
56
11
18
90
1995





NUC
RQLLWFHISCL
98
11
18
90
1996





POL
RVAEDLNLGNL
36
11
18
90
1997





POL
RVHFASPLHVA
818
11
15
75
1998





POL
SIPWTHKVGNF
49
11
20
100
1999





ENV
SLDSWWTSLNF
194
11
19
95
2000





ENV
SLLVPFVQWFV
337
11
19
95
2001





NUC
SPEHCSPHHTA
44
11
20
100
2002





POL
SPFLLAQFTSA
511
11
19
95
2003





NUC
SPHHTALRQAI
49
11
20
100
2004





ENV
SPTVWLSVIWM
350
11
15
75
2005





ENV
SVRFSWLSLLV
330
11
16
80
2006





POL
SVVLSRKYTSF
739
11
17
85
2007





POL
SVVRRAFPHCL
524
11
19
95
2008





POL
TPARVTGGVFL
354
11
18
90
2009





POL
TQCGYPALMPL
636
11
19
95
2010





NUC
TVQASKLCLGW
16
11
16
80
2011





ENV
VLLDYQGMLPV
259
11
18
90
2012





POL
VLSRKYTSFPW
741
11
17
85
2013





POL
VPNLQSLTNLL
398
11
19
95
2014





NUC
VQASKLCLGWL
17
11
16
80
2015





ENV
VQWFVGLSPTV
343
11
19
95
2016





POL
VVLGAKSVQHL
542
11
16
80
2017





POL
VVRRAFPHCLA
525
11
19
95
2018





POL
WILRGTSFVYV
759
11
16
80
2019





POL
WLLGCAANWIL
751
11
16
80
2020





POL
WLSLDVSAAFY
414
11
19
95
2021





ENV
WLSLLVPFVQW
335
11
20
100
2022





ENV
WMCLRRFIIFL
237
11
19
95
2023





ENV
WMMWYWGPSLY
359
11
17
85
2024





POL
YLHTLWKAGIL
147
11
20
100
2025





POL
YLPLDKGIKPY
122
11
20
100
2026





POL
YPALMPLYACI
640
11
19
95
2027
















TABLE XV







HBV A01 Motif with Binding information














Conservancy
Freq.
Protein
Position
Sequence
AA
A*0101
SEQ ID NO:


















100
20
POL
166
ASFCGSPY
8

2028






90
18
POL
737
DNSVVLSRKY
10
0.0001
2029





95
19
POL
631
FAAPFTQCGY
10
0.0680
2030





95
19
POL
630
GFAAPFTQCGY
11

2031





75
15
NUC
140
GRETVLEY
8

2032





85
17
POL
579
GYSLNFMGY
9

2033





100
20
POL
149
HTLWKAGILY
10
0.1100
2034





95
19
POL
653
KQAFTFSPTY
10
0.0001
2035





85
17
NUC
30
LLDTASALY
9
12.0000
2036





95
19
POL
415
LSLDVSAAFY
10
0.0150
2037





75
15
NUC
137
LTFGRETVLEY
11

2038





85
17
ENV
360
MMWYWGPSLY
10
0.0810
2039





75
15
X
103
MSTTDLEAY
9
0.8500
2040





90
18
POL
738
NSVVLSRKY
9
0.0005
2041





100
20
POL
124
PLDKGIKPY
9

2042





100
20
POL
124
PLDKGIKPYY
10
0.1700
2043





85
17
POL
797
PTTGRTSLY
9
0.2100
2044





100
20
POL
165
SASFCGSPY
9

2045





95
19
POL
416
SLDVSAAFY
9
5.2000
2046





75
15
X
104
STTDLEAY
8

2047





85
17
POL
798
TTGRTSLY
8

2048





95
19
POL
414
WLSLDVSAAFY
11

2049





85
17
ENV
359
WMMWYWGPSLY
11
0.3200
2050





95
19
POL
640
YPALMPLY
8

2051





85
17
POL
580
YSLNFMGY
8

2052
















TABLE XVI







HBV A03 Motif With Binding














Conservancy
Freq.
Protein
Position
Sequence
AA
A′0301
SeqID Num


















85
17
POL
721
AACFARSR
8
0.0004
2053






85
17
POL
721
AACFARSRSGA
11

2054





95
19
POL
632
AAPFTQCGY
9

2055





95
19
POL
632
AAPFTQCGYPA
11

2056





85
17
POL
722
ACFARSRSGA
10

2057





80
16
POL
688
ADATPTGWGLA
11

2058





90
18
POL
776
ADDPSRGR
8

2059





95
19
POL
529
AFPHCLAF
8

2060





95
19
POL
529
AFPHCLAFSY
10

2061





95
19
X
62
AFSSAGPCA
9

2062





90
18
X
62
AFSSAGPCALR
11

2063





95
19
POL
655
AFTFSPTY
8

2064





95
19
POL
655
AFTFSPTYK
9
0.2600
2065





95
19
POL
655
AFTFSPTYKA
10

2066





95
19
POL
655
AFTFSPTYKAF
11

2067





80
16
ENV
180
AGFFLLTR
8

2068





90
18
X
66
AGPCALRF
8

2069





90
18
X
66
AGPCALRFTSA
11

2070





95
19
POL
18
AGPLEEELPR
10
0.0004
2071





95
19
POL
521
AICSVVRR
8
−0.0002
2072





95
19
POL
521
AICSVVRRA
9

2073





95
19
POL
521
AICSVVRRAF
10

2074





95
19
NUC
41
ALESPEHCSPH
11

2075





90
18
POL
772
ALNPADDPSR
10
0.0003
2076





85
17
X
70
ALRFTSAR
8
0.0047
2077





80
16
ENV
108
AMQWNSTTF
9

2078





80
16
ENV
108
AMQWNSTTFH
10

2079





75
15
X
102
AMSITDLEA
9

2080





85
17
NUC
34
ASALYREA
8

2081





100
20
POL
166
ASFCGSPY
8
0.0460
2082





80
16
POL
822
ASPLHVAWR
9

2083





75
15
ENV
84
ASTNRQSGR
9
0.0009
2084





80
16
POL
690
ATPTGWGLA
9

2085





80
16
POL
755
CAANWILR
8

2086





95
19
X
61
CAFSSAGPCA
10

2087





90
18
X
69
CALRFTSA
8

2088





85
17
X
69
CALRFTSAR
9
0.0034
2089





80
16
X
6
CCQLDPAR
8

2090





85
17
POL
723
CFARSRSGA
9

2091





75
15
POL
607
CFRKLPVNR
9

2092





95
19
POL
638
CGYPALMPLY
10

2093





95
19
POL
638
CGYPALMPLYA
11

2094





100
20
ENV
312
CIPIPSSWA
9

2095





100
20
ENV
312
CIPIPSSWAF
10

2096





80
16
EVN
312
CIPIPSSWAFA
11

2097





95
19
ENV
253
CLIFLLVLLDY
11
0.0083
2098





90
18
X
17
CLRPVGAESR
10
0.0011
2099





95
19
ENV
239
CLRRFIIF
8

2100





75
15
ENV
239
CLRRFIIFLF
10

2101





100
20
NUC
48
CSPHHTALR
9
0.0029
2102





100
20
NUC
48
CSPHHTALRQA
11

2103





95
19
POL
523
CSVVRRAF
8

2104





95
19
POL
523
CSVVRRAFPH
10

2105





100
20
ENV
310
CTCIPIPSSWA
11

2106





80
16
POL
689
DATPTGWGLA
10

2107





90
18
POL
540
DDVVLGAK
8

2108





90
18
NUC
31
DIDPYKEF
8

2109





90
18
NUC
31
DIDPYKEFGA
10

2110





85
17
NUC
29
DLLDTASA
8

2111





85
17
NUC
29
DLLDTASALY
10
0.0001
2112





85
17
NUC
29
DLLDTASALYR
11
0.0042
2113





95
19
ENV
196
DSWWTSLNF
9
0.0006
2114





85
17
NUC
32
DTASALYR
8
0.0004
2115





80
16
NUC
32
DTASALYREA
10

2116





95
19
X
14
DVLCLRPVGA
10

2117





95
19
POL
418
DVSAAFYH
8

2118





90
18
POL
541
DVVLGAKSVQH
11

2119





95
19
POL
17
EAGPLEEELPR
11
−0.0009
2120





90
18
NUC
40
EALESPEH
8

2121





90
18
POL
718
ELLAACFA
8

2122





90
18
POL
718
ELLAACFAR
9
0.0002
2123





85
17
POL
718
ELLAACFARSR
11
0.0082
2124





95
19
NUC
43
ELLSFLPSDF
10

2125





95
19
NUC
43
ELLSFLPSDFF
11

2126





95
19
NUC
43
ESPEHCSPH
9

2127





95
19
NUC
43
ESPEHCSPHH
10

2128





95
19
POL
374
ESRLVVDF
8

2129





95
19
POL
374
ESRLVVDFSQF
11

2130





95
19
NUC
174
ETTVVRRR
8
0.0003
2131





80
16
NUC
174
ETTVVRRRGR
10
0.0003
2132





95
19
POL
631
FAAPFTQCGY
10

2133





85
17
POL
724
FARSRSGA
8

2134





80
16
POL
821
FASPLHVA
8

2135





80
16
POL
821
FASPLHVAWR
10

2136





90
18
ENV
13
FFPDHQLDPA
10

2137





85
17
ENV
13
FFPDHQLDPAF
11

2138





75
15
NUC
139
FGRETVLEY
9

2139





75
15
POL
244
FGVEPSGSGH
10

2140





95
19
NUC
122
FGVWIRTPPA
10

2141





95
19
NUC
122
FGVWIRTPPAY
11

2142





80
16
ENV
248
FILLLCLIF
9

2143





80
16
ENV
246
FLFILLLCLIF
11

2144





75
15
ENV
171
FLGPLLVLQA
10

2145





95
19
POL
513
FLLAQFTSA
9
0.0006
2146





95
19
POL
562
FLLSLGIH
8

2147





95
19
ENV
256
FLLVLLDY
8
0.0050
2148





100
20
POL
363
FLVDKNPH
8

2149





95
19
POL
658
FSPTYKAF
8

2150





95
19
X
63
FSSAGPCA
8

2151





90
18
X
63
FSSAGPCALR
10

2152





90
18
X
63
FSSAGPCALRF
11

2153





100
20
ENV
333
FSWLSLLVPF
10
0.0004
2154





90
18
POL
536
FSYMDDVVLGA
11

2155





95
19
POL
656
FTFSPTYK
8
0.0100
2156





95
19
POL
656
FTFSPTYKA
9

2157





95
19
POL
656
FTFSPTYKAF
10
0.0004
2158





95
19
POL
635
FTOCGYPA
8

2159





95
19
POL
518
FTSAICSVVR
10
0.0003
2160





95
19
POL
518
FTSAICSVVRR
11
0.0065
2161





95
19
X
132
FVLGGCRH
8

2162





90
18
X
132
FVLGGCRHK
9
0.0430
2163





90
18
POL
766
FVYVPSALNPA
11

2164





80
16
POL
754
GCAANWILR
9

2165





95
19
POL
630
GFAAPFTOCGY
11

2166





90
18
ENV
12
GFFPDHOLDPA
11

2167





75
15
ENV
170
GFLGPLLVLOA
11

2168





85
17
ENV
61
GGLLGWSPQA
10

2169





100
20
POL
360
GGVFLVDK
8

2170





100
20
POL
360
GGVFLVDKNPH
11

2171





75
15
POL
567
GIHLNPNK
8

2172





75
15
POL
567
GIHLNPNKTK
10
0.0025
2173





75
15
POL
567
GIHLNPNKTKR
11

2174





85
17
POL
682
GLCQVFADA
9
0.0001
2175





95
19
POL
627
GLLGFAAPF
9
0.0006
2176





85
17
ENV
62
GLLGWSPOA
9

2177





95
19
X
57
GLPVCAFSSA
10

2178





95
19
POL
509
GLSPFLLA
8

2179





95
19
POL
509
GLSPFLLAQF
10

2180





85
17
NUC
29
GMDIDPYK
8
0.0006
2181





85
17
NUC
29
GMDIDPYKEF
10
−0.0003
2182





90
18
POL
735
GTDNSVVLSR
10
0.0010
2183





90
18
POL
735
GTDNSVVLSRK
11
0.0140
2184





80
16
POL
763
GTSFVYVPSA
10

2185





80
16
POL
245
GVEPSGSGH
9

2186





100
20
POL
361
GVFLVDKNPH
10

2187





80
16
POL
507
GVGLSPFLLA
10

2188





95
19
NUC
123
GVWIRTPPA
9

2189





95
19
NUC
123
GVWIRTPPAY
10
0.0047
2190





95
19
NUC
123
GVWIRTPPAYR
11
0.1900
2191





100
20
NUC
47
HCSPHHTA
8

2192





100
20
NUC
47
HCSPHHTALR
10

2193





80
16
POL
820
HFASPLHVA
9

2194





80
16
POL
820
HFASPLHVAWR
11

2195





95
19
X
49
HGAHLSLR
8

2196





85
17
ENV
60
HGGLLGWSPQA
11

2197





90
18
NUC
104
HISCLTFGR
9

2198





75
15
POL
569
HLNPNKTK
8

2199





75
15
POL
569
HLNPNKTKR
9

2200





90
18
X
52
HLSLRGLPVCA
11

2201





80
16
POL
491
HLYSHPIILGF
11

2202





85
17
POL
715
HTAELLAA
8

2203





85
17
POL
715
HTAELLAACF
10

2204





85
17
POL
715
HTAELLAACFA
11

2205





100
20
POL
149
HTLWKAGILY
10
0.0440
2206





100
20
POL
149
HTLWKAGILYK
11
0.5400
2207





95
19
POL
522
ICSVVRRA
8

2208





95
19
POL
522
ICSVVRRAF
9

2209





95
19
POL
522
ICSVVRRAFPH
11

2210





90
18
NUC
32
IDPYKEFGA
9

2211





90
18
POL
617
IDWKVCQR
8

2212





100
20
ENV
381
IFFCLWVY
8

2213





95
19
ENV
255
IFLLVLLDY
9

2214





80
16
POL
734
IGTDNSVVLSR
11

2215





100
20
ENV
249
ILLLCLIF
8

2216





80
16
POL
760
ILRGTSFVY
9
0.0440
2217





90
18
NUC
105
ISCLTFGR
8
0.0004
2218





90
18
POL
625
IVGLLGFA
8

2219





90
18
POL
625
IVGLLGFAA
9

2220





90
18
POL
625
IVGLLGFAAPF
11

2221





100
20
POL
153
KAGILYKR
8
0.0002
2222





80
16
POL
503
KIPMGVGLSPF
11

2223





75
15
POL
108
KLIMPARF
8

2224





75
15
POL
108
KLIMPARFY
9

2225





80
16
POL
610
KLPVNRPIDWK
11

2226





85
17
POL
574
KTKRWGYSLNF
11

2227





75
15
X
130
KVFVLGGCR
9
0.0420
2228





75
15
X
130
KVFVLGGCRH
10

2229





95
19
POL
55
KVGNFTGLY
9
0.2100
2230





85
17
POL
720
LAACFARSR
9
0.0058
2231





95
19
X
16
LCLRPVGA
8

2232





90
18
X
16
LCLRPVGAESR
11

2233





95
19
POL
683
LCQVFADA
8

2234





100
20
POL
125
LDKGIKPY
8

2235





100
20
POL
125
LDKGIKPYY
9

2236





80
16
X
9
LDPARDVLCLR
11

2237





95
19
ENV
195
LDSWWTSLNF
10

2238





85
17
NUC
31
LDTASALY
8

2239





85
17
NUC
31
LDTASALYR
9
0.0004
2240





80
16
NUC
31
LDTASALYREA
11

2241





95
19
POL
417
LDVSAAFY
8

2242





95
19
POL
417
LDVSAAFYH
9

2243





80
16
ENV
247
LFILLLCLIF
10

2244





95
19
POL
544
LGAKSVQH
8

2245





80
16
POL
753
LGCAANWILR
10

2246





75
15
POL
566
LGIHLNPNK
9

2247





75
15
POL
566
LGIHLNPNKTK
11

2248





95
19
ENV
172
LGPLLVLQA
9

2249





95
19
ENV
172
LGPLLVLQAGF
11

2250





95
19
EMI
254
LIFLLVLLDY
10
0.0022
2251





100
20
POL
109
LIMPARFY
8
−0.0002
2252





90
18
POL
719
LLAACFAR
8
0.0024
2253





85
17
POL
719
LLAACFARSR
10

2254





95
19
POL
514
LLAQFTSA
8

2255





85
17
NUC
30
LLDTASALY
9
0.0013
2256





85
17
NUC
30
LLDTASALYR
10
0.0050
2257





80
16
POL
752
LLGCAANWILR
11

2258





95
19
POL
628
LLGFAAPF
8

2259





85
17
EMI
63
LLGWSPQA
8

2260





100
20
ETV
378
LLPIFFCLWVY
11
0.0230
2261





95
19
NUC
44
LLSFLPSDF
9

2262





95
19
NUC
44
LLSFLPSDFF
10

2263





95
19
ENV
175
LLVLQAGF
8

2264





95
19
ENV
175
LLVLQAGFF
9
0.0006
2265





100
20
ENV
336
LLVPFVQWF
9

2266





85
17
NUC
100
LLWFHISCLTF
11

2267





95
19
NUC
45
LSFLPSDF
8

2268





95
19
NUC
45
LSFLPSDFF
9
0.0006
2269





95
19
POL
415
LSLDVSAA
8

2270





95
19
POL
415
LSLDVSAAF
9
0.0004
2271





95
19
POL
415
LSLDVSAAFY
10

2272





95
19
POL
415
LSLDVSAAFYII
11

2273





75
15
POL
564
LSLGIHLNPNK
11

2274





100
20
ENV
336
LSLLVPFVQWF
11

2275





95
19
X
53
LSLRGLPVCA
10

2276





95
19
X
53
LSLRGLPVCAF
11

2277





95
19
POL
510
LSPFLLAQF
9

2278





95 85
17
POL
742
LSRKYTSF
8

2279





95
19
NUC
169
LSTLPETTVVR
11
−0.0009
2280





75
15
ENV
16
LSVPNPLGF
9

2281





100
20
POL
412
LSWLSLDVSA
10
0.0048
2282





100
19
POL
412
LSWLSLDVSAA
11

2283





95
15
POL
3
LSYQIIFRK
8

2284





75
15
NUC
137
LTFGRETVLEY
11

2285





85
17
POL
99
LTVNEKRR
8
−0.0002
2286





95
19
ENV
176
LVLQAGFF
8

2287





100
20
ENV
339
LVPFVQWF
8
0.0028
2288





90
18
NUC
119
LVSFGVWIR
9

2289





100
20
POL
377
LWDFSQF
8
0.0016
2290





100
20
POL
377
LWDFSQFSR
10

2291





95
19
ENV
238
MCLRRFIIF
9

2292





75
15
ENV
238
MCLRRFIIFLF
11

2293





90
18
POL
539
MDDWLGA
8

2294





90
18
POL
539
MDDWLGAK
9

2295





90
18
NUC
30
MDIDPYKEF
9

2296





90
18
NUC
30
MDIDPYKEFGA
11

2297





80
16
POL
506
MGVGLSPF
8

2298





80
16
POL
506
MGVGLSPFLLA
11

2299





85
17
ENV
360
MMWYWGPSLY
10
0.0500
2300





80
16
X
103
MSTTDLEA
8

2301





75
15
X
103
MSTTDLEAY
9
0.0008
2302





75
15
X
103
MSTTDLEAYF
10

2303





75
15
X
103
MSTTDLEAYFK
11

2304





95
19
POL
561
NFLLSLGIH
9

2305





90
18
NUC
75
NLEDPASR
8
−0.0002
2306





95
19
POL
45
NLNVSIPWTH
10

2307





95
19
POL
45
NLNVSIPWTHK
11
−0.0009
2308





95
15
ENV
15
NLSVPNPLGF
10

2309





90
18
POL
411
NLSWLSLDVSA
11

2310





75
15
ENV
215
NSQSPTSNH
9

2311





90
18
POL
738
NSVVLSRK
8
0.0006
2312





90
18
POL
738
NSVVLSRKY
9
0.0020
2313





100
20
POL
47
NVSIPWTH
8

2314





100
20
POL
47
NVSIPWTHK
9
0.0820
2315





90
18
POL
775
PADDPSRGR
9
0.0008
2316





95
19
POL
641
PALMPLYA
8

2317





75
15
X
145
PAPCNFFTSA
10

2318





80
16
X
11
PARDVLCLR
9
0.0002
2319





90
18
POL
355
PARVTGGVF
9

2320





75
15
ENV
83
PASTNRQSGR
10

2321





95
19
NUC
130
PAYRPPNA
8

2322





90
18
X
68
PCALRFTSA
9

2323





85
17
X
68
PCALRFTSAR
10

2324





75
15
X
147
PCNFFTSA
8

2325





95
19
ENV
15
PDHQLDPA
8

2326





90
18
ENV
15
PDHQLDPAF
9

2327





95
19
POL
512
PFLLAQFTSA
10

2328





95
19
POL
634
PFTQCGYPA
9

2329





100
20
ENV
233
PGYRWMCLR
9
0.0008
2330





95
19
ENV
233
PGYRWMCLRR
10
0.0048
2331





95
19
ENV
233
PGYRWMCLRRF
11

2332





90
18
POL
616
PIDWKVCQR
9
0.0002
2333





100
20
ENV
380
PIFFCLWVY
9
0.0011
2334





85
17
POL
713
PIHTAELLA
9

2335





85
17
POL
713
PIHTAELLAA
10

2336





80
16
POL
496
PIILGFRK
8

2337





100
20
ENV
314
PIPSSWAF
8

2338





80
16
ENV
314
PIPSSWAFA
9

2339





100
20
POL
124
PLDKGIKPY
9
0.0001
2340





100
20
POL
124
PLDKGIKPYY
10
0.0002
2341





95
19
POL
20
PLEEELPR
8
0.0002
2342





90
16
POL
20
PLEEELPRLA
10

2343





90
19
ENV
10
PLGFFPDH
8

2344





100
20
POL
427
PLHPAAMPH
9
0.0012
2345





95
19
ENV
174
PLLVLQAGF
9

2346





95
19
ENV
174
PLLVLQAGFF
10

2347





80
16
POL
711
PLPIHTAELLA
11

2348





100
20
POL
2
PLSYQHFR
8
−0.0002
2349





75
15
POL
2
PLSYQHFRK
9
0.0011
2350





85
17
POL
98
PLTVNEKR
8
0.0002
2351





85
17
POL
98
PLTVNEKRR
9
0.0008
2352





80
16
POL
505
PMGVGLSPF
9

2353





85
17
POL
797
PTTGRTSLY
9
0.0001
2354





85
17
POL
797
PTTGRTSLYA
10

2355





95
19
X
59
PVCAFSSA
8

2356





90
18
X
20
PVGAESRGR
9
0.0002
2357





85
17
POL
612
PVNRPIDWK
9
0.0310
2358





95
19
POL
654
QAFTFSPTY
9
0.0030
2359





95
19
POL
654
QAFTFSPTYK
10
0.0450
2360





95
19
POL
654
QAFTFSPTYKA
11

2361





80
16
ENV
179
QAGFFLLTR
9

2362





80
16
ENV
107
QAMQWNSTTF
10

2363





80
16
ENV
107
QAMQWNSTTFH
11

2364





95
19
POL
637
QCGYPALMPLY
11

2365





95
19
POL
517
QFTSAICSVVR
11

2366





75
15
NUC
169
QSPRRRRSQSR
11

2367





80
16
POL
189
QSSGILSR
8

2368





95
19
POL
528
RAFPHCLA
8

2369





95
19
POL
528
RAFPHCLAF
9
0.0015
2370





95
19
POL
528
RAFPHCIAFSY
11
0.1200
2371





85
17
NUC
28
RDLLDTASA
9

2372





85
17
NUC
28
RDLLDTASALY
11

2373





95
19
X
13
RDVLCLRPVGA
11

2374





100
20
ENV
332
RFSWLSLLVPF
11

2375





95
19
X
56
RGLPVCAF
8

2376





95
19
X
56
RGLPVCAFSSA
11

2377





100
20
NUC
152
RGRSPRRR
8

2378





80
16
POL
762
RGTSFVYVPSA
11

2379





90
18
POL
624
RNGLLGF
8

2380





90
18
POL
624
RIVGLLGFA
9

2381





90
18
POL
624
RIVGLLGFAA
10

2382





75
15
POL
106
RLKLIMPA
8

2383





75
15
POL
106
RLKLIMPAR
9
0.0950
2384





75
15
POL
106
RLKLIMPARF
10

2385





75
15
POL
106
RLKLIMPARFY
11

2386





75
15
X
128
RLKVFVLGGCR
11

2387





95
19
POL
376
RLVVDFSQF
9
0.0006
2388





95
19
POL
376
RLVVDFSQFSR
11
0.2800
2389





95
19
NUC
163
RSPRRRTPSPR
11
−0.0007
2390





75
15
NUC
167
RSQSPRRR
8

2391





75
15
NUC
167
RSQSPRRRR
9

2392





90
18
POL
353
RTPARVTGGVF
11

2393





95
19
NUC
127
RTPPAYRPPNA
11

2394





95
19
NUC
188
RTPSPRRR
8
−0.0002
2395





95
19
NUC
188
RTPSPRRRR
9
0.0054
2396





95
16
POL
818
RVHFASPLH
9

2397





75
15
POL
818
RVHFASPLHVA
11

2398





100
20
POL
357
RVTGGVFIVDK
11
0.0190
2399





90
18
X
65
SAGPCALR
8
−0.0002
2400





90
18
X
65
SAGPCALRF
9
−0.0003
2401





95
19
POL
520
SAICSVVR
8
−0.0002
2402





95
19
POL
520
SAICSVVRR
9
0.0058
2403





95
19
POL
520
SAICSWRRA
10

2404





95
19
POL
520
SAICSWRRAF
11

2405





95
18
POL
771
SALNPADDPSR
11
−0.0004
2406





90
20
POI
165
SASFCGSPY
9

2407





100
18
NUC
121
SFGVWIRTPPA
11

2408





90
19
NUC
46
SFLPSDFF
8

2409





95
15
POL
748
SFPWLIGCA
9

2410





75
15
POL
740
SFPWLLGCAA
10

2411





75
16
POL
765
SFVWPSA
8

2412





80
20
POL
49
SIPWTHKVGNF
11

2413





100
19
ENV
194
SLDSWWTSINF
11

2414





95
19
POL
416
SLDVSAAF
8

2415





95
19
POL
416
SIQVSAAFY
9
0.0016
2416





95
19
POL
416
SLDVSAAFYH
10

2417





75
15
POL
565
SIGIHLNPNK
10

2418





100
20
ENV
337
SLLVPFVQWF
10

2419





95
19
X
54
SLRGLPVCA
9

2420





95
19
X
54
SLRGLPVCAF
10
0.0004
2421





95
18
X
64
SSAGPCALR
9
0.0080
2422





90
18
X
64
SSAGPCALRF
10
−0.0003
2423





90
19
NUC
170
STIPETTVVR
10
0.0007
2424





95
19
NUC
170
STLPETTWRR
11
0.0150
2425





95
16
ENV
85
STNRQSGR
8

2426





80
15
X
104
STTDLEAY
8

2427





75
15
X
104
STTDLEAYF
9

2428





75
15
X
104
STTDLEAYFK
10
0.0066
2429





75
15
ENV
17
SVPNPLGF
8

2430





90
18
POL
739
SVVLSRKY
8
−0.0002
2431





85
17
POL
739
SVVLSRKYTSF
11

2432





95
19
POL
524
SVVRRAFPH
9
0.1100
2433





85
17
POL
716
TAELLAACF
9

2434





85
17
POL
716
TAELLAACFA
10

2435





85
17
POL
716
TAELLAACFAR
11
0.0006
2436





80
16
NUC
33
TASALYREA
9

2437





100
20
ENV
311
TCIPIPSSWA
10

2438





100
20
ENV
311
TCIPIPSSWAF
11

2439





80
16
X
106
TDLEAYFK
8

2440





90
18
POL
736
TDNSVVLSR
9

2441





90
18
POL
736
TDNSVVLSRK
10
0.0006
2442





90
18
POL
736
TDNSVVLSRKY
11

2443





75
15
NUC
138
TFGRETVLEY
10

2444





95
19
POL
657
TFSPTYKA
8

2445





95
19
POL
857
TFSPTYKAF
9

2446





100
20
POL
359
TGGVFLVQK
9
0.0007
2447





85
17
POL
799
TGRTSLYA
6

2448





95
19
NUC
171
TLPETTVVR
9
0.0008
2449





95
19
NUC
171
TLPETTVVRR
10
0.0007
2450





95
19
NUC
171
TLPETTVVRRR
11
0.0005
2451





100
20
POL
150
TLWKAGILY
9
0.1300
2452





100
20
POL
150
TLWKAGILYK
10
5.3000
2453





100
20
POL
150
TLWKAGILYKR
11
0.0082
2454





95
19
POL
519
TSAICSVVR
9
0.0005
2455





95
19
POL
519
TSAICSVVRR
10
0.0018
2456





95
19
POL
519
TSAICSVVRRA
11

2457





75
15
POL
747
TSFPWLLGCA
10

2458





75
15
POL
747
TSFPWLLGCAA
11

2459





80
16
POL
764
TSFVYVPSA
9

2460





75
15
X
105
TTDLEAYF
8

2461





75
15
X
105
TTDLEAYFK
9
0.0006
2462





85
17
POL
798
TTGRTSLY
8
0.0004
2463





85
17
POL
798
TTGRTSLYA
9

2464





75
15
ENV
278
TTSTGPCK
8

2465





80
16
NUC
175
TTVVRRRGR
9
0.0008
2466





80
16
NUC
176
TVVRRRGR
8
0.0003
2467





80
16
NUC
176
TVVRRRGRSPR
11

2468





95
19
X
60
VCAFSSAGPCA
11

2469





85
17
POL
621
VCQRNGLLGF
11

2470





100
20
POL
379
VDFSQFSR
8

2471





100
20
POL
362
VFLVDKNPH
9

2472





80
16
X
131
VFVLGGCR
8

2473





80
16
X
131
VFVLGGCRH
9

2474





75
15
X
131
VFVLGGCRHK
10

2475





95
19
X
21
VGAESRGR
8

2476





95
19
POL
626
VGLLGFAA
8

2477





95
19
POL
626
VGLLGFAAPF
10

2478





80
16
POL
508
VGLSPFLLA
9

2479





80
16
POL
508
VGLSPFLLAQF
11

2480





95
19
PO[-
56
VGNFTGLY
8

2481





85
17
POL
96
VGPLTVNEK
9
0.0007
2482





85
17
POL
96
VGPLTVNEKR
10

2483





85
17
POL
96
VGPLTVNEKRR
11

2484





95
19
X
15
VLCLRPVGA
9

2485





95
19
POL
543
VLGAKSVQH
9

2486





90
18
X
133
VLGGCRHK
8
0.0150
2487





80
16
ENV
177
VLQAGFFLLTR
11

2488





85
17
POL
741
VLSRKYTSF
9

2489





90
18
NUC
120
VSFGVWIR
8
0.0040
2490





100
20
POL
48
VSIPWTHK
8
0.0130
2491





100
20
POL
358
VTGGVFLVDK
10
0.0390
2492





100
20
POL
378
VVDFSQFSR
9
0.0015
2493





90
18
POL
542
VVLGAKSVQH
10

2494





85
17
POL
740
VVLSRKYTSF
10
0.0004
2495





95
19
POL
525
VVRRAFPH
8

2496





95
19
POL
525
VVRRAFPHCLA
11

2497





80
16
NUC
177
VVRRRGRSPR
10
0.0027
2498





80
16
NUC
177
VVRRRGRSPRR
11

2499





90
18
NUC
102
WFHISCLTF
9

2500





90
16
NUC
102
WFHISCLTFGR
11

2501





85
17
NUC
28
WGMDIDPY
8

2502





85
17
NUC
28
WGMDIDPYK
9
−0.0003
2503





85
17
NUC
28
WGMDIDPYKEF
11

2504





85
17
POL
578
WGYSLNFMGY
10

2505





80
16
POL
759
WILRGTSF
8

2506





80
16
POL
759
WILRGTSFVY
10
0.0076
2507





95
19
NUC
125
WIRTPPAY
8
−0.0002
2508





95
19
NX
125
WIRTPPAYR
9
0.0008
2509





90
18
POL
314
WLQFRNSK
8
−0.0002
2510





100
20
POL
414
WLSLDVSA
8

2511





95
19
POL
414
WLSLDVSAA
9

2512





95
19
POL
414
WLSLDVSAAF
10

2513





95
19
POL
414
WLSLDVSAAFY
11
0.0034
2514





100
20
ENV
335
WLSLLVPF
8

2515





85
17
RUC
26
WLWGMDIDPY
10
0.0002
2516





85
17
NUC
26
WLWGMDIDPYK
11
0.0030
2517





95
19
ENV
237
WMCLRRFIIF
10
0.0004
2518





85
17
ENV
359
WMMWYWGPSLY
11
0.0009
2519





100
20
POI
52
WTHKVGNF
8

2520





100
20
POL
147
YLHTLWKA
8

2521





100
20
POL
122
YLPLDKGIK
9
0.0001
2522





100
20
POL
122
YLPLDKGIKPY
11
−0.0004
2523





90
18
NUC
118
YLVSFGVWIR
10
0.0005
2524





90
18
POL
538
YMDDVVLGA
9
0.0001
2525





90
18
POL
538
YMDDWLGAK
10
0.0330
2526





80
16
POL
493
YSHPIILGF
9

2527





80
16
FOL
493
YSHPIILGFR
10

2528





80
16
POL
493
YSHIPIILGFRK
11

2529





85
17
POL
580
YSLNFMGY
8
−0.0002
2530





75
15
POL
746
YTSFPWLLGCA
11

2531





90
18
POL
768
YVPSALNPA
9

2532
















TABLE XVII







A11 Motif With Binding Information














Conservancy
Frequency
Protein
Position
Sequence
AA
A*1101
SeqID Num


















85
17
POL
721
AACFARSR
8

2533






95
19
POL
632
AAPFTQCGY
9

2534





90
18

776
ADDPSRGR
8

2535





95
19
POL
529
AFPHCLAFSY
10

2536





90
19
X
62
AFSSAGPCALR
11

2537





95
19
POL
655
AFTFSPTY
8

2538





95
19
POL
655
AFTFSPTYK
9

2539





80
16
ENV
180
AGFFLLTR
8

2540





95
19
POL
18
AGPLEEELPR
10

2541





95
19
POL
521
AICSVVRR
8

2542





95
19
NUC
41
ALESPEHCSPH
11

2543





90
18
POL
772
ALNPADDPSR
10

2544





85
17
X
70
ALRFTSAR
8

2545





80
16
ENV
108
AMQWNSTTFH
10

2546





80
8
POL
166
ASFCGSPY
8

2547





80
16
POL
822
ASPLHVAWR
9

2548





75
15
ENV
84
ASTNRQSGR
9

2549





80
16
POL
755
CAANWILR
8

2550





85
17
X
69
CALRFTSAR
9

2551





80
16
X
6
CCQLDPAR
8

2552





75
15
POL
607
CFRKLPVNR
9

2553





95
19
POL
638
CGYPALMPLY
10

2554





95
19
ENV
253
CLIFLLVLLDY
11

2555





90
18
X
17
CLRPVGAESR
10

2556





100
20
NUC
48
CSPHHTALR
9

2557





95
19
POL
523
CSVVRRAFPH
10

2558





90
18
POL
540
DDVVLGAK
8

2559





85
17
NUC
29
DLLDTASALY
10

2560





85
17
NUC
29
DLLDTASALYR
11

2561





90
18
POL
737
DNSVVLSR
8

2562





90
18
POL
737
DNSVVLSRK
9

2562





90
18
POL
737
DNSVVLSRKY
10

2533





85
17
NUC
32
DTASALYR
8

2534





95
19
POL
418
DVSAAFYH
8

2535





90
18
POL
541
DVVLGAKSVQH
11

2536





95
19
POL
17
EAGPLEEELPR
11

2537





90
18
NUC
40
EALESPEH
8

2538





90
18
POL
718
ELLAACFAR
9

2539





85
17
POL
718
ELLAACFARSR
11

2540





95
19
NUC
43
ESPEHCSPH
9

2541





95
19
NUC
43
ESPEHCSPHH
10

2542





95
19
NUC
174
ETTVVRRR
8

2543





80
16
NUC
174
ETTVVRRRGR
10

2544





95
19
POL
631
FAAPFTQCGY
10

2545





80
16
POL
821
FASPLHVAWR
10

2546





75
15
NUC
139
FGRETVLEY
9

2547





75
15
POL
244
FGVEPSGSGH
10

2548





95
19
NUC
122
FGVWIRTPPAY
11

2549





95
19
POL
562
FLLSLGIH
8

2550





95
19
ENV
256
FLLVLLDY
8

2551





100
20
POL
363
FLVDKVPH
8

2552





90
18
X
63
FSSAGPCALR
10

2553





95
19
POL
656
FTFSPTYK
8

2554





95
19
POL
518
FTSAICSVVR
10

2555





95
19
POL
518
FTSAICSVVRR
11

2556





95
19
X
132
FVLGGCRH
8

2557





90
18
X
132
FVLGGCRHK
9

2558





80
16
POL
754
GCAANWILR
9

2559





95
19
POL
630
GFAAPFTQCGY
11

2560





100
20
POL
360
GGVFLVDK
8

2561





100
20
POL
360
GGVFLVDKNPH
11

2562





75
15
POL
567
GIHLNPNK
8

2563





75
15
POL
567
GIHLNPNKTK
10

2564





75
15
POL
567
GIHLNPNKTKR
11

2565





85
17
NUC
29
GMDIDPYK
8

2566





95
19
POL
44
GNLNVSIPWTH
11

2567





90
18
POL
735
GTDNSVVLSR
10

2568





90
18
POL
735
GTDNSVVLSRK
11

2569





80
16
POL
245
GVEPSGSGH
9

2570





100
20
POL
361
GVFLVDKNPH
10

2571





95
19
NUC
123
GVWIRTPPAY
10

2572





95
19
NUC
123
GVWIRTPPAYR
11

2573





100
20
NUC
47
HCSPHHTALR
10

2574





80
16
POL
820
HFASPLHVAWR
11

2575





95
19
X
49
HGAHLSLR
8

2576





90
18
NUC
104
HISCLTFGR
9

2577





75
15
POL
569
HLNPNKTK
8

2578





75
15
POL
569
HLNPNKTKR
9

2579





100
20
POL
149
HTLWKAGILY
10

2580





100
20
POL
149
HTLWKAGILYK
11

2581





95
19
POL
522
ICSVVRRAFPH
11

2582





90
18
POL
617
IDWKVCQR
8

2583





100
20
ENV
381
IFFCLWVY
8

2584





95
19
ENV
255
IFLLVLLDY
9

2585





80
16
POL
734
IGTDNSVVLSR
11

2586





80
16
POL
760
ILRGTSFVY
9

2587





90
18
NUC
105
ISCLTFGR
8

2588





100
20
POL
153
KAGILYKR
8

2589





75
15
POL
108
KLIMPARFY
9

2590





80
16
POL
610
KLPVNRPIDWK
11

2591





75
15
X
130
KVFVLGGCR
9

2592





75
15
X
130
KVFVLGGCRH
10

2593





95
19
POL
55
KVGNFTGLY
9

2594





85
17
POL
720
LAACFARSR
9

2595





90
18
X
16
LCLRPVGAESR
11

2596





100
20
POL
125
LDKGIKPY
8

2597





100
20
POL
125
LDKGIKPYY
9

2598





80
16
x
9
LDPARDVLCLR
11

2599





85
17
NUC
31
LDTASALY
8

2600





85
17
NUC
31
LDTASALYR
9

2601





95
19
POL
417
LDVSAAFY
8

2602





95
19
POL
417
LDVSAAFYH
9

2603





95
19
POL
544
LGAKSVQH
8

2604





80
16
POL
753
LGCAANWILR
10

2605





75
15
POL
566
LGIHLNPNK
9

2606





75
15
POL
566
LGIHLNPNKTK
11

2607





95
19
ENV
254
LIFLLVLLDY
10

2608





100
20
POL
109
LIMPARFY
8

2609





90
18
POL
719
LLAACFAR
8

2610





85
17
POL
719
LLAACFARSR
10

2611





85
17
NUC
30
LLDTASALY
9

2612





85
17
NUC
30
LLDTASALYR
10

2613





80
16
POL
752
LLGCAANWILR
11

2614





100
20
ENV
378
LLPIFFCLWVY
11

2615





90
18
POL
773
LNPADDPSR
9

2616





90
18
POL
773
LNPADDPSRGR
11

2617





75
15
POL
570
LNPNKTKA
8

2618





75
15
POL
570
LNPNKTKRWGY
11

2619





95
19
POL
46
LNVSIPWTH
11

2620





95
19
POL
46
LNVSIPWTHK
10

2621





95
19
POL
415
LSLDVSAAFY
10

2622





95
19
POL
415
LSLDVSAAFYH
11

2623





75
15
POL
564
LSLGIHLNPNK
11

2624





95
19
NUC
169
LSTLPETTVVR
11

2625





75
15
POL
3
LSYQHFRK
8

2626





75
15
NUC
137
LTFGRETVLEY
11

2627





85
17
POL
99
LTVNEKRR
8

2628





90
18
NUC
119
LVSFGVWIR
9

2629





100
20
POL
377
LVVDFSQFSR
10

2630





90
18
POL
539
MDDVVLGAK
9

2631





85
17
ENV
360
MMWYWGPSLY
10

2632





75
15
X
103
MSTTDLEAY
9

2633





75
15
X
103
MSTTDLEAYFK
11

2634





95
19
POL
561
NFLLSLGIH
9

2635





90
18
NUC
75
NLEDPASR
8

2636





95
19
POL
45
NLNVSIPWTH
10

2637





95
19
POL
45
NLNVSIPWTHK
11

2638





75
15
ENV
215
NSQSPTSNH
9

2639





90
18
POL
738
NSVVLSRK
8

2640





90
18
POL
738
NSVVLSRKY
9

2641





100
20
POL
47
NVSIPWTH
8

2642





100
20
POL
47
NVSIPWTHK
9

2643





90
18
POL
775
PADDPSRGR
9

2644





80
16
X
11
PARDVLCLR
9

2645





75
15
ENV
83
PASTNRQSGR
10

2646





85
17
X
68
PCALRFTSAR
10

2647





100
20
ENV
233
PGYRWMCLR
9

2648





95
19
ENV
233
PGYRWMCLRR
10

2649





90
18
POL
616
PIDWKVCQR
9

2650





100
20
ENV
380
PIFFCLWVY
9

2651





80
16
POL
496
PIILGFRK
8

2652





100
20
POL
124
PLDKGIKPY
9

2653





100
20
POL
124
PLDKGIKPYY
10

2654





95
19
POL
20
PLEEELPR
8

2655





95
19
POL
10
PLGFFPDH
8

2656





100
20
POL
427
PLHPAAMPH
9

2657





100
20
POL
2
PLSYQHFR
8

2658





75
15
POL
2
PLSYQHFRK
9

2659





85
17
POL
98
PLTVNEKR
8

2660





85
17
POL
98
PLTVNEKRR
9

2661





75
15
POL
572
PNKTKRWGY
9

2662





85
17
POL
797
PTTGRTSLY
9

2663





90
18
X
20
PVGAESRGR
9

2664





85
17
POL
612
PVNRPIDWK
9

2665





95
19
POL
654
QAFTFSPTY
9

2666





95
19
POL
654
QAFTFSPTYK
10

2667





80
16
ENV
179
QAGFFLLTR
9

2668





80
16
ENV
107
QAMQMNSTTFH
10

2669





95
19
POL
637
QCGYPALMPLY
11

2670





95
19
POL
517
QFTSAICSVVR
11

2671





75
15
NUC
169
QSPRRRRSQSR
11

2672





80
16
POL
189
QSSGILSR
8

2673





95
19
POL
528
RAFPHCLAFSY
11

2674





85
17
NUC
28
RDLLDTASALY
11

2675





100
20
NUC
152
RGRSPRRR
8

2676





75
15
POL
108
RLKLIMPAR
9

2677





75
15
POL
106
RIKLIMPARFY
11

2678





75
15
X
128
RLKVFVLGGCR
11

2679





95
19
POL
376
RLVVDFSQFSR
11

2680





95
19
NUC
183
RSPRRRTPSPR
11

2681





75
15
NUC
167
RSQSPRRR
8

2682





75
15
NUC
167
RSQSPRRRR
9

2683





95
19
NUC
188
RTPSPRRR
8

2684





95
19
NUC
188
RTPSPRRRR
9

2685





80
16
POL
818
RVHFASPLH
9

2686





100
20
POL
357
RVTGGVFLVDK
11

2687





90
18
X
65
SAGPCALR
8

2688





95
19
POL
520
SAICSVVR
8

2689





95
19
POL
520
SAICSVVRR
9

2690





90
18
POL
771
SALNPADDPSR
11

2691





100
20
POL
165
SASFCGSPY
9

2692





95
19
POL
416
SLDVSAAFY
9

2693





95
19
POL
416
SLDVSAAFYH
10

2694





75
15
POL
565
SLGIHLNPNK
10

2695





90
18
X
64
SSAGPCALR
9

2696





95
19
NUC
170
STLPETTVVR
10

2697





95
19
NUC
170
STLPETTVVRR
11

2698





80
16
ENV
85
STNRQSGR
8

2699





75
15
X
104
STTDLEAY
8

2700





75
15
X
104
STTDLEAYFK
10

2701





90
18
POL
739
SVVLSRKY
8

2702





95
19
POL
524
SVVRRAFPH
9

2703





85
17
POL
716
TAELLAACFAR
11

2704





80
16
X
106
TDLEAYFK
8

2705





90
18
POL
736
TDNSVVLSR
9

2706





90
18
POL
736
TDNSVVLSRK
10

2707





90
18
POL
736
TDNSVVLSRKY
11

2708





75
15
NUC
138
TFGRETVLEY
10

2709





100
20
POL
359
TGGVFLVDK
9

2710





95
19
NUC
171
TLPETTVVR
9

2711





95
19
NUC
171
TLPETTVVRR
10

2712





95
19
NUC
171
TLPETTVVRRR
11

2713





100
20
POL
150
TLWKAGILY
9

2714





100
20
POL
150
TLWKAGILYK
10

2715





100
20
POL
150
TLWKAGILYKR
11

2716





95
19
POL
560
TNFLLSLGIH
10

2717





95
19
POL
519
TSAICSVVR
9

2718





95
19
POL
519
TSAICSVVRR
10

2719





75
15
X
105
TTDLEAYFK
9

2720





85
17
POL
798
TTGRTSLY
8

2721





75
15
ENV
278
TTSTGPCK
8

2722





80
16
NUC
175
TTVVRRRGR
9

2723





80
16
NUC
176
TVVRRRGR
8

2724





80
16
NUC
176
TVVRRRGRSPR
11

2725





100
20
POL
379
VDFSQFSR
8

2726





100
20
POL
362
VFLVDKNPH
9

2727





80
16
X
131
VFVLGGCR
8

2728





80
16
X
131
VFVLGGCRH
9

2729





75
15
X
131
VFVLGGCRHK
10

2730





95
19
X
21
VGAESRGR
8

2731





95
19
POL
56
VGNFTGLY
8

2732





85
17
POL
96
VGPLTVNEK
9

2733





85
17
POL
96
VGPLTVNEKR
10

2734





85
17
POL
96
VGPLTVNEKRR
11

2735





95
19
POL
543
VLGAKSVQH
9

2736





90
18
X
133
VLGGCRHK
8

2737





80
16
ENV
177
VLQAGFFLLTR
11

2738





85
17
POL
613
VNRPIDWK
8

2739





90
18
NUC
120
VSFGVWIR
8

2740





100
20
POL
48
VSIPWTHK
8

2741





100
20
POL
358
VTGGVFLVDK
10

2742





100
20
POL
378
VVDFSQFSR
9

2743





90
18
POL
542
VVLGAKSVQH
10

2744





95
19
POL
525
VVRRAFPH
8

2745





80
16
NUC
177
VVRRRGRSPR
10

2746





80
16
NUC
177
VVRRRGRSPRR
11

2747





90
18
NUC
102
WFHISCLTFGR
11

2748





85
17
NUC
28
WGMDIDPY
8

2749





85
17
NUC
28
WGMDIDPYK
9

2750





85
17
POL
578
WGYSLNFMGY
10

2751





80
16
POL
759
WILRGTSFVY
10

2752





95
19
NUC
125
WIRTPPAY
8

2753





95
19
NUC
125
WIRTPPAYR
9

2754





90
18
POL
314
WLQFRNSK
8

2755





95
19
POL
414
WLSLDVSAAFY
11

2756





85
17
NUC
26
WLWGMDIDPY
10

2757





85
17
NUC
26
WLWGMDIDPYK
11

2758





85
17
ENV
359
WMMWYWGPSLY
11

2759





100
20
POL
122
YLPLDKGIK
9

2760





100
20
POL
122
YLPLDKGIKPY
11

2761





90
18
NUC
118
YLVSFGVWIR
10

2762





90
18
POL
538
YMDDVVLGAK
10

2763





80
16
POL
493
YSHPIILGFR
10

2764





80
16
POL
493
YSHPIILGFRK
11

2765





85
17
POL
580
YSLNFMGY
8

2766
















TABLE XVIII







HBV A24 Motif With Binding Information





















SEQ ID






Conservancy
Freq.
Protein
Position
Sequence
NO:
AA
Filed
A*2401



















95
19
POL
529
AFPHCLAF
2767
8








95
19
X
62
AFSSAGPCAL
2768
10

0.0012





90
18
POL
535
AFSYMDDVVL
2769
10

0.0009





95
19
POL
655
AFTFSPTYKAF
2770
11





80
16
ENV
108
AMQWNSTTF
2771
9





100
20
NUC
131
AYRPPNAPI
2772
9
*
0.0310





100
20
NUC
131
AYRPPNAPIL
2773
10
*
0.0042





75
15
POL
607
CFRKLPVNRPI
2774
11





85
17
POL
618
DWKVCQRI
2775
8





85
17
POL
618
DWKVCQRIVGL
2776
11





90
18
ENV
262
DYQGMLPVCPL
2777
11

0.0002





90
18
NUC
117
EYLVSFGVW
2778
9





90
18
NUC
117
EYLVSFGVWI
2779
10
*





100
20
ENV
382
FFCLWVYI
2780
8





80
16
ENV
182
FFLLTRIL
2781
8





80
16
ENV
182
FFLLTRILTI
2782
10





85
17
ENV
13
FFPDHQLDPAF
2783
11





80
16
ENV
181
GFFLLTRI
2784
8





80
16
ENV
181
GFFLLTRIL
2785
9





80
16
ENV
181
GFFLLTRILTI
2786
11





95
19
ENV
12
GFFPDHQL
2787
8





75
15
ENV
170
GFLGPLLVL
2788
9





80
16
POL
500
GFRKIPMGVGL
2789
11





85
17
NUC
29
GMDIDPYKEF
2790
10





90
18
ENV
265
GMLPVCPL
2791
8





85
17
NUC
25
GWLWGMDI
2792
8





85
17
ENV
65
GWSPQAQGI
2793
9

0.0024





85
17
ENV
65
GWSPQAQGIL
2794
10

0.0003





95
19
POL
639
GYPALMPL
2795
8





95
19
ENV
234
GYRWMCLRRF
2796
10
*
0.0007





95
19
ENV
234
GYRWMCLRRFI
2797
11





75
15
POL
579
GYSLNFMGYVI
2798
11





80
16
POL
820
HFASPLHVAW
2799
10





75
15
POL
7
HFRKLLLL
2800
8





100
20
POL
146
HYLHTLWKAGI
2801
11





100
20
ENV
381
IFFCLWVYI
2802
9

0.0087





80
16
ENV
245
IFLFILLL
2803
8





80
16
ENV
245
IFLFILLLCL
2804
10





80
16
ENV
245
IFLFILLLCLI
2805
11





85
17
ENV
358
IWMMWYWGPSL
2806
11

0.0004





95
19
POL
395
KFAVPNLQSL
2807
10

0.0020





100
20
POL
121
KYLPLDKGI
2808
9





85
17
POL
745
KYTSFPWL
2809
8





85
17
POL
745
KYTSFPWLL
2810
9
*
5.3000





80
16
ENV
247
LFILLLCL
2811
8





80
16
ENV
247
LFILLLCLI
2812
9





80
16
ENV
247
LFILLLCLIF
2813
10





80
16
ENV
247
LFILLLCLIFL
2814
11





95
19
POL
643
LMPLYACI
2815
8





90
18
NUC
101
LWFHISCL
2816
8





85
17
NUC
101
LWFHISCLTF
2817
10





80
16
POL
492
LYSHPIIL
2818
8





80
16
POL
492
LYSHPIILGF
2819
10
*
1.1000





85
17
ENV
360
MMWYWGPSL
2820
9
*
0.0060





85
17
ENV
361
MWYWGPSL
2821
8

0.0005





95
19
POL
561
NFLLSLGI
2822
8





95
19
POL
561
NFLLSLGIHL
2823
10

0.0099





80
16
POL
758
NWILRGTSF
2824
9





95
19
POL
512
PFLLAQFTSAI
2825
11





95
19
POL
634
PFTQCGYPAL
2826
10

0.0002





95
19
ENV
341
PFVQWFVGL
2827
9

0.0003





80
16
POL
505
PMGVGLSPF
2828
9





80
16
POL
505
PMGVGLSPFL
2829
10





80
16
POL
505
PMGVGLSPFLL
2830
11





80
16
POL
750
PWLLGCAANW
2831
10





80
16
POL
750
PWLLGCAANWI
2832
11





100
20
POL
51
PWTHKVGNF
2833
9
*
0.0290





95
19
ENV
344
QWFVGLSPTVW
2834
11





75
15
ENV
242
RFIIFLFI
2835
8





75
15
ENV
242
RFIIFLFIL
2836
9





75
15
ENV
242
RFIIFLFILL
2837
10





75
15
ENV
242
RFIIFLFILLL
2838
11





100
20
ENV
332
RFSWLSLL
2839
8





100
20
ENV
332
RFSWLSLLVPF
2840
11





85
17
POL
577
RWGYSLNF
2841
8





95
19
ENV
236
RWMCLRRF
2842
8





95
19
ENV
236
RWMCLRRFI
2843
9
*
0.0710





95
19
ENV
236
RWMCLRRFII
2844
10
*
1.1000





95
19
ENV
236
RWMCLRRFIIF
2845
11





100
20
POL
167
SFCGSPYSW
2846
9
*
0.0710





95
19
NUC
46
SFLPSDFF
2847
8





80
16
POL
765
SFVYVPSAL
2848
9





95
19
POL
413
SWLSLDVSAAF
2849
11





100
20
ENV
334
SWLSLLVPF
2850
9
*
0.3900





95
19
POL
392
SWPKFAVPNL
2851
10
*
5.6000





100
20
ENV
197
SWWTSLNF
2852
8





95
19
ENV
197
SWWTSLNFL
2853
9
*
0.3800





90
18
POL
537
SYMDDVVL
2854
8





75
15
POL
4
SYQHFRKL
2855
8





75
15
POL
4
SYQHFRKLL
2856
9

0.0051





75
15
POL
4
SYQHFRKLLL
2857
10
*
0.0660





75
15
POL
4
SYQHFRKLLLL
2858
11





75
15
NUC
138
TFGRETVL
2859
8





75
15
NUC
138
TFGRETVLEYL
2860
11





95
19
POL
657
TFSPTYKAF
2861
9

0.0060





95
19
POL
657
TFSPTYKAFL
2862
10

0.0043





95
19
POL
686
VFADATPTGW
2863
10
*
0.0180





75
15
X
131
VFVLGGCRHKL
2864
11





90
18
NUC
102
WFHISCLTF
2865
9
*
0.0300





95
19
ENV
345
WFVGLSPTVW
2866
10
*
0.0120





95
19
ENV
345
WFVGLSPTVWL
2867
11





95
19
ENV
237
WMCLRRFI
2868
8





95
19
ENV
237
WMCLRRFII
2869
9
*





95
19
ENV
237
WMCLRRFIIF
2870
10

0.0013





95
19
ENV
237
WMCLRRFIIFL
2871
11





85
17
ENV
359
WMMWYWGPSL
2872
10
*





95
19
ENV
198
WWTSLNFL
2873
8
















TABLE XIXa







HBV DR-SUPER MOTIF























Position

Exemplary






Core


In HBV
Exemplary
Sequence



Core SEQ
Core
Core
Conservancy
Exemplary
Exemplary
Poly-
Sequence
Conservancy


Protein
ID NO:
Sequence
Freq.
(%)
SEQ ID NO:
Sequence
Protein
Frequency
(%)



















POL
2874
FAAPFTQCG
19
95
3021
LLGFAAPFTQCGYPA
628
19
95





POL
2875
FADATPTGW
19
95
3022
CQVFADATPTGWGLA
684
16
80





POL
2876
FAVPNLQSL
19
95
3023
WPKFAVPNLQSLTNL
393
19
95





NUC
2877
FGRETVLEY
15
75
3024
CLTFGRETVLEYLVS
136
14
70





POL
2878
FGVEPSGSG
15
75
3025
RRSFGVEPSGSGHID
252
6
30





NUC
2879
FHISCLTFG
18
90
3026
LLWFHISCLTFGRET
100
17
85





NUC
2880
FHLCLIISC
16
80
3027
MQLFHLCLIISCSCP
1
10
50





ENV
2881
FILLLCLIF
16
80
3028
IFLFILLLCLIFLLV
245
16
80





ENV
2882
FLFILLLCL
16
80
3029
FIIFLFILLLCLIFL
243
16
80





ENV
2883
FLGPLLVLQ
15
75
3030
TSGFLGPLLVLQAGF
168
15
75





ENV
2884
FLLTRILTI
16
80
3031
AGFFLLTRILTIPQS
180
16
80





ENV
2885
FLLVLLDYQ
19
95
3032
CLIFLLVLLDYQGML
253
19
95





ENV
2886
FPAGGSSSG
15
75
3033
GLYFPAGGSSSGTVN
127
11
55





ENV
2887
FPDHQLDPA
18
90
3034
LGFFPDHQLDPAFGA
22
9
45





POL
2888
FPHCLAFSY
19
95
3035
RRAFPHCLAFSYMDD
527
19
95





POL
2889
FRKIPMGVG
16
80
3036
ILGFRKIPMGVGLSP
498
13
65





POL
2890
FRKLPVNRP
16
80
3037
KQCFRKLPVNRPIDW
616
9
45





X
2891
FSSAGPCAL
19
95
3038
VCAFSSAGPCALRFT
60
18
90





ENV
2892
FSWLSLLVP
20
100
3039
SVRFSWLSLLVPFVQ
330
16
80





POL
2893
FTFSPTYKA
19
95
3040
KQAFTFSPTYKAFLC
653
12
60





POL
2894
FTGLYSSTV
18
90
3041
VGNFTGLYSSTVPVF
56
11
55





POL
2895
FTSAICSVV
19
95
3042
LAQFTSAICSVVRRA
515
19
95





ENV
2896
FVGLSPTVW
19
95
3043
VQWFVGLSPTVWLSV
343
14
70





X
2897
FVLGGCRHK
18
90
3044
LKVFVLGGCRHKLVC
129
14
70





ENV
2898
FVQWFVGLS
19
95
3045
LVPFVQWFVGLSPTV
339
19
95





POL
2899
FVYVPSALN
18
90
3046
GTSFVYVPSALNPAD
763
16
80





POL
2900
IDWKVCQRI
17
85
3047
NRPIDWKVCQRIVGL
614
16
80





ENV
2901
IFLFILLLC
16
80
3048
RFIIFLFILLLCLIF
242
15
75





ENV
2902
IFLLVLLDY
19
95
3049
LCLIFLLVLLDYQGM
252
19
95





POL
2903
IGTDNSVVL
16
80
3050
AKLIGTDNSVVLSRK
731
13
65





POL
2904
IHTAELLAA
17
85
3051
PLPIHTAELLAACFA
711
16
80





ENV
2905
IIFLFILLL
16
80
3052
RRFIIFLFILLLCLI
241
15
75





ENV
2906
ILLLCLIFL
20
100
3053
FLFILLLCLIFLLVL
246
16
80





POL
2907
ILRGTSFVY
16
80
3054
ANWILRGTSFVYVPS
757
16
80





NUC
2908
ILSTLPETT
20
100
3055
NAPILSTLPETTVVR
165
19
95





ENV
2909
IPIPSSWAF
20
100
3056
CTCIPIPSSWAFARF
321
8
40





NUC
2910
IRTPPAYRP
19
95
3057
GVWIRTPPAYRPPNA
123
19
95





POL
2911
LAACFARSR
17
85
3058
AELLAACFARSRSGA
717
16
80





POL
2912
LAFSYMDDV
18
90
3059
PHCLAFSYMDDVVLG
531
18
90





POL
2913
LAQFTSAIC
19
$$5
3060
PFLLAQFTSAICSVV
512
19
95





NUC
2914
LCLGWLWGM
17
85
3061
ASKLCLGWLWGMDID
19
17
85





ENV
2915
LCLIFLLVL
20
100
3062
ILLLCLIFLLVLLDY
249
19
95





X
2916
LCLRPVGAE
19
95
3063
RDVLCLRPVGAESRG
13
18
90





POL
2917
LCQVFADAT
19
95
3064
RPGLCQVFADATPTG
680
11
55





ENV
2918
LDSWWTSLN
19
95
3065
PQSLDSWWTSLNFLG
192
17
85





NUC
2919
LDTASALYR
17
85
3066
RDLLDTASALYREAL
28
16
80





POL
2920
LDVSAAFYH
19
95
3067
WLSLDVSAAFYHIPL
425
11
55





ENV
2921
LDYQGMLPV
18
90
3068
LVLLDYQGMLPVCPL
258
18
90





POL
2922
LEEELPRLA
18
90
3069
AGPLEEELPRLADEG
18
13
65





ENV
2923
LFILLLCLI
16
80
3070
IIFLFILLLCLIFLL
244
16
80





POL
2924
LGAKSVQHL
17
85
3071
DVVLGAKSVQHLESL
541
16
80





POL
2925
LGFAAPFTQ
19
95
3072
VGLLGFAAPFTQCGY
626
19
95





POL
2926
LGFRKIPMG
19
95
3073
PIILGFRKIPMGVGL
496
13
65





POL
2927
LGNLNVSIP
19
95
3074
DUNLGNLNVSIPWTH
40
19
95





ENV
2928
LGPLLVLQA
19
95
3075
SGFLGPLLVLQAGFF
169
15
75





POL
2929
LHPAAMPHL
20
100
3076
HLPLHPAAMPHLLVG
425
9
45





ENV
2930
LIFLLVLLD
19
95
3077
LLCLIFLLVLLDYQG
251
19
95





POL
2931
LKLIMPARF
15
75
3078
KRRLKLIMPARFYPN
104
7
35





X
2932
LKVFVLGGC
15
75
3079
EIRLKVFVLGGCRHK
126
13
65





POL
2933
LLAQFTSAI
19
95
3080
SPFLLAQFTSAICSV
511
19
95





NUC
2934
LLDTASALY
17
85
3081
IRDLLDTASALYREA
56
9
45





POL
2935
LLGCAANWI
16
80
3082
FPWLLGCAANWILRG
749
15
75





POL
2936
LLGFAAPFT
19
95
3083
IVGLLGFAAPFTQCG
625
18
90





ENV
2937
LLGWSPQAQ
17
85
3084
HGGLLGWSPQAQGIL
60
15
75





ENV
2938
LLLCLIFLL
20
100
3085
LFILLLCLIFLLVLL
247
16
80





NUC
2939
LLSFLPSDF
19
95
3086
SVELLSFLPSDFFPS
41
11
55





POL
2940
LLSLGIHLN
19
95
3087
TNFLLSLGIHLNPNK
560
15
75





POL
2941
LLSSNLSWL
18
90
3088
LTNLLSSNLSWLSLD
404
18
90





ENV
2942
LLTRILTIP
16
80
3089
GFFLLTRILTIPQSL
181
16
80





ENV
2943
LLVLQAGFF
19
95
3090
LGPLLVLQAGFFLLT
172
18
90





ENV
2944
LLVPFVQWF
20
100
3091
WLSLLVPFVQWFVGL
335
19
95





NUC
2945
LLWFHISCL
18
90
3092
IRQLLWFHISCLTFG
126
13
65





POL
2946
LMPLYACIQ
19
95
3093
YPALMPLYACIQSKQ
640
11
55





POL
2947
LNLGNLNVS
19
95
3094
AEDLNLGNLNVSIPW
38
19
95





POL
2948
LNPNKTKRW
15
75
3095
GIHLNPNKTKRWGYS
567
15
75





POL
2949
LNRRVAEDL
17
85
3096
DEGLNRRVAEDLNLG
30
12
60





POL
2950
LNVSIPWTH
19
95
3097
LGNLNVSIPWTHKVG
43
19
95





NUC
2951
LPETTVVRR
19
95
3098
LSTLPETTVVRRRGR
169
16
80





ENV
2952
LPIFFCLWV
20
100
3099
LPLLPIFFCLWVYIZ
376
13
65





POL
2953
LPIHTAELL
17
85
3100
VAPLPIHTAELLAAC
709
9
45





POL
2954
LPVNRPIDW
16
80
3101
FRKLPVNRPIDWKVC
608
15
75





POL
2955
LQFRNSKPC
18
90
3102
CWWLQFRNSKPCSDY
312
10
50





X
2956
LRGLPVCAF
19
95
3103
HLSLRGLPVCAFSSA
52
18
90





X
2957
LRPVGAESR
18
90
3104
VLCLRPVGAESRGRP
15
18
90





NUC
2958
LRQAILCWG
18
90
3105
HTALRQAILCWGELM
52
18
90





ENV
2959
LRRFIIFLF
15
75
3106
WMCLRRFIIFLFILL
237
15
75





NUC
2960
LSFLPSDFF
19
95
3107
VELLSFLPSDFFPSI
42
10
50





POL
2961
LSLDVSAAF
19
95
3108
LSWLSLDVSAAFYHI
423
11
55





ENV
2962
LSLLVPFVQ
20
100
3109
FSWLSLLVPFVQWFV
333
19
95





X
2963
LSLRGLPVC
19
95
3110
GAHLSLRGLPVCAFS
50
18
90





POL
2964
LSPFLLAQF
19
95
3111
GVGLSPFLLAQFTSA
507
16
80





POL
2965
LSRKYTSFP
17
85
3112
SVVLSRKYTSFPWLL
739
17
85





POL
2966
LSSNLSWLS
18
90
3113
TNLLSSNLSWLSLDV
405
18
90





ENV
2967
LSVPNPLGF
15
75
3114
GTNLSVPNPLGFFPD
13
14
70





POL
2968
LSWLSLDVS
20
100
3115
SSNLSWLSLDVSAAF
409
17
85





ENV
2969
LTIPQSLDS
18
90
3116
TRILTIPQSLDSWWT
186
15
75





POL
2970
LTNLLSSNL
18
90
3117
LQSLTNLLSSNLSWL
401
18
90





ENV
2971
LTRILTIPQ
16
80
3118
FFLLTRILTIPQSLD
182
15
75





POL
2972
LVDKNPHNT
20
100
3119
GVFLVDKNPHNTTES
372
11
55





NUC
2973
LVSFGVWIR
18
90
3120
LEYLVSFGVWIRTPP
145
14
70





POL
2974
LVVDFSQFS
20
100
3121
ESRLVVDFSQFSRGN
374
9
45





NUC
2975
LWFHISCLT
17
85
3122
RQLLWFHISCLTFGR
98
17
85





NUC
2976
LWGMDIDPY
17
85
3123
LGWLWGMDIDPYKEF
24
17
85





POL
2977
LWKAGILYK
20
100
3124
LHTLWKAGILYKRET
148
18
90





NUC
2978
LYREALESP
17
85
3125
ASALYREALESPEHC
34
17
85





POL
2979
LYSHPIILG
16
80
3126
KLHLYSHPIILGFRK
489
16
80





POL
2980
MDDVVLGAK
18
90
3127
FSYMDDVVLGAKSVQ
536
18
90





POL
2981
MGVGLSPFL
16
80
3128
KIPMGVGLSPFLLAQ
503
16
80





POL
2982
MPHLLVGSS
17
85
3129
PAAMPHLLVGSSGLS
430
8
40





ENV
2983
MQWNSTTFH
16
80
3130
PQAMQWNSTTFHQTL
106
8
40





X
2984
MSTTDLEAY
15
75
3131
LSAMSTTDLEAYFKD
100
9
45





ENV
2985
MWYWGPSLY
17
85
3132
IWMMWYWGPSLYNIL
369
9
45





X
2986
VCAFSSAGP
19
95
3133
GLPVCAFSSAGPCAL
57
18
90





POL
2987
VCQRIVGLL
17
85
3134
DWKVCQRIVGLLGFA
618
17
85





POL
2988
VFADATPTG
19
95
3135
LCQVFADATPTGWGL
683
19
95





ENV
2989
VGLSPTVWL
19
95
3136
QWFVGLSPTVWLSVI
344
14
70





POL
2990
VGPLTVNEK
17
85
3137
QQYVGPLTVNEKRRL
93
8
40





POL
2991
VHFASPLHV
16
80
3138
PDRVHFASPLHVAWR
816
12
60





X
2992
VLCLRPVGA
19
95
3139
ARDVLCLRPVGAESR
12
14
70





POL
2993
VLGAKSVQH
19
95
3140
DDVVLGAKSVQHLES
540
16
80





X
2994
VLHKRTLGL
17
85
3141
LPKVLHKRTLGLSAM
89
11
55





POL
2995
VPNLQSLTN
19
95
3142
KFAVPNLQSLTNLLS
395
19
95





NUC
2996
VQASKLCLG
16
80
3143
CPTVQASKLCLGWLW
14
15
75





ENV
2997
VRFSWLSLL
16
80
3144
WASVRFSWLSLLVPF
328
13
65





POL
2998
VRRAFPHCL
19
95
3145
CSVVRRAFPHCLAFS
523
19
95





POL
2999
VSIPWTHKV
20
100
3146
NLNVSIPWTHKVGNF
45
19
95





NUC
3000
VWIRTPPAY
19
95
3147
SFGVWIRTPPAYRPP
121
18
90





POL
3001
VYVPSALNP
18
90
3148
TSFVYVPSALNPADD
764
16
80





NUC
3002
WFHISCLTF
18
90
3149
QLLWFHISCLTFGRE
99
17
85





ENV
3003
WFVGLSPTV
19
95
3150
FVQWFVGLSPTVWLS
342
19
95





POL
3004
WILRGTSFV
16
80
3151
AANWILRGTSFVYVP
756
14
70





NUC
3005
WIRTPPAYR
19
95
3152
FGVWIRTPPAYRPPN
122
19
95





POL
3006
WKAGILYKR
20
100
3153
HTLWKAGILYKRETT
149
18
90





POL
3007
WLLGCAANW
16
80
3154
SFPWLLGCAANWILR
748
15
75





POL
3008
WLSLDVSAA
19
95
3155
NLSWLSLDVSAAFYH
411
17
85





ENV
3009
WLSLLVPFV
20
100
3156
RFSWLSLLVPFVQWF
332
20
100





POL
3010
WPKFAVPNL
19
95
3157
RVSWPKFAVPNLQSL
390
11
55





POL
3011
YMDDVVLGA
18
90
3158
AFSYMDDVVLGAKSV
535
18
90





POL
3012
YPALMPLYA
19
95
3159
QCGYPALMPLYACIQ
637
19
95





ENV
3013
YQGMLPVCP
18
90
3160
LLDYQGMLPVCPLIP
260
10
50





NUC
3014
YRPPNAPIL
20
100
3161
PPAYRPPNAPILSTL
129
19
95





ENV
3015
YRWMCLRRF
19
95
3162
CPGYRWMCLRRFIIF
232
19
95





POL
3016
YSHPIILGF
16
80
3163
LHLYSHPIILGFRKI
490
16
80





POL
3017
YSLNFMGYV
15
75
3164
RWGYSLNFMGYVIGS
588
11
55





POL
3018
YVPSALNPA
18
90
3165
SFVYVPSALNPADDP
765
16
80





ENV
3019
FFCLWVYIZ
20



382







ENV
3020
MGTNLSVPN
15



12
















TABLE XIXB







HBV DR-SUPER MOTIF With Binding Data























Core


















SEQ ID

SEQ ID
















NO:
Core Sequence
NO:
Exemplary Sequence
DR1
DR2w2β1
DR2w2β2
DR3
DR4w4
DR4w15
DR5w11
DR5w12
DR6w19
DR7
DR8W2
DR9
Drw53


























2874
FAAPFTQCG
3021
LLGFAAPFTQCGYPA


















2875
FADATPTGW
3022
CQVFADATPTGWGLA





0.2800












2876
FAVPNLQSL
3023
WPKFAVPNLQSLTNL
0.0007

0.0013

0.0023

0.0002


0.0008


0.0180





2877
FGRETVLEY
3024
CLTFGRETVLEYLVS


















2878
FGVEPSGSG
3025
RRSFGVEPSGSGHID


















2879
FHISCLTFG
3026
LLWFHISCLTFGRET


















2880
FHLCLIISC
3027
MQLFHLCLIISCSCP


















2881
FILLLCLIF
3028
IFLFILLLCLIFLLV
0.0005



0.0041




0.0018








2882
FLFILLLCL
3029
FIIFLFILLLCLIFL


















2883
FLGPLLVLQ
3030
TSGFLGPLLVLQAGF


















2884
FLLTRILTI
3031
AGFFLLTRILTIPQS
4.6000
0.0420
0.0190
0.0040
5.3000
0.1500
3.6000
0.0700
0.3700
3.1000
0.2600
1.3000






2885
FLLVLLDYQ
3032
CLIFLLVLLDYQGML


















2886
FPAGGSSSG
3033
GLYFPAGGSSSGTVN


















2887
FPDHQLDPA
3034
LGFFPDHQLDPAFGA


















2888
FPHCLAFSY
3035
RRAFPHCLAFSYMDD
0.0010

0.0010

−0.0009

0.0010


0.0017








2889
FRKIPMGVG
3036
ILGFRKIPMGVGLSP


















2890
FRKLPVNRP
3037
KQCFRKLPVNRPIDW
1.5000
0.0022
0.0210
−0.0006
1.2000
0.8500
0.0130
0.0013
0.0043
0.4000
0.0580
0.0250






2891
FSSAGPCAL
3038
VCAFSSAGPCALRFT
0.2100

0.2600

0.0023

0.0003


0.0200


0.0150





2892
FSWLSLLVP
3039
SVRFSWLSLLVPFVQ
0.9000



0.0099




0.0037








2893
FTFSPTYKA
3040
KQAFTFSPTYKAFLC
0.5300
0.2400
0.1400
0.0090
1.1000
0.2200
0.2400
0.0024
0.0200
0.3300
0.1200
0.5400






2894
FTGLYSSTV
3041
VGNFTGLYSSTVPVF
1.7000
0.0100
0.0016

0.0140
0.1700
0.0035

0.0580
0.5800
0.0044
0.3100






2895
FTSAICSVV
3042
LAQFTSAICSVVRRA
0.0120
0.0065
0.1500
−0.0009
0.0150
0.0280
0.0076
0.0091
0.0010
0.0280
0.0150
0.0880
0.0190





2896
FVGLSPTVW
3043
VQWFVGLSPTVWLSV


















2897
FVLGGCRHK
3044
LKVFVLGGCRHKLVC


















2898
FVQWFVGLS
3045
LVPFVQWFVGLSPTV
0.0130
0.6900
0.0140
−0.0013
0.1500
1.4000
0.3800
0.6600
0.0018
0.0092
0.6600
2.5000
2.6000





2899
FVYVPSALN
3046
GTSFVYVPSALNPAD
0.3500
0.0140
0.0500
−0.0006
0.3800
0.4100
0.0470
−0.0001
0.0001
0.2700
0.0610
0.3400






2900
IDWKVCQRI
3047
NRPIDWKVCQRIVGL


















2901
IFLFILLLC
3048
RFIIFLFILLLCLIF


















2902
IFLLVLLDY
3049
LCLIFLLVLLDYQGM
0.0016

0.0060

0.0230

0.0017


0.0044








2903
IGTDNSVVL
3050
AKLIGTDNSVVLSRK


















2904
IHTAELLAA
3051
PLPIHTAELLAACFA
0.0046



0.0490




−0.0003








2905
IIFLFILLL
3052
RRFIIFLFILLLCLI


















2906
ILLLCLIFL
3053
FLFILLLCLIFLLVL


















2907
ILRGTSFVY
3054
ANWILRGTSFVYVPS


















2908
ILSTLPETT
3055
NAPILSTLPETTVVR
0.0009

0.0009

−0.0007

−0.0002


0.0005


0.1600





2909
IPIPSSWAF
3056
CTCIPIPSSWAFARF


















2910
IRTPPAYRP
3057
GVWIRTPPAYRPPNA
0.3700
0.0420
7.2000
0.0120
3.4000
0.5700
0.4800
0.0140
−0.0004
0.2200
0.5300
0.0450






2911
LAACFARSR
3058
AELLAACFARSRSGA


















2912
LAFSYMDDV
3059
PHCLAFSYMDDVVLG


















2913
LAQFTSAIC
3060
PFLLAQFTSAICSVV
0.1800
0.0270
0.0042
−0.0013
0.0800
0.1200
0.0120
0.0016
0.0800
0.0770
0.0580
0.0590






2914
LCLGWLWGM
3061
ASKLCLGWLWGMDID
0.0002

−0.0005

0.0017

−0.0002


0.0013


0.0010





2915
LCLIFLLVL
3062
ILLLCLIFLLVLLDY
0.0026

0.0069

0.0320

0.0018


0.0047








2916
LCLRPVGAE
3063
RDVLCLRPVGAESRG


















2917
LCQVFADAT
3064
RPGLCQVFADATPTG


















2918
LDSWWTSLN
3065
PQSLDSWWTSLNFLG


















2919
LDTASALYR
3066
RDLLDTASALYREAL
0.0001



0.0092




0.0770








2920
LDVSAAFYH
3067
WLSLDVSAAFYHIPL


















2921
LDYQGMLPV
3068
LVLLDYQGMLPVCPL
0.0034



−0.0013




0.0011








2922
LEEELPRLA
3069
AGPLEEELPRLADEG



0.0022














2923
LFILLLCLI
3070
IIFLFILLLCLIFLL


















2924
LGAKSVQHL
3071
DVVLGAKSVQHLESL


















2925
LGFAAPFTQ
3072
VGLLGFAAPFTQCGY
0.0470
0.3100
0.0008

−0.0014

−0.0004

−0.0001
0.0014

0.5700






2926
LGFRKIPMG
3073
PIILGFRKIPMGVGL


















2927
LGNLNVSIP
3074
DLNLGNLNVSIPWTH
0.0038



0.0240




0.0010








2928
LGPLLVLQA
3075
SGFLGPLLVLQAGFF


















2929
LHPAAMPHL
3076
HLPLHPAAMPHLLVG


















2930
LIFLLVLLD
3077
LLCLIFLLVLLDYQG


















2931
LKLIMPARF
3078
KRRLKLIMPARFYPN


















2932
LKVFVLGGC
3079
EIRLKVFVLGGCRHK


















2933
LLAQFTSAI
3080
SPFLLAQFTSAICSV
0.1200
0.0200
0.0085
−0.0013
0.0740
0.0190
−0.0002
−0.0013
0.0540
0.0330
0.0014
0.0380
0.2000





2934
LLDTASALY
3081
IRDLLDTASALYREA


















2935
LLGCAANWI
3082
FPWLLGCAANWILRG


















2936
LLGFAAPFT
3083
IVGLLGFAAPFTQCG
0.0200

−0.0005

−0.0007

−0.0002


0.0009


0.0067





2937
LLGWSPQAQ
3084
HGGLLGWSPQAQGIL


















2938
LLLCLIFLL
3085
LFILLLCLIFLLVLL


















2939
LLSFLPSDF
3086
SVELLSFLPSDFFPS


















2940
LLSLGIHLN
3087
TNFLLSLGIHLNPNK
3.5000
0.0410
0.1200

0.0220
0.0360
0.0053

0.0160
0.2200
0.0032
0.3800






2941
LLSSNLSWL
3088
LTNLLSSNLSWLSLD
0.0010

0.0083

0.0160

0.0013


0.0019


0.0200





2942
LLTRILTIP
3089
GFFLLTRILTIPQSL
0.4300
0.0150
0.0110

3.1000
0.4500
2.3000

0.0780
3.5000
1.6000
0.5500






2943
LLVLQAGFF
3090
LGPLLVLQAGFFLLT


















2944
LLVPFVQWF
3091
WLSLLVPFVQWFVGL


















2945
LLWFHISCL
3092
IRQLLWFHISCLTFG


















2946
LMPLYACIQ
3093
YPALMPLYACIQSKQ
0.2400



0.0014




0.0011








2947
LNLGNLNVS
3094
AEDLNLGNLNVSIPW
0.0001

−0.0005

−0.0007

−0.0002


−0.0003


0.0170





2948
LNPNKTKRW
3095
GIHLNPNKTKRWGYS


















2949
LNRRVAEDL
3096
DEGLNRRVAEDLNLG


















2950
LNVSIPWTH
3097
LGNLNVSIPWTHKVG


















2951
LPETTVVRR
3098
LSTLPETTVVRRRGR


















2952
LPIFFCLWV
3099
LPLLPIFFCLWVYIZ


















2953
LPIHTAELL
3100
VAPLPIHTAELLAAC


















2954
LPVNRPIDW
3101
FRKLPVNRPIDWKVC


















2955
LQFRNSKPC
3102
CWWLQFRNSKPCSDY


















2956
LRGLPVCAF
3103
HLSLRGLPVCAFSSA
1.3000



0.0028




0.0130








2957
LRPVGAESR
3104
VLCLRPVGAESRGRP


















2958
LRQAILCWG
3105
HTALRQAILCWGELM


















2959
LRRFIIFLF
3106
WMCLRRFIIFLFILL


















2960
LSFLPSDFF
3107
VELLSFLPSDFFPSI


















2961
LSLDVSAAF
3108
LSWLSLDVSAAFYHI


















2962
LSLLVPFVQ
3109
FSWLSLLVPFVQWFV


















2963
LSLRGLPVC
3110
GAHLSLRGLPVCAFS
0.7800

0.0042
−0.0041
0.0011

0.0025


0.0077


0.0150





2964
LSPFLLAQF
3111
GVGLSPFLLAQFTSA


















2965
LSRKYTSFP
3112
SVVLSRKYTSFPWLL
0.0005

0.0057
0.2100
−0.0016

0.5300


0.0130








2966
LSSNLSWLS
3113
TNLLSSNLSWLSLDV
0.0016

−0.0005

0.1300

0.0006


0.0019


0.0410





2967
LSVPNPLGF
3114
GTNLSVPNPLGFFPD


















2968
LSWLSLDVS
3115
SSNLSWLSLDVSAAF
0.1400
0.0030
−0.0005
1.5000
0.2700

0.0046
0.0180
0.1000
0.0039
0.0460
0.0110
6.2000





2969
LTIPQSLDS
3116
TRILTIPQSLDSWWT


















2970
LTNLLSSNL
3117
LQSLTNLLSSNLSWL
2.5000
0.4400
0.0200
−0.0013
4.8000
0.8100
0.0680
0.7500
0.0260
0.1500
0.0880
0.1100






2971
LTRILTIPQ
3118
FFLLTRILTIPQSLD


















2972
LVDKNPHNT
3119
GVFLVDKNPHNTTES


















2973
LVSFGVWIR
3120
LEYLVSFGVWIRTPP


















2974
LVVDFSQFS
3121
ESRLVVDFSQFSRGN
0.0007
0.0074
−0.0010
2.6000


−0.0004

0.0040
−0.0014
0.0029







2975
LWFHISCLT
3122
RQLLWFHISCLTFGR
0.0002

0.0009

0.0140

0.0011


0.0061


0.0096





2976
LWGMDIDPY
3123
LGWLWGMDIDPYKEF
0.0004

0.0006
0.0200
0.0280

−0.0002


0.0004


0.0430





2977
LWKAGILYK
3124
LHTLWKAGILYKRET


















2978
LYREALESP
3125
ASALYREALESPEHC


















2979
LYSHPIILG
3126
KLHLYSHPIILGFRK


















2980
MDDVVLGAK
3127
FSYMDDVVLGAKSVQ


















2981
MGVGLSPFL
3128
KIPMGVGLSPFLLAQ


















2982
MPHLLVGSS
3129
PAAMPHLLVGSSGLS


















2983
MQWNSTTFH
3130
PQAMQWNSTTFHQTL
0.0012



0.0300




0.1200








2984
MSTTDLEAY
3131
LSAMSTTDLEAYFKD


















2985
MWYWGPSLY
3132
IWMMWYWGPSLYNIL


















2986
VCAFSSAGP
3133
GLPVCAFSSAGPCAL


















2987
VCQRIVGLL
3134
DWKVCQRIVGLLGFA
0.0120

−0.0026

0.0030

0.2500


0.0018


0.0130





2988
VFADATPTG
3135
LCQVFADATPTGWGL
0.0020



0.9600




0.0013








2989
VGLSPTVWL
3136
QWFVGLSPTVWLSVI


















2990
VGPLTVNEK
3137
QQYVGPLTVNEKRRL


















2991
VHFASPLHV
3138
PDRVHFASPLHVAWR
0.0510
0.0290
0.0008

0.0008
0.0054
0.0008

0.0190
0.0810
0.0035
0.2400






2992
VLCLRPVGA
3139
ARDVLCLRPVGAESR


















2993
VLGAKSVQH
3140
DDVVLGAKSVQHLES


















2994
VLHKRTLGL
3141
LPKVLHKRTLGLSAM


















2995
VPNLQSLTN
3142
KFAVPNLQSLTNLLS
0.0180
0.0005
−0.0003

0.1300

0.0043

0.0088
−0.0003

0.0056






2996
VQASKLCLG
3143
CPTVQASKLCLGWLW


















2997
VRFSWLSLL
3144
WASVRFSWLSLLVPF


















2998
VRRAFPHCL
3145
CSVVRRAFPHCLAFS
0.1000
0.1024
0.0770
0.0032
0.0016
−0.0022
0.0008
−0.0013
0.0540
0.0590
0.0250
1.2000
0.0460





2999
VSIPWTHKV
3146
NLNVSIPWTHKVGNF
0.0001

−0.0005
−0.0041
−0.0007

−0.0002


0.0005


0.0009





3000
VWIRTPPAY
3147
SFGVWIRTPPAYRPP
0.0094
0.0110
0.4300
−0.0009
0.0780
0.0630
0.0260
0.0071
0.0002
0.0240
0.2500
0.0800
0.0016





3001
VYVPSALNP
3148
TSFVYVPSALNPADD


















3002
WFHISCLTF
3149
QLLWFHISCLTFGRE


















3003
WFVGLSPTV
3150
FVQWFVGLSPTVWLS
0.4700
0.0035
0.0160
−0.0013
0.0130

0.0072
0.0021
0.0190
0.0690
0.0180
0.0410
0.0044





3004
WILRGTSFV
3151
AANWILRGTSFVYVP
0.0920
0.0240
0.0061
0.0023
0.0510
0.0250
0.0140
0.3700
0.0250
0.5800
0.2500
0.2700






3005
WIRTPPAYR
3152
FGVWIRTPPAYRPPN


















3006
WKAGILYKR
3153
HTLWKAGILYKRETT


















3007
WLLGCAANW
3154
SFPWLLGCAANWILR


















3008
WLSLDVSAA
3155
NLSWLSLDVSAAFYH
0.1400
0.0003
−0.0005
1.3000
0.2900

0.0033
0.0022
0.0330
0.0041
0.0150
0.0620
2.4000





3009
WLSLLVPFV
3156
RFSWLSLLVPFVQWF
0.0430

0.0009

−0.0007

0.0002


0.0005


0.0031





3010
WPKFAVPNL
3157
RVSWPKFAVPNLQSL


















3011
YMDDVVLGA
3158
AFSYMDDVVLGAKSV
0.0027

−0.0005
0.0130
2.9000

0.0006


−0.0003


−0.0005





3012
YPALMPLYA
3159
QCGYPALMPLYACIQ
0.0062

0.0018

0.0068

0.0023


0.0006








3013
YQGMLPVCP
3160
LLDYQGMLPVCPLIP


















3014
YRPPNAPIL
3161
PPAYRPPNAPILSTL
0.0056

−0.0005

0.0038

0.0022


0.0024


0.0015





3015
YRWMCLRRF
3162
CPGYRWMCLRRFIIF


















3016
YSHPIILGF
3163
LHLYSHPIILGFRKI
0.0220
0.0340
0.0400
0.0040
0.6800
0.1600
0.0410
0.0310
0.0002
0.0006
0.0610
0.0490






3017
YSLNFMGYV
3164
RWGYSLNFMGYVIGS


















3018
YVPSALNPA
3165
SFVYVPSALNPADDP


















3019
FFCLWVYIZ




















3020
MGTNLSVPN
















TABLE XXa







HBV DR-3A Motif

















Core







Exemplary



SEQ


Core



Exemplary
Sequence



ID
Core
Core
Conservancy
Exemplary
Exemplary
Position in
Sequence
Conservancy


Protein
NO:
Sequence
Freq.
(%)
SEQ ID NO:
Sequence
Poly-Protein
Frequency
(%)



















ENV
3166
FFPDHQLDP
19
95
3181
PLGFFPDHQLDPAFG
10
9
95





NUC
3167
FGRETVLEY
15
75
3182
CLTFGRETVLEYLVS
136
14
75





POL
3168
FGVEPSGSG
15
75
3183
RRSFGVEPSGSGHD
241
6
75





POL
3169
FLVDKNPHN
20
100
3184
GGVFLVDKNPHNTTE
360
11
100





POL
3170
IGTDNSVVL
16
80
3185
AKLIGTDNSVVLSRK
731
13
80





POL
3171
LEEELPRLA
18
90
3186
AGPLEEELPRLADEG
18
13
90





POL
3172
LPLDKGIKP
20
100
3187
TKYLPLDKGIKPYYP
120
20
100





POL
3173
LSLDVSAAF
19
95
3188
LSWLSLDVSAAFYHI
412
11
95





POL
3174
LVVDFSQFS
20
100
3189
ESRLVVDFSQFSRGN
374
9
100





NUC
3175
LYREALESP
17
85
3190
ASALYREALESPEHC
34
17
85





NUC
3176
MQIDPYKEF
17
85
3191
LWGMDIDPYKEFGAS
27
9
85





POL
3177
VAEDLNLGN
20
100
3192
NRRVAEDLNLGNLNV
34
17
100





POL
3178
VFADATPTG
19
95
3193
LCQVFADATPTGWGL
683
19
95





ENV
3179
VLLDYQGML
19
95
3194
FLLVLLDYQGMLPVC
256
18
95





POL
3180
YMDDVVLGA
18
90
3195
AFSYMDDVVLGAKSV
535
18
90
















TABLE XXb





HCV DR 3A Motif























Core SEQ
Core
SEQ ID
Exemplary







ID NO:
Sequence
NO:
Sequence
DR1
DR2w2β1
DR2w2β2
DR3
DR4w4





3166
FFPDHQLDP
3181
PLGFFPDHQLDPAFG










3167
FGRETVLEY
3182
CLTFGRETVLEYLVS










3168
FGVEPSGSG
3183
RRSFGVEPSGSGHID










3169
FLVDKNPHN
3184
GGVFLVDKNPHNTTE



0.0790






3170
IGTDNSVVL
3185
AKLIGTDNSVVLSRK










3171
LEEELPRLA
3186
AGPLEEELPRLADEG



0.0022






3172
LPLDKGIKP
3187
TKYLPLDKGIKPYYP



−0.0017






3173
LSLDVSAAF
3188
LSWLSLDVSAAFYHI










3174
LVVDFSQFS
3189
ESRLVVDFSQFSRGN
0.0007
0.0074
−0.0010
2.6000






3175
LYREALESP
3190
ASALYREALESPEHC










3176
MDIDPYKEF
3191
LWGMDIDPYKEFGAS










3177
VAEDLNLGN
3192
NRRVAEDLNLGNLNV



0.1400






3178
VFADATPTG
3193
LCQVFADATPTGWGL
0.0020



0.9600





3179
VLLDYQGML
3194
FLLVLLDYQGMLPVC



0.0170






3180
YMDDVVLGA
3195
AFSYMDDVVLGAKSV
0.0027

−0.0005
0.0130
2.9000




















Core SEQ











ID NO:
DR4w15
DR5w11
DR5w12
DR6w19
DR7
DR8W2
DR9
DRW53






3166














3167














3168














3169














3170














3171














3172














3173














3174

−0.0004

0.4000
−0.0014
0.0029








3175














3176














3177














3178




0.0013









3179














3180

0.0006


−0.0003


−0.0005
















TABLE XXc







HBV DR-3B Motif




















Core
SEQ


Exemplary




Core SEQ
Core
Core
Conservancy
ID

Position in
Sequence
Exemplary


Protein
ID NO:
Sequence
Freq.
(%)
NO:
Exemplary Sequence
HBV Poly-Protein
Frequency
Sequence



















X
3196
AHLSLRGLP
18
90
3202
DHGAHLSLRGLPVCA
48
18
90.00





POL
3197
FSPTYKAFL
19
95
3203
AFTFSPTYKAFLCKQ
655
11
55.00





POL
3198
IPWTHKVGN
20
100
3204
NVSIPWTHKVGNFTG
47
20
100.00





POL
3199
LTVNEKRRL
17
85
3205
VGPLTVNEKRRLKLI
96
12
60.00





X
3200
VGAESRGRP
19
95
3206
LRPVGAESRGRPVSG
18
7
35.00





POL
3201
VVLSRKYTS
18
90
3207
DNSVVLSRKYTSFPW
737
17
85.00
















TABLE XXd





HBV DR-3B Motif With Binding Information























Core SEQ
Core
SEQ ID








ID NO:
Sequence
NO:
Exemplary Sequence
DR1
DR2w2β1
DR2w2β2
DR3
DR4w4





3196
AHLSLRGLP
3202
DHGAHLSLRGLPVCA










3197
FSPTYKAFL
3203
AFTFSPTYKAFLCKQ



0.0035






3198
IPWTHKVGN
3204
NVSIPWTHKVGNFTG










3199
LTVNEKRRL
3205
VGPLTVNEKRRLKLI
0.0006
0.0022
0.0047
2.2000






3200
VGAESRGRP
3206
LRPVGAESRGRPVSG



−0.0017






3201
VVLSRKYTS
3207
DNSVVLSRKYTSFPW




















Core SEQ











ID NO:
DR4w15
DR5w11
DR5w12
DR6w19
DR7
DR8w2
DR9
DRw53






3196














3197














3198














3199

0.0030

0.0009
−0.0014
0.0092








3200














3201
















TABLE XXI







Population coverage with combined HLA Supertypes









PHENOTYPIC FREQUENCY















North








American


HLA-SUPERTYPES
Caucasian
Black
Japanese
Chinese
Hispanic
Average
















a. Individual Supertypes








A2
45.8
39.0
42.4
45.9
43.0
43.2


A3
37.5
42.1
45.8
52.7
43.1
44.2


B7
38.6
52.7
48.8
35.5
47.1
44.7


A1
47.1
16.1
21.8
14.7
26.3
25.2


A24
23.9
38.9
58.6
40.1
38.3
40.0


B44
43.0
21.2
42.9
39.1
39.0
37.0


B27
28.4
26.1
13.3
13.9
35.3
23.4


B62
12.6
4.8
36.5
25.4
11.1
18.1


B58
10.0
25.1
1.6
9.0
5.9
10.3


b. Combined Supertypes


A2, A3, B7
83.0
86.1
87.5
88.4
86.3
86.2


A2, A3, B7, A24, B44, A1
99.5
98.1
100.0
99.5
99.4
99.3


A2, A3, B7, A24, B44, A1,
99.9
99.6
100.0
99.8
99.9
99.8


B27, B62, B58
















TABLE XXII







HBV ANALOGS





















A2
A3

B7







Fixed
A1
Super
Super
A24
Super
Anchor

SEQ ID


AA
Sequence
Nomen.
Motif
Motif
Motif
Motif
Motif
Fixer
Analog
NO:




















10
CILLLCLIFL

N
Y
N
N
N
No
A
3208





9
RMTGGVFLV
VM2.V9
N
Y
N
N
N
1
A
3209





9
LMPFVQWFV
VM2.V9
N
Y
N
N
N
1
A
3210





9
RLTGGVFLV
VL2.V9
N
Y
N
N
N
1
A
3211





9
GLCQVFADV
L2.AV9
N
Y
N
N
N
1
A
3212





9
WLLRGTSFV
IL2.V9
N
Y
N
N
N
1
A
3213





9
NLGNLNVSV
L2.IV9
N
Y
N
N
N
1
A
3214





9
YLPSALNPV
VL2.AV9
N
Y
N
N
N
1
A
3215





9
GLWIRTPPV
VL2.AV9
N
Y
N
N
N
1
A
3216





9
RLSWPKFAV
VL2.V9
N
Y
N
N
N
1
A
3217





9
ILGLLGFAV
VL2.AV9
N
Y
N
N
N
1
A
3218





9
RMLTIPQSV
IM2.LV9
N
Y
N
N
N
1
A
3219





9
SLDSWWTSV
L2.LV9
N
Y
N
N
N
1
A
3220





10
FMLLLCLIFL
IM2.L10
N
Y
N
Y
N
1
A
3221





10
LMLQAGFFLV
VM2.LV
N
Y
N
N
N
1
A
3222





10
SMLSPFLPLV
IM2.LV1
N
Y
N
N
N
1
A
3223





10
LMLLDYQGMV
VM2.LV
N
Y
N
N
N
1
A
3224





10
FLGLSPTVWV
VL2.LV1
N
Y
N
N
N
1
A
3225





8
FPAAMPHL

N
N
N
N
Y

A
3226





8
HPFAMPHL

N
N
N
N
Y

A
3227





8
HPAAMPHI

N
N
N
N
Y

A
3228





8
FMFSPTYK

N
N
Y
N
N

A
3229





8
FVFSPTYK

N
N
Y
N
N

A
3230





9
FLLTRILTV
L2.IV9
N
Y
N
N
N
1
A
3231





9
ALMPLYACV
L2.IV9
N
Y
N
N
N
1
A
3232





9
LLAQFTSAV
L2.IV9
N
Y
N
N
N
1
A
3233





9
LLPFVQWFV
VL2.V9
N
Y
N
N
N
1
A
3234





9
FLLAQFTSV
L2.AV9
N
Y
N
N
N
1
A
3235





9
KLHLYSHPV
L2.IV9
N
Y
N
N
N
1
A
3236





9
KLFLYSHPI

N
Y
N
N
N
No
A
3237





9
LLSSNLSWV
L2.LV9
N
Y
N
N
N
1
A
3238





9
FLLSLGIHV
L2.LV9
N
Y
N
N
N
1
A
3239





9
MMWYWGPSV
M2.LV9
N
Y
N
N
N
1
A
3240





9
VLQAGFFLV
L2.LV9
N
Y
N
N
N
1
A
3241





9
PLLPIFFCV
L2.LV9
N
Y
N
N
N
1
A
3242





9
FLLPIFFCL

N
Y
N
N
N
No
A
3243





9
VLLDYQGMV
L2.LV9
N
Y
N
N
N
1
A
3244





9
YMFDVVLGA

N
Y
N
N
N
No
A
3245





9
GLLGWSPQV
L2.AV9
N
Y
N
N
N
1
A
3246





9
FPAAMPHLL

N
N
N
N
Y

A
3247





9
HPFAMPHLL

N
N
N
N
Y

A
3248





9
HPAAMPHLI

N
N
N
N
Y

A
3249





9
FPVCAFSSA

N
N
N
N
Y

A
3250





9
LPFCAFSSA

N
N
N
N
Y

A
3251





9
LPVCAFSSI

N
N
N
N
Y

A
3252





9
FPALMPLYA

N
N
N
N
Y

A
3253





9
YPFLMPLYA

N
N
N
N
Y

A
3254





9
YPALMPLYI

N
N
N
N
Y

A
3255





9
FPSRGRLGL

N
N
N
N
Y

A
3256





9
DPFRGRLGL

N
N
N
N
Y

A
3257





9
DPSRGRLGI

N
N
N
N
Y

A
3258





9
SMICSVVRR

N
N
Y
N
N

A
3259





9
SVICSVVRR

N
N
Y
N
N

A
3260





9
KVGNFTGLK

N
N
Y
N
N

A
3261





9
KVGNFTGLR

N
N
Y
N
N

A
3262





9
VVFFSQFSR

N
N
Y
N
N

A
3263





9
SVNRPIDWK

N
N
Y
N
N

A
3264





9
TLWKAGILK

N
N
Y
N
N

A
3265





9
TLWKAGILR

N
N
Y
N
N

A
3266





9
TMWKAGILY

Y
N
Y
N
N

A
3267





9
TVWKAGILY

N
N
Y
N
N

A
3268





9
RMYLHTLWK

N
N
Y
N
N

A
3269





9
RVYLHTLWK

N
N
Y
N
N

A
3270





9
AMTFSPTYK

N
N
Y
N
N

A
3271





9
SVVRRAFPR

N
N
Y
N
N

A
3272





9
SVVRRAFPK

N
N
Y
N
N

A
3273





9
SAIXSVVRR

N
N
Y
N
N

A
3274





9
LPVXAFSSA

N
N
N
N
Y

A
3275





10
FLLAQFTSAV
L2.IV10
N
Y
N
N
N
1
A
3276





10
YLFTLWKAGI

N
Y
N
N
N
No
A
3277





10
YLLTLWKAGI

N
Y
N
N
N
No
A
3278





10
LLFYQGMLPV

N
Y
N
N
N
No
A
3279





10
LLLYQGMLPV

N
Y
N
N
N
No
A
3280





10
LLVLQAGFFV
L2.LV10
N
Y
N
N
N
1
A
3281





10
ILLLCLIFLV
L2.LV10
N
Y
N
N
N
1
A
3282





10
FPFCLAFSYM

N
N
N
N
Y

A
3283





10
FPHCLAFSYI

N
N
N
N
Y

A
3284





10
FPARVTGGVF

N
N
N
N
Y

A
3285





10
TPFRVTGGVF

N
N
N
N
Y

A
3286





10
TPARVTGGVI

N
N
N
N
Y

A
3287





10
FPCALRFTSA

N
N
N
N
Y

A
3288





10
GPFALRFTSA

N
N
N
N
Y

A
3289





10
GPCALRFTSI

N
N
N
N
Y

A
3290





10
FPAAMPHLLV

N
N
N
N
Y

A
3291





10
HPFAMPHLLV

N
N
N
N
Y

A
3292





10
HPAAMPHLLI

N
N
N
N
Y

A
3293





10
QMFTFSPTYK

N
N
Y
N
N

A
3294





10
QVFTFSPTYK

N
N
Y
N
N

A
3295





10
TMWKAGILYK

N
N
Y
N
N

A
3296





10
TVWKAGILYK

N
N
Y
N
N

A
3297





10
VMGGVFLVDK

N
N
Y
N
N

A
3298





10
VVGGVFLVDK

N
N
Y
N
N

A
3299





10
SMLPETTVVR

N
N
Y
N
N

A
3300





10
SVLPETTVVR

N
N
Y
N
N

A
3301





10
TMPETTVVRR

N
N
Y
N
N

A
3302





10
TVPETTVVRR

N
N
Y
N
N

A
3303





10
HTLWKAGILK

N
N
Y
N
N

A
3304





10
HTLWKAGILR

N
N
Y
N
N

A
3305





10
HMLWKAGILY

Y
N
Y
N
N

A
3306





10
HVLWKAGILY

N
N
Y
N
N

A
3307





10
GMDNSVVLSR

N
N
Y
N
N

A
3308





10
GVDNSVVLSR

N
N
Y
N
N

A
3309





10
GTFNSVVLSR

N
N
Y
N
N

A
3310





10
YMFDVVLGAK

N
N
Y
N
N

A
3311





10
MMWYWGPSLK

N
N
Y
N
N

A
3312





10
MMWYWGPSLR

N
N
Y
N
N

A
3313





9
ILLLXLIFL

N
Y
N
N
N

A
3314





9
LLLXLIFLL

N
Y
N
N
N

A
3315





9
LLXLIFLLV

N
Y
N
N
N

A
3316





9
PLLPIFFXL

N
Y
N
N
N

A
3317





9
ALMPLYAXI

N
Y
N
N
N

A
3318





9
GLXQVFADA

N
Y
N
N
N

A
3319





9
HISXLTFGR

N
N
Y
N
N

A
3320





9
FVLGGXRHK

N
N
Y
N
N

A
3321





10
FILLLXLIFL

N
Y
N
N
N

A
3322





10
ILLLXLIFLL

N
Y
N
N
N

A
3323





10
LLLXLIFLLV

N
Y
N
N
N

A
3324





10
LLPIFFXLWV

N
Y
N
N
N

A
3325





10
QLLWFHISXL

N
Y
N
N
N

A
3326





10
LLGXAANWIL

N
Y
N
N
N

A
3327





10
TSAIXSVVRR

N
N
Y
N
N

A
3328





10
GYRWMXLRRF

N
N
N
Y
N

A
3329





10
GPXALRFTSA

N
N
N
N
Y

A
3330





10
FPHXLAFSYM

N
N
N
N
Y

A
3331





11
HMLWKAGILYK

N
N
Y
N
N

A
3332





11
HVLWKAGILYK

N
N
Y
N
N

A
3333





11
SMLPETTVVRR

N
N
Y
N
N

A
3334





11
SVLPETTVVRR

N
N
Y
N
N

A
3335





11
GMDNSVVLSRK

N
N
Y
N
N

A
3336





11
GVDNSVVLSRK

N
N
Y
N
N

A
3337





11
GTFNSVVLSRK

N
N
Y
N
N

A
3338





8
MPLSYQHI

N
N
N
N
Y

A
3339





8
LPIFFCLI

N
N
N
N
Y

A
3340





8
SPFLLAQI

N
N
N
N
Y

A
3341





8
YPALMPLI

N
N
N
N
Y

A
3342





8
VPSALNPI

N
N
N
N
Y

A
3343





9
LPIFFCLWI

N
N
N
N
Y

A
3344





9
LPIHTAELI

N
N
N
N
Y

A
3345





10
VPFVQWFVGI

N
N
N
N
Y

A
3346





11
NPLGFFPDHQI

N
N
N
N
Y

A
3347





11
LPIHTAELLAI

N
N
N
N
Y

A
3348





9
FLPSYFPSA
L2.FV5.
N
Y
N
N
N
Rev3
A
3349





10
YLHTLWKAGV
L2.IV10
N
Y
N
N
N
1
A
3350





11
STLPETYVVRR

N
N
Y
N
N

A
3351





9
YMDDVVLGV
M2.AV9
N
Y
N
N
N
1
A
3352





9
FPIPSSWAF

N
N
N
N
Y

A
3353





9
IPITSSWAF

N
N
N
N
Y

A
3354





9
IPILSSWAF

N
N
N
N
Y

A
3355





9
FPVCLAFSY

N
N
N
N
Y

A
3356





9
FPHCLAFAY

N
N
N
N
Y

A
3357





9
FPHCLAFSL

N
N
N
N
Y

A
3358





9
IPIPMSWAF

N
N
N
N
Y

A
3359





9
FPHCLAFAL

N
N
N
N
Y

A
3360





10
FLPSZFFPSV

N
Y
N
N
N
No
A
3361





10
FLPSZFFPSV

N
Y
N
N
N
No
A
3362





9
IPFPSSWAF

N
N
N
N
Y

A
3363





9
IPIPSSWAI

N
N
N
N
Y

A
3364





9
FPFCLAFSY

N
N
N
N
Y

A
3365





9
FPHCLAFSI

N
N
N
N
Y

A
3366





9
FPHCLAFSA

N
N
N
N
Y

A
3367





10
FQPSDYFPSV

N
Y
N
N
N
Rev
A
3368





9
YLLTRILTI

N
Y
N
N
N

A
3369





9
FLYTRILTI

N
Y
N
N
N

A
3370





9
FLLTYILTI

N
Y
N
N
N

A
3371





9
FLLTRILYI

N
Y
N
N
N

A
3372





11
FLPSDFFPSVR

N
N
Y
N
N

A
3373





9
FLPSDFFPS

N
N
N
N
N

A
3374





8
FLPSDFFP

N
N
N
N
N

A
3375





10
FLPSDFFPSI
L2.VI10
N
Y
N
N
N
Rev
A
3376





10
FLPSDYFPSV

N
Y
N
N
N
No
A
3377





12
YSFLPSDFFPSV

N
N
N
N
N

A
3378





10
YNMGLKFRQL

N
N
N
N
N

A
3379





9
NMGLKYRQL

N
Y
N
Y
N
No
A
3380





10
FLPS(X)YFPSV

N
N
N
N
N

A
3381





10
FLPSD(X)FPSV

N
N
N
N
N

A
3382





11
FLPSDLLPSVR

N
N
Y
N
N

A
3383





12
FLPSDFFPSVRD

N
N
N
N
N

A
3384





12
LSFLPSDFFPSV

N
N
N
N
N

A
3385





11
SFLPSDFFPSV

N
N
N
N
N

A
3386





8
PSDFFPSV

N
N
N
N
N

A
3387





9
FLMSYFPSV

N
Y
N
N
N
No
A
3388





9
FLPSYFPSV
L2.FY5.
N
Y
N
N
N
3
A
3389





10
FLMSDYFPSV

N
Y
N
N
N
No
A
3390





11
CILLLCLIFLL

N
Y
N
N
N
No
A
3391





10
FLPNDFFPSA
L2.SN4.
N
Y
N
N
N
Rev
A
3392





10
FLPDDFFPSA
L2.SD4.
N
Y
N
N
N
Rev
A
3393





10
FLPNDFFPSV

N
Y
N
N
N
No
A
3394





10
FLPSDFFPSA
L2.VA10
N
Y
N
N
N
Rev
A
3395





10
FLPDDFFPSV

N
Y
N
N
N
No
A
3396





10
FLPADFFPSV

N
Y
N
N
N
No
A
3397





10
FLPVDFFPSV

N
Y
N
N
N
No
A
3398





10
FLPADFFPSI
L2.SA4.
N
Y
N
N
N
Rev
A
3399





10
FLPVDFFPSI
L2.SV4.
N
Y
N
N
N
Rev
A
3400





10
FLPSDAFPSV

N
Y
N
N
N
No
A
3401





10
FLPSAFFPSV

N
Y
N
N
N
No
A
3402





10
FLPSDFAPSV

N
Y
N
N
N
No
A
3403





10
FLPSDFFASV

N
Y
N
N
N
No
A
3404





10
FLPSDFFPAV

N
Y
N
N
N
No
A
3405





10
FLASDFFPSV

N
Y
N
N
N
No
A
3406





10
FAPSDFFPSV
LA2.V10
N
Y
N
N
N
Rev
A
3407





10
ALPSDFFPSV

N
Y
N
N
N
No
A
3408





10
YLPSDFFPSV

N
Y
N
N
N
No
A
3409





10
FMPSDFFPSV
LM2.V1
N
Y
N
N
N
1
A
3410





10
FLKSDFFPSV

N
Y
N
N
N
No
A
3411





10
FLPSEFFPSV

N
Y
N
N
N
No
A
3412





10
FLPSDFYPSV

N
Y
N
N
N
No
A
3413





10
FLPSDFFKSV

N
Y
N
N
N
No
A
3414





10
FLPSDFFPKV

N
Y
N
N
N
No
A
3415






FLPSDFFPSV(CONH2)








3416






VLEYLVSFGV(NH2)








3417






ATVELLSFLPSDFFPSV-NH2








3418






TVELLSFLPSDFFPSV-NH2








3419






VELLSFLPSDFFPSV-NH2








3420






ELLSFLPSDFFPSV-NH2








3421






LLSFLPSDFFPSV-NH2








3422






LSFLPSDFFPSV-NH2








3423






SFLPSDFFPSV-NH2








3424






FLPSDFFPSV-NH2








3425






LPSDFFPSV-NH2








3426






PSDFFPSV-NH2








3427






FLPSDFFPS-NH2








3428






FLPSDFFP-NH2








3429






FLPSDFF-NH2








3430






ALPSDFFPSV-NH2








3431






SLNFLGGTTV(NH2)








3432






FLPSDFFPSVR-NH2








3433






ALFKDWEEL








3434






VLGGSRHKL








3435






KIKESFRKL








3436






ALMPLYASI








3437






FLSKQYLNL








3438






LLGSAANWI








3439






NLNNLNVSI








3440






IIKKSEQFV








3441






ALSLIVNLL








3442






RIPRTPRSV








3443















3444















3445
















TABLE XXIII







Immunogenicity of HBV-derived peptides









Immunogenicity
















Supermotif
Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1



















A2 supermotif
924.07
FLPSDFFPSV
3492
HBV core 18
5
10/10
6/6
25/32a
+






1069.06
LLVPFVQWFV
3493
HBV env 338
5
3/4
6/9

+






1147.13
FLLAQFTSAI
3494
HBV pol 513
5

0/3

unk






1090.77
YMDDVVLGV
3495
HBV pol 538
5

9/9

+






777.03
FLLTRILTI
3496
HBV env 183
4


14/23a
+






927.15
ALMPLYACI
3497
HBV pol 642
4
10/12
3/5
 2/15a
+






1013.01
WLSLLVPFV
3498
HBV env 335
4
2/6
5/9
23/29a
+






1069.05
LLAQFTSAI
3499
HBV pol 504
4
0/4
0/5

unk






1132.01
LVPFVQWFV
3500
HBV env 339
4
0/3
0/4

unk






1147.14
VLLDYQGMLPV
3501
HBV env 259
4
4/4
6/6

+






927.41
LLSSNLSWL
3502
HBV pol 992
3
0/4
0/3

unk






927.42
NLSWLSLDV
3503
HBV pol 411
3

2/8

+






927.46
KLHLYSHPI
3504
HBV pol 489
3
0/4
4/6

+






1069.07
FLLAQFTSA
3505
HBV pol 503
3
1/2
0/3

+






1168.02
GLSRYVARL
3506
HBV pol 455
3


 9/13a
+





A2 supermotif
927.11
FLLSLGIHL
3507
HBV pol 562
2
15/22
12/13
 9/15a
+






927.47
HLYSHPIIL
3508
HBV pol 1076
2

10/14

+






1039.03
MMWYWGPSL
3509
HBV env 360
2
3/4
0/4

+






1069.12
YLHTLWKAGV
3510
HBV pol 147
2
2/4


+






1137.02
LLDYQGMLPV
3511
HBV env 260
2
1/2
0/4

+






1142.07
GLLGWSPQA
3512
HBV env 62
2
3/4
5/6

+






1.0573
ILRGTSFVYV
3513
HBV pol 773
1


3/7b
+






1013.14
VLQAGFFLL
3514
HBV env 177
1
0/4
 5/12

+






1069.10
LLPIFFCLWV
3515
HBV env 378
1
3/3
0/4
2/5c
+






1069.13
PLLPIFFCL
3516
HBV env 377
1
0/4
 7/12

+






1090.06
LLVLQAGFFL
3517
HBV env 175
1
1/5
0/4

+






1090.12
YLVSFGVWI
3518
HBV nuc 118
1
9/9


+






1.0518
GLSPTVWLSV
3519
HBV env 338
1


3/9c
+






1090.14
YMDDVVLGA
3520
HBV pol 538
1
2/7
2/5
2/7b
+





A3 supermotif
1147.16
HTLWKAGILYK
3521
HBV POL 149
5
0/6
3/3
1/22
+






1083.01
STLPETTVVRR
3522
HBV core 141
4
3/5
6/6
8/32
+






1150.51
GSTHVSWPK
3523
HBV pol 398
4

3/6

+






1.0219
FVLGGCRHK
3524
HBV adr “X” 1550
3
0/4


unk






1069.16
NVSIPWTHK
3525
HBV pol 47
3
0/8
0/3
1/21
+






1069.20
LVVDFSQFSR
3526
HBV pol 388
3
0/4
6/6
1/22
+






1090.10
QAFTFSPTYK
3527
HBV pol 665
3
3/6
0/3
3/21
+






1090.11
SAICSVVRR
3528
HBV pol 531
3
1/4

2/22
+





A3 supermotif
1069.15
TLWKAGILYK
3529
HBV pol 150
2
3/8
0/3
5/28
+






1142.05
KVGNFTGLY
3530
HBV adr POL 629
2

0/3
2/22
+





B7 supermotif
1147.05
FPHCLAFSYM
3531
HBV POL 530
5
1/3

0/12
+






988.05
LPSDFFPSV
3532
HBV core 19-27
4


2/16
+






1145.04
IPIPSSWAF
3533
HBV ENV 313
4
0/4

1/12
+






1147.02
HPAAMPHLL
3534
HBV POL 429
4
0/5

0/12
unk






1147.06
LPVCAFSSA
3535
HBV X 58
4
1/4


+






1147.08
YPALMPLYA
3536
HBV POL 640
4


0/12
unk






1145.08
FPHCLAFSYM
3537
HBV POL 541
3
0/4


unk





B7 supermotif
1147.04
TPARVTGGVF
3538
HBV POL 354
2


2/12
+





Immunogenicity evaluation derived from primary cultures, acute patients (aBertoni et al, J Clin Invest 100: 503, bRehermann et al., J. Clin. Invest 97: 1655, cNayersina et al., J Immunol 150: 4659) or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems.


Unk = unknown













TABLE XXIV





MHC-peptide binding assays: cell lines and radiolabeled ligands.







A. Class I binding assays









Radiolabeled peptide













Species
Antigen
Allele
Cell line
Source
Sequence
SEQ ID NO:





Human
A1
A*0101
Steinlin
Hu. J chain 102-110
YTAVVPLVY
3539






A2
A*0201
JY
HBVc 18-27 F6->Y
FLPSDYFPSV
3540






A2
A*0202
P815
HBVc 18-27 F6->Y
FLPSDYFPSV
3540





(transfected)









A2
A*0203
FUN
HBVc 18-27 F6->Y
FLPSDYFPSV
3540






A2
A*0206
CLA
HBVc 18-27 F6->Y
FLPSDYFPSV
3540






A2
A*0207
721.221
HBVc 18-27 F6->Y
FLPSDYFPSV
3540





(transfected)









A3

GM3107
non-natural (A3CON1)
KVFPYALINK
3541






A11

BVR
non-natural (A3CON1)
KVFPYALINK
3541






A24
A*2402
KAS116
non-natural (A24CON1)
AYIDNYNKF
3542






A31
A*3101
SPACH
non-natural (A3CON1)
KVFPYALINK
3541






A33
A*3301
LWAGS
non-natural (A3CON1)
KVFPYALINK
3541






A28/68
A*6801
C1R
HBVc 141-151 T7->Y
STLPETYVVRR
3543






A28/68
A*6802
AMAI
HBV pol 646-654 C4->A
FTQAGYPAL
3544






B7
B*0702
GM3107
A2 sigal seq. 5-13 (L7->Y)
APRTLVYLL
3545






B8
B*0801
Steinlin
HIVgp 586-593 Y1->F, Q5->Y
FLKDYQLL
3546






B27
B*2705
LG2
R 60s
FRYNGLIHR
3547






B35
B*3501
C1R, BVR
non-natural (B35CON2)
FPFKYAAAF
3548






B35
B*3502
TISI
non-natural (B35CON2)
FPFKYAAAF
3548






B35
B*3503
EHM
non-natural (B35CON2)
FPFKYAAAF
3548






B44
B*4403
PITOUT
EF-1 G6->Y
AEMGKYSFY
3549






B51

KAS116
non-natural (B35CON2)
FPFKYAAAF
3550






B53
B*5301
AMAI
non-natural (B35CON2)
FPFKYAAAF
3550






B54
B*5401
KT3
non-natural (B35CON2)
FPFKYAAAF
3550






Cw4
Cw*0401
C1R
non-natural (C4CON1)
QYDDAVYKL
3551






Cw6
Cw*0602
721.221
non-natural (C6CON1)
YRHDGGNVL
3552





transfected









Cw7
Cw*0702
721.221
non-natural (C6CON1)
YRHDGGNVL
3552





transfected








Mouse
Db

EL4
Adenovirus E1A P7->Y
SGPSNTYPEI
3553






Kb

EL4
VSV NP 52-59
RGYVFQGL
3554






Dd

P815
HIV-IIIB ENV G4->Y
RGPYRAFVTI
3555






Kd

P815
non-natural (KdCON1)
KFNPMKTYI
3556






Ld

P815
HBVs 28-39
IPQSLDSYWTSL
3557










B. Class II binding assays









Radiolabeled peptide













Species
Antigen
Allele
Cell line
Source
Sequence
SEQ ID NO:





Human
DR1
DRB1*0101
LG2
HA Y307-319
YPKYVKQNTLKLAT
3558






DR2
DRB1*1501
L466.1
MBP 88-102Y
VVHFFKNIVTPRTPPY
3559






DR2
DRB1*1601
L242.5
non-natural (760.16)
YAAFAAAKTAAAFA
3560






DR3
DRB1*0301
MAT
MT 65 kD Y3-13
YKTIAFDEEARR
3561






DR4w4
DRB1*0401
Preiss
non-natural (717.01)
YARFQSQTTLKQKT
3562






DR4w10
DRB1*0402
YAR
non-natural (717.10)
YARFQRQTTLKAAA
3563






DR4w14
DRB1*0404
BIN 40
non-natural (717.01)
YARFQSQTTLKQKT
3562






DR4w15
DRB1*0405
KT3
non-natural (717.01)
YARFQSQTTLKQKT
3562






DR7
DRB1*0701
Pitout
Tet. tox. 830-843
QYIKANSKFIGITE
3564






DR8
DRB1*0802
OLL
Tet. tox. 830-843
QYIKANSKFIGITE
3564






DR8
DRB1*0803
LUY
Tet. tox. 830-843
QYIKANSKFIGITE
3564






DR9
DRB1*0901
HID
Tet. tox. 830-843
QYIKANSKFIGITE
3564






DR11
DRB1*1101
Sweig
Tet. tox. 830-843
QYIKANSKFIGITE
3564






DR12
DRB1*1201
Herluf
unknown eluted peptide
EALIHQLKINPYVLS
3565






DR13
DRB1*1302
H0301
Tet. tox. 830-843 S->A
QYIKANAKFIGITE
3566






DR51
DRB5*0101
GM3107
Tet. tox. 830-843
QYIKANAKFIGITE
3566





or L416.3









DR51
DRB5*0201
L255.1
HA 307-319
PKYVKQNTLKLAT
3567






DR52
DRB3*0101
MAT
Tet. tox. 830-843
NGQIGNDPNRDIL
3568






DR53
DRB4*0101
L257.6
non-natural (717.01)
YARFQSQTTLKQKT
3569






DQ3.1
QA1*0301/DQB1*03text missing or illegible when filed
PF
non-natural (ROIV)
AHAAHAAHAAHAAHAA
3570





Mouse
IAb

DB27.4
non-natural (ROIV)
AHAAHAAHAAHAAHAA
3570






IAd

A20
non-natural (ROIV)
AHAAHAAHAAHAAHAA
3570






IAk

CH-12
HEL 46-61
YNTDGSTDYGILQINSR
3571






IAg

LS102.9
non-natural (ROIV)
AHAAHAAHAAHAAHAA
3570






IAu

91.7
non-natural (ROIV)
AHAAHAAHAAHAAHAA
3570






IEd

A20
Lambda repressor 12-26
YLEDARRKKAIYEKKK
3572






IEk

CH-12
Lambda repressor 12-26
YLEDARRKKAIYEKKK
3572






text missing or illegible when filed indicates data missing or illegible when filed














TABLE XXV







Monoclonal antibodies used in MHC purifi










Monoclonal antibody
Specificity







W6/32
HLA-class I



B123.2
HLA-B and C



IVD12
HLA-DQ



LB3.1
HLA-DR



M1/42
H-2 class I



28-14-8S
H-2 Db and Ld



34-5-8S
H-2 Dd



B8-24-3
H-2 Kb



SF1-1.1.1
H-2 Kd



Y-3
H-2 Kb



10.3.6
H-2 IAk



14.4.4
H-2 IEd, IEK



MKD6
H-2 IAd



Y3JP
H-2 IAb, IAs, IAu

















TABLE XXVI







in vitro binding of conserved HBV-derived peptides to


HLA-A2-supertype alleles.










A2-supertype binding capacity




(IC50 nM)
Alleles



















Peptide
AA
Molecule
1st Pos
Sequence
SEQ ID NO:
Consv.1
A*0201
A*0202
A*0203
A*0206
A*6802
bound2






















924.07
10
Core
18
FLPSDFFPSV
3492
95
2.5
2.1
6.0
3.0
36
5





1069.06
10
ENV
349
LLVPFVQWFV
3493
95
7.5
11
5.9
13
286
5





1147.13
10
POL
524
FLLAQFTSAI
3494
95
24
134
1.4
34
455
5





1013.0102
9
ENV
346
WLSLLVPFV
3498
100
4.6
113
1.4
10
1290
4





777.03
9
ENV
183
FLLTRILTI
3496
80
9.8
100
1.3
19
3
4





927.15
9
POL
653
ALMPLYACI
3497
95
10
126
3.0
160
851
4





1069.05
9
POL
525
LLAQFTSAI
3499
95
50
16
3.0
1538
51
4





1132.01
9
ENV
350
LVPFVQWFV
3500
95
119
287
2083
463
14
4





1147.14
11
ENV
259
VLLDYQGMLPV
3501
90
8.6
20
2.0
13
2353
4





1090.77
9
POL
538 (a)
YMDDVVLGV
3495
90
5.1
90
6.7
71
1905
4





1069.071
9
POL
524
FLLAQFTSA
3505
95
6.0
1654
9.1
39
870
3





927.46
9
POL
500
KLHLYSHPI
3504
95
72
126
3.7
627
26667
3





927.42
9
POL
422
NLSWLSLDV
3503
90
77
843
16
2313
404
3





1168.02
9
POL
455
GLSRYVARL
3506
90
79
391
18
12333

3





927.41
9
POL
418
LLSSNLSWL
3502
90
455
55
2.6
1370
4000
3





1039.031
9
ENV
360
MMWYWGPSL
3509
85
5.6
5375
833
112
3636
2





927.11
9
POL
573
FLLSLGIHL
3507
95
7.7
4300
1000
34
11429
2





1142.07
9
ENV
73
GLLGWSPQA
3512
85
13
14333
286
1429

2





927.47
9
POL
502
HLYSHPIIL
3508
80
23
14333
11
2176
755
2





1137.02
10
ENV
271
LLDYQGMLPV
3511
90
51

500
552

2





1069.09
9
ENV
270
VLLDYQGML
3573
95
114

476
4111

2





1069.14
10
NUC
168
ILSTLPETTV
3574
100
238
506
130
1194
5970
2





1069.11
10
POL
147
YLHTLWKAGI
3575
100
313
8600
18
4000
1250
2





1142.01
9
NUC
129
LLWFHISCL
3576
90
385
21500
238
1194
4082
2





1090.12
9
NUC
147
YLVSFGVWI
3518
90
13




1





1.0518
10
ENV
359
GLSPTVWLSV
3519
75
18




1





1013.1402
9
ENV
177
VLQAGFFLL
3514
95
33
2389
3704
1947
6349
1





1069.13
9
ENV
388
PLLPIFFCL
3516
100
77

5556
3364
8511
1





1069.10
10
ENV
389
LLPIFFCLWV
3515
100
156
5375
667
5000

1





1090.06
10
ENV
175
LLVLQAGFFL
3517
90
161
1162
2222
2467
3636
1





1.0895
10
ENV
248
FILLLCLIFL
3577
80
179




1





927.24
9
POL
770
WILRGTSFV
3578
80
185




1





1090.14
9
POL
538
YMDDVVLGA
3520
90
200

4167


1





3.0205
10
ENV
171
FLGPLLVLQA
3579
75
263




1





1069.08
10
ENV
260
ILLLCLIFLL
3580
100
263


2846
26667
1





1.0573
10
POL
773
ILRGTSFVYV
3581
80
313




1






1Frequency of entire sequence amongst isolates scanned.




2Number of supertpe alleles bound. Peptides binding 3 or more alleles are considered degenerate.




3A dash (—) indicates IC50














TABLE XXVII







in vitro binding of conserved HBV-derived peptides to


HLA-A3-supertype alleles.










A3-supertype binding capacity




(IC50 nM)
Alleles



















Peptide
AA
Molecule
1st Pos
Sequence
SEQ ID NO:
Consv.1
A*03
A*11
A*3101
A*3301
A*6801
bound






















26.0535
11
X NUC FUS
299
GVWIRTPPAYR
3582
95
58
35
3.0
40
12
5





1147.16
11
pol
149
HTLWKAGILYK
3583
100
20
14
486
403
42
5





26.0539
11
POL
376
RLVVDFSQFSR
3584
95
39
2.0
7.0
24
1.0
5





26.0149
9
X
69
CALRFTSAR
3585
85
3235
261
12
3.6
11
4





1.0993
9
X
130
KVFVLGGCR
3586
75
262
73
30
408
2667
4





26.0153
9
X
64
SSAGPCALR
3587
90
1375
43
55
181
11
4





1083.01
11
Core
141
STLPETTVVRR
3588
95
733
4.0
180
181
26
4





20.0130
9
pol
655
AFTFSPTYK
3589
95
42
150
3103
13182
296
3





26.0008
8
POL
656
FTFSPTYK
3590
95
193
136
1286
1000
73
3





1.0219
9
X
1550
FVLGGCRHK
3591
80
169
316
1500
744
103
3





1069.20
10
POL
388
LVVDFSQFSR
3592
100
6875
17
692
126
16
3





1069.16
9
POL
47
NVSIPWTHK
3593
100
134
105
3
2900
250
3





1090.10
10
POL
665
QAFTFSPTYK
3594
95
244
11
18000
5088
6.7
3





1090.11
9
POL
531
SAICSVVRR
3595
95
1897
29
1200
446
21
3





20.0131
9
pol
524
SVVRRAFPH
3596
95
100
10
621

500
3





26.0545
11
X NUC FUS
318
TLPETTVVRRR
3597
95
22000
375
2951
408
13
3





26.0023
8
X NUC FUS
296
VSFGVWIR
3598
90
2750
207
240
1074
222
3





1142.05
9
POL
55
KVGNFTGLY
3599
95
52
353



2





1142.06
9
POL
623
PVNRPIDWK
3600
85
355
43


8889
2





1.0975
9
POL
106
RLKLIMPAR
3601
75
116

5.8
592

2





1.0562
10
POL
576
SLGIHLNPNK
3602
75
55
77



2





1069.21
10
NUC
170
STLPETTVVR
3603
95
15714
100
2250
1208
320
2





1069.22
10
NUC
171
TLPETTVVRR
3604
95
15714
261

2417
182
2





1069.15
10
POL
150
TLWKAGILYK
3605
100
2.1
17
3529
29000
615
2





1.0215
9
X
105
TTDLEAYFK
3606
75
18333
6.5

24167
471
2





1069.17
10
POL
369
VTGGVFLVDK
3607
100
282
65


3636
2





1069.19
9
POL
389
VVDFSQFSR
3608
100
7333
80
13846
1706
242
2





26.0026
8
POL
168
ASFCGSPY
3609
100
239
26


20000
2





26.0549
11
ENV
389
LLPIFFCLWVY
3610
100
478
10000
2609
644
82
2





26.0550
11
POL
528
RAFPHCLAFSY
3611
95
92
15
667
26364
2667
2





1090.04
10
POL
746
GTDNSVVLSR
3612
90
11000
143
6000
15263
10000
1





1069.04
10
POL
149
HTLWKAGILY
3613
100
250
7500

8529
6667
1





1.0205
9
POL
771
ILRGTSFVY
3614
80
250




1





1090.08
9
NUC
148
LVSFGVWIR
3615
90
3929
500



1





1039.01
10
ENV
360
MMWYWGPSLY
3616
85
220
7500


26667
1





1.0584
10
X
104
STTDLEAYFK
3617
75
1667
2.2



1





1147.17
11
pol
735
GTDNSVVLSRK
3618
90
786
11



1





1147.18
11
pol
357
RVTGGVFLVDK
3619
100
578
207



1





1099.03
9
POL
150
TLWKAGILY
3620
100
85
7500



1





1090.15
10
POL
549
YMDDVVLGAK
3621
90
333
1395



1





26.0024
8
POL
50
VSIPWTHK
3622
100
846
353
5806
22308
20000
1






1Frequency or entire sequence amongst isolates scanned.




2Number supertpe alleles bound. Peptides binding 3 or more alleles are considered degenerate.




3A dash (—) indicates IC50














TABLE XXVIII







in vitro binding of conserved HBV-derived peptides to


HLA-B7 supertype alleles.














B7-supertype binding capacity




SEQ ID

(IC50 nM)
Alleles



















Peptide
AA
Molecule
1st Pos
Sequence
NO:
Consv.1
B*0702
B*3501
B*5101
B*5301
B*5401
bound2






















1147.05
10
POL
541
FPHCLAFSYM
3623
95
56
33
61
118
208
5





1145.04
9
ENV
324
IPIPSSWAF
3624
100
42
2.6
2.3
12
2941
4





1147.02
9
POL
440
HPAAMPHLL
3625
100
56
267
500
186
833
4





1147.06
9
X
58
LPVCAFSSA
3626
95
115
101
500
10333
0.53
4





1147.08
9
POL
651
YPALMPLYA
3627
95
306
150
162
664
0.63
4





988.05
9
CORE
19
LPSDFFPSV
3628
95
1774
343
9.0
120
4.8
4





1145.08
9
POL
541
FPHCLAFSY
3629
95
3
14
83
17
503
3





19.0014
8
POL
640
YPALMPLY
3630
190
13750
28
13
207
1786
3





26.0570
11
pol
640
YPALMPLYACI
3631
95
1375

117
291
143
3





1147.04
10
POL
365
TPARVTGGVF
3632
90
17
72

939
16667
2





15.0034
9
ENV
390
LPIFFCLWV
3633
100


57
2325
53
2





20.0140
9
POL
723
LPIHTAELL
3634
85
1375
114
1058
30
20000
2





19.0006
8
ENV
340
VPFVQWFV
3635
95
5500

0.29

91
2





19.0007
8
ENV
379
LPIFFCLW
3636
100


153
66
2857
2





19.0010
8
POL
1
MPLSYQHF
3637
100

742
458
251
526
2





19.0011
8
POL
429
HPAAMPHL
3638
100
85
18000
18
2514
625
2





19.0012
8
POL
511
SPFLLAQF
3639
95
10
8000
306
10333
1075
2





26.0566
11
pol
511
SPFLLAQFTSA
3640
95
67



0.83
2





1147.01
9
POL
789
DPSRGRLGL
3641
90
458




1





16.0182
10
X
67
GPCALRFTSA
3642
90
61



2857
1





20.0273
10
POL
440
HPAAMPHLLV
3643
85
344
3600
705
664
588
1





15.0030
9
ENV
191
IPQSLDSWW
3644
90


27500
62

1





15.0210
10
POL
123
LPLDKGIKPY
3645
100

248
27500


1





16.0006
9
ENV
25
FPDHQLDPA
3646
90

8000


12
1





16.0177
10
ENV
324
IPIPSSWAFA
3647
80
4231
3000

6643
22
1





16.0180
10
POL
644
APFTQCGYPA
3648
95
1897



7.1
1





16.0181
10
POL
723
LPIHTAELLA
3649
85
3056
6545

5813
30
1





19.0003
8
ENV
173
GPLLVLQA
3650
95
18333

500

1538
1





19.0005
8
ENV
313
IPIPSSWA
3651
100
13750
18000
2895

167
1





19.0009
8
NUC
133
RPPNAPIL
3652
100
724

196


1





19.0015
8
POL
659
SPTYKAFL
3653
95
14

2895


1





19.0016
8
POL
769
VPSALNPA
3654
90
3000

786

10
1





26.0554
11
pol
633
APFTQCGYPAL
3655
95
24
7200
13750

1075






26.0559
11
pol
712
LPIHTAELLAA
3656
85
611
2667

775
3.6
1





26.0561
11
pol
774
NPADDPSRGRL
3657
90
458




1





26.0564
11
Core
133
RPPNAPILSTL
3658
100
42

3056


1





26.0567
11
Core
49
SPHHTALRQAI
3659
100
9.5

13750
18600

1





26.0568
11
pol
354
TPARVTGGVFL
3660
90
58


18600
20000
1






1Frequency of entire sequence amongst isolates scanned.




2Number of supertpe alleles bound. Peptides binding 3 or more alleles are considered degenerate.




3A dash (—) indicates IC50














TABLE XXIX





HBV derived A1- and A24-motif containing peptides







a. A1-motif peptides



















HLA-A*0101


Peptide
Molecule
Position
Sequence
SEQ ID NO:
Conserv.
binding (IC50 nM)





1069.01
Core
59
LLDTASALY
3661
75
2.1





1.0519
Core
419
DLLDTASALY
3662
75
2.3





1069.02
pol
427
SLDVSAAFY
3663
95
4.8





2.0239

1000
LSLDVSAAFY
3664
95
6.0





2.0126

1521
MSTTDLEAY
3665
75
29





1039.06
ENV
359
WMMWYWGPSLY
3666
85
78





1090.14
pol
538
YMDDVVLGA
3667
90
96





1090.09
pol
808
PTTGRTSLY
3668
85
119





1069.03
pol
124
PLDKGIKPYY
3669
100
147





1069.08
env
249
ILLLCLIFLL
3670
100
192





1069.04
pol
149
HTLWKAGILY
3671
100
381





1039.01

360
MMWYWGPSLY
3672
85
309





1.0774
Core
416
WLWGMDIDPY
3673
75
309





20.0254
pol
631
FAAPFTQCGY
3674
95
368





1.0166
pol
629
KVGNFTGLY
3675
95
368










b. A24-motif peptides



















HLA-A*2402


Peptide
Molecule
Position
Sequence
SEQ ID NO:
Conserv.
binding (IC50 nM)





20.0271
POL
392
SWPKFAVPNL
3676
95
2.1





1069.23
POL
745
KYTSFPWLL
3677
85
2.3





2.0181
POL
492
LYSHPIILGF
3678
80
11





20.0269
ENV
236
RWMCLRRFII
3679
95
11





20.0136
ENV
334
SWLSLLVPF
3680
100
31





20.0137
ENV
197
SWWTSLNFL
3681
95
32





20.0135
ENV
236
RWMCLRRFI
3682
95
169





20.0139
POL
167
SFCGSPYSW
3683
100
169





2.0173
POL
4
SYQHFRKLLL
3684
75
182





2.0060

1224
GYPALMPLY
3685
95
245





13.0129
NUC
117
EYLVSFGVWI
3686
90
353





1090.02
core
131
AYRPPNAPI
3687
90
387





13.0073
NUC
102
WFHISCLTF
3688
80
400





20.0138
POL
51
PWTHKVGNF
3689
100
414





A dash indicates IC50 nM.













TABLE XXXa







Immunogenicity of HBV-derived A2-supermotif cross-reactive peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1


















924.07
FLPSDFFPSV
3690
HBV core 18
5
10/10
6/6
25/32a
+





1069.06
LLVPFVQWFV
3691
HBV env 338
5
3/4
6/9

+





1147.13
FLLAQFTSAI
3692
HBV pol 513
5

0/3







1090.77
YMDDVVLGV
3693
HBV pol 538
5

9/9

+





777.03
FLLTRILTI
3694
HBV env 183
4


14/23a
+





927.15
ALMPLYACI
3695
HBV pol 642
4
10/12
3/5
 2/15a
+





1013.01
WLSLLVPFV
3696
HBV env 335
4
2/6
5/9
23/29a
+





1069.05
LLAQFTSAI
3697
HBV pol 504
4
0/4
0/5







1132.01
LVPFVQWFV
3698
HBV env 339
4
0/3
0/4







1147.14
VLLDYQGMLPV
3699
HBV env 259
4
4/4
6/6

+





927.41
LLSSNLSWL
3700
HBV pol 992
3
0/4
0/3







927.42
NLSWLSLDV
3701
HBV pol 411
3

2/8

+





927.46
KLHLYSHPI
3702
HBV pol 489
3
0/4
4/6

+





1069.07
FLLAQFTSA
3703
HBV pol 503
3
1/2
0/3

+





1168.02
GLSRYVARL
3704
HBV pol 455
3


 9/13a
+





Immunogenicity evaluation derived from primary cultures, acute patients (aBertoni et al, J Clin Invest 100: 503, bRehermann et al., J. Clin. Invest 97: 1655, c- Nayersina et al., J Immunol 150: 4659) or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems.













TABLE XXXb







Immunogenicity of non-crossreactive HBV A2-supermotif peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1



















927.11
FLLSLGIHL
3705
HBV pol 562
2
15/22
12/13
9/15a
+






927.47
HLYSHPIIL
3706
HBV pol 1076
2

10/14

+





1039.03
MMWYWGPSL
3707
HBV env 360
2
3/4
0/4

+





1069.12
YLHTLWKAGV
3708
HBV pol 147
2
2/4


+





1137.02
LLDYQGMLPV
3709
HBV env 260
2
1/2
0/4

+





1142.07
GLLGWSPQA
3710
HBV env 62
2
3/4
5/6

+





1.0573
ILRGTSFVYV
3711
HBV pol 773
1


3/7b
+





1013.14
VLQAGFFLL
3712
HBV env 177
1
0/4
 5/12

+





1069.10
LLPIFFCLWV
3713
HBV env 378
1
3/3
0/4
2/5c
+





1069.13
PLLPIFFCL
3714
HBV env 377
1
0/4
 7/12

+





1090.06
LLVLQAGFFL
3715
HBV env 175
1
1/5
0/4

+





1090.12
YLVSFGVWI
3716
HBV nuc 118
1
9/9


+





1.0518
GLSPTVWLSV
3717
HBV env 338
1


3/9c
+





1090.14
YMDDVVLGA
3718
HBV pol 538
1
2/7
2/5
2/7b
+





Immunogenicity evaluation derived from primary cultures, acute patients (aBertoni et al, J Clin Invest 100: 503, bRehermann et al., J. Clin. Invest 97: 1655, cNayersina et al., J Immunol 150: 4659) or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems.













TABLE XXXc







Cross-recognition of HBV pol 538 and a Lamivudine


induced pol 538 variant by CTL induced with a


pol 538 analoga.









Day 6 CTL response (ΔLU)










HBV pol 538
HBV pol 538 mutant


Stimulating peptide
(YMDDVVLGA)b
(YVDDVVLGA)





HBV pol 538
27.8
54.2





HBV pol 538 mutant
35.3
27.9






aCTLs were induced using the 1090.77 analog of HBV pol 538 (peptide 1090.14). 1090.77 was encoded in the DNA minigene pEP2.AOS.




bValues shown represent the geometric mean of ΔLU from 2 independent cultures. Peptides loaded onto target cells were 1090.14 (HBV pol 538) or 1353.02 (a Lamivudine induced mutant of pol 538).














TABLE XXXIa







Immunogenicity of HBV-derived A3-supermotif cross-reactive peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1



















1147.16
HTLWKAGILYK
3719
HBV POL 149
5
0/6
3/3
1/22
+






1083.01
STLPETTVVRR
3720
HBV core 141
4
3/5
6/6
8/32
+





1150.51
GSTHVSWPK
3721
HBV pol 398
4

3/6

+





1.0219
FVLGGCRHK
3722
HBV adr “X” 1550
3
0/4








1069.16
NVSIPWTHK
3723
HBV pol 47
3
0/8
0/3
1/21
+





1069.20
LVVDFSQFSR
3724
HBV pol 388
3
0/4
6/6
1/22
+





1090.10
QAFTFSPTYK
3725
HBV pol 665
3
3/6
0/3
3/21
+





1090.11
SAICSVVRR
3726
HBV pol 531
3
1/4

2/22
+






1Immunogenicity evaluation derived from primary cultures, Bertoni et al, J Clin Invest 100: 503 or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems. A negative assessment (−) indicates that no responders when examined.














TABLE XXXIb







Immunogenicity of non-crossreactive HBV A3-supermotif peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1



















1069.15
TLWKAGILYK
3727
HBV pol 150
2
3/8
0/3
5/28
+






1142.05
KVGNFTGLY
3728
HBV adr POL 629
2

0/3
2/22
+






1Immunogenicity evaluation derived from primary cultures, Bertoni et al, J Clin Invest 100: 503 or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems. A negative assessment (−) indicates that no responders when examined.














TABLE XXXIIa







Immunogenicity of HBV B7-supermotif cross-reactive peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1



















1147.05
FPHCLAFSYM
3729
HBV POL 530
5
1/3

0/12
+






988.05
LPSDFFPSV
3730
HBV core 19-27
4


2/16
+





1145.04
IPIPSSWAF
3731
HBV ENV 313
4
0/4

1/12
+





1147.02
HPAAMPHLL
3732
HBV POL 429
4
0/5

0/12






1147.06
LPVCAFSSA
3733
HBV X 58
4
1/4


+





1147.08
YPALMPLYA
3734
HBV POL 640
4


0/12






1145.08
FPHCLAFSY
3735
HBV POL 541
3
0/4









1Immunogenicity evaluation derived from primary cultures, Bertoni et at, J Clin Invest 100: 503 or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems. A negative assessment (−) indicates that no responders when examined.














TABLE XXXIIb







Immunogenicity of non-crossreactive HBV B7-supermotif peptides









Immunogenicity















Peptide
Sequence
SEQ ID NO:
Protein
XRN
primary
transgenic
patients
overall1





1147.04
TPARVTGGVF
3736
HBV POL 354
2


2/12
+






1Immunogenicity evaluation derived from primary cultures, Bertoni et al, J Clin Invest 100: 503 or transgenic mice. A positive assessment (+) is assigned when responders have been noted in one of these systems. A negative assessment (−) indicates that no responders when examined.














TABLE XXXIII







Candidate HBV-derived HTL epitopes










Selection
Conservancy

SEQ














criteria
Peptide
Mol
1st Pos
Core
Total
Sequence
ID NO:


















DR-supermotif
F107.01
ENV
249
100
95
ILLLCLIFLLVLLDY
3737







F107.02
ENV
252
95
95
LCLIFLLVLLDYQGM
3738






1280.17
ENV
258
90
90
LVLLDYQGMLPVCPL
3739






1186.22
ENV
332
100
100
RFSWLSLLVPFVQWF
3740






1186.15
ENV
339
95
95
LVPFVQWFVGLSPTV
3741






1186.06
ENV
342
95
95
FVQWFVGLSPTVWLS
3742






1186.03
NUC
19
85
85
ASKLCLGWLWGMDID
3743






1186.12
NUC
24
85
85
LGWLWGMDIDPYKEF
3744






857.02
NUC
50

90
PHHTALRQAILCWGELMTLA
3745






1186.23
NUC
98
85
85
RQLLWFHISCLTFGR
3746






27.0279
NUC
117

90
EYLVSFGVWIRTPPA
3747






27.0280
NUC
123
95
95
GVWIRTPPAYRPPNA
3748






1186.20
NUC
129
100
95
PPAYRPPNAPILSTL
3749






1186.16
NUC
136
100
95
NAPILSTLPETTVVR
3750






1186.01
POL
38
95
95
AEDLNLGNLNVSIPW
3751






1186.17
POL
45
100
95
NLNVSIPWTHKVGNF
3752






27.0281
POL
145
100
100
RHYLHTLWKAGILYK
3753






1280.13
POL
406
95
95
KFAVPNLQSLTNLLS
3754






27.0283
POL
409

85
VPNLQSLTNLLSSNL
3755






F107.03
POL
412
90
90
LQSLTNLLSSNLSWL
3756






1186.28
POL
416
90
90
TNLLSSNLSWLSLDV
3757






1186.27
POL
420
100
85
SSNLSWLSLDVSAAF
3758






F107.04
POL
523
95
95
PFLLAQFTSAICSVV
3759






1186.10
POL
526
95
95
LAQFTSAICSVVRRA
3760






1186.04
POL
534
95
95
CSVVRRAFPHCLAFS
3761






F107.05
POL
538
95
95
RRAFPHCLAFSYMDD
3762






1186.02
POL
546
90
90
AFSYMDDVVLGAKSV
3763






1186.05
POL
629
85
85
DWKVCQRIVGLLGFA
3764






1280.21
POL
637
95
95
VGLLGFAAPFTQCGY
3765






27.0278
POL
643

95
AAPFTQCGYPALMPL
3766






1186.21
POL
648
95
95
QCGYPALMPLYACIQ
3767






1280.14
POL
694
95
95
LCQVFADATPTGWGL
3768






27.0282
POL
750
85
85
SVVLSRKYTSFPWLL
3769







X
13
95
90
RDVLCLRPVGAESRG
3770






1186.07
X
50
95
90
GAHLSLRGLPVCAFS
3771






1186.29
X
60
95
90
VCAFSSAGPCALRFT
3772





Algorithm
1280.20
ENV
330
100
80
SVRFSWLSLLVPFVQ
3773






1280.19
NUC
28
85
80
RDLLDTASALYREAL
3774






1298.02
POL
56
90
55
VGNFTGLYSSTVPVF
3775






1298.03
POL
571
95
75
TNFLLSLGIHLNPNK
3776






1298.05
POL
651
95
55
YPALMPLYACIQSKQ
3777






1298.06
POL
664
95
60
KQAFTFSPTYKAFLC
3778






1280.181
POL
722
85
80
PLPIHTAELLAACFA
3779






1280.09
POL
774
90
80
GTSFVYVPSALNPAD
3780





DR3-motif
795.05
ENV
10

95
PLGFFPDHQLDP
3781






35.0090
ENV
312
95
90
FLLVLLDYQGMLPVC
3782






CF-03
NUC
28
85
80
RDLLDTASALYREALESPEH
3783






35.0091
POL
18
90
65
AGPLEEELPRLADEG
3784






35.0092
POL
34
100
85
NRRVAEDLNLGNLNV
3785






35.0093
POL
96
85
60
VGPLTVNEKRRLKLI
3786






35.0094
POL
120
100
100
TKYLPLDKGIKPYYP
3787






35.0095
POL
371
100
55
GGVFLVDKNPHNTTE
3788






35.0096
POL
385
100
45
ESRLVVDFSQFSRGN
3789






1186.18
POL
422
95
85
NLSWLSLDVSAAFYH
3790






35.0099
POL
666
95
55
AFTFSPTYKAFLCKQ
3791






35.0101
X
18
95
35
LRPVGAESRGRPVSG
3792





Lower conservancy or miscellaneous
799.01
ENV
11
80
75
PLLVLQAGFFLLTRILTIPQ
3793






799.02
ENV
31
95

SLDSWWTSLNFLGGTTVCLG
3794






799.04
ENV
71
95
75
GYRWMCLRRFIIFLFILLLC
3795






1298.01
ENV
117
80
40
PQAMQWNSTTFHQTL
3796






1280.06
ENV
180
80
80
AGFFLLTRILTIPQS
3797






1280.11
ENV
245
80
80
IFLFILLLCLIFLLV
3798






CF-08
NUC
120

90
VSFGVWIRTPPAYRPPNAPI
3799






1186.25
NUC
121
95
90
SFGVWIRTPPAYRPP
3800






1280.15
POL
501
80
80
LHLYSHPIILGFRKI
3801






1298.04
POL
618
80
45
KQCFRKLPVNRPIDW
3802






1298.07
POL
767
80
70
AANWILRGTSFVYVP
3803






1298.08
POL
827
80
60
PDRVHFASPLHVAWR
3804
















TABLE XXXIV







HLA-DR screening panels









Screening
Representative Assay
Phenotypic Frequencies

















Panel
Antigen
Alleles
Allele
Alias
Cauc.
Blk.
Jpn.
Chn.
Hisp.
Avg.




















Primary
DR1
DRB1*0101-03
DRB1*0101
(DR1)
18.5
8.4
10.7
4.5
10.1
10.4



DR4
DRB1*0401-12
DRB1*0401
(DR4w4)
23.6
6.1
40.4
21.9
29.8
24.4



DR7
DRB1*0701-02
DRB1*0701
(DR7)
26.2
11.1
1.0
15.0
16.6
14.0



Panel total



59.6
24.5
49.3
38.7
51.1
44.6


Secondary
DR2
DRB1*1501-03
DRB1*1501
(DR2w2 β1)
19.9
14.8
30.9
22.0
15.0
20.5



DR2
DRB5*0101
DRB5*0101
(DR2w2 β2)









DR9
DRB1*09011, 09012
DRB1*0901
(DR9)
3.6
4.7
24.5
19.9
6.7
11.9



DR13
DRB1*1301-06
DRB1*1302
(DR6w19)
21.7
16.5
14.6
12.2
10.5
15.1



Panel total



42.0
33.9
61.0
48.9
30.5
43.2


Tertiary
DR4
DRB1*0405
DRB1*0405
(DR4w15)









DR8
DRB1*0801-5
DRB1*0802
(DR8w2)
5.5
10.9
25.0
10.7
23.3
15.1



DR11
DRB1*1101-05
DRB1*1101
(DR5w11)
17.0
18.0
4.9
19.4
18.1
15.5



Panel total



22.0
27.8
29.2
29.0
39.0
29.4


Quarternary
DR3
DRB1*0301-2
DRB1*0301
(DR3w17)
17.7
19.5
0.4
7.3
14.4
11.9



DR12
DRB1*1201-02
DRB1*1201
(DR5w12)
2.8
5.5
13.1
17.6
5.7
8.9



Panel total



20.2
24.4
13.5
24.2
19.7
20.4
















TABLE XXXV





HBV-derived cross-reactive HLA-DR binding peptides






















HLA-DR binding




Conservancy

capacity (IC50 nM)
















Peptide
Mol
1st Pos
Core
Total
Sequence
SEQ ID NO:
DR1
DR2w2 β1
DR2w2 β2





F107.03
POL
412
90
90
LQSLTNLLSSNLSWL
3805
2.0
21
1000





1298.06
POL
664
95
60
KQAFTFSPTYKAFLC
3806
9.4
38
143





1280.06
ENV
180
80
80
AGFFLLTRILTIPQS
3807
1.1
217
1053





1280.09
POL
774
90
80
GTSFVYVPSALNPAD
3808
14
650
400





1186.25
NUC
121
95
90
SFGVWIRTPPAYRPP
3809
532
827
47





  27.0280
NUC
123
95
95
GVWIRTPPAYRPPNA
3810
14
217
2.8





CF-08
NUC
120

90
VSFGVWIRTPPAYRPPNAPI
3811
192

105





  27.0281
POL
145
100
100
RHYLHTLWKAGILYK
3812
17
5.4
35





1186.15
ENV
339
95
95
LVPFVQWFVGLSPTV
3813
385
13
1429





1280.15
POL
501
80
80
LHLYSHPIILGFRKI
3814
227
268
500





F107.04
POL
523
95
95
PFLLAQFTSAICSVV
3815
28
337
4762





1298.04
POL
618
80
45
KQCFRKLPVNRPIDW
3816
3.3
4136
952





1298.07
POL
767
80
70
AANWILRGTSFVYVP
3817
54
379
3279





 857.02
NUC
50

90
PHHTALRQAILCWGELMTLA
3818
70
9.1
211













HLA-DR binding capacity (IC50 nM)
Total DR


















Peptide
DR3
DR4w4
DR4w15
DR5w11
DR6
DR7
DR8
DR9
alleles bound







F107.03
a
9.4
47
294
135
167
557
682
10







1298.06

41
173
83
175
76
408
139
10







1280.06

8.5
253
5.6
9.5
8.1
188
58
9







1280.09

118
93
426

93
803
221
9







1186.25

577
603
769
17500
1042
196
938
8







  27.0280

13
67
42

114
92
1667
8







CF-08

300

426

124


5







  27.0281

2250
1462
42
745
61
27
174
8







1186.15

300
27
53
1944
2717
74
30
7







1280.15

66
238
488
17500

803
1531
7







F107.04

563
317
1667
44
325
845
1271
7







1298.04

38
45
1538
814
63
845
3000
7







1298.07

882
1520
1429
140
43
196
278
7







 857.02

85

263
193000
676
196
2273
7








aA dash (—) indicates IC50 nM > 20,000.














TABLE XXXVI







HBV-derived DR3-binding peptides










Conservancy















Peptide
Mol
1st Pos
Core
Total
Sequence
SEQ ID NO:
DR3


















1280.14*
POL
694
95
95
LCQVFADATPTGWGL
3819
67






35.0096
POL
385
100
45
ESRLVVDFSQFSRGN
3820
115





35.0093
POL
96
85
60
VGPLTVNEKRRLKLI
3821
136





1186.27
POL
420
100
85
SSNLSWLSLDVSAAF
3822
200





1186.18
POL
422
95
85
NLSWLSLDVSAAFYH
3823
231





*tested as peptide 35.0100













TABLE XXXVIIa







HBV Preferred CTL Epitopes











Peptide
Sequence
SEQ ID NO:
Protein
HLA














924.07
FLPSDFFPSV
3824
core 18
A2





777.03
FLLTRILTI
3825
env 183
A2





927.15
ALMPLYACI
3826
pol 642
A2





1013.01
WLSLLVPFV
3827
env 335
A2





1090.77
YMDDVVLGV
3828
pol 538
A2/A1





1168.02
GLSRYVARL
3829
pol 455
A2





927.11
FLLSLGIHL
3830
pol 562
A2





1069.10
LLPIFFCLWV
3831
env 378
A2





1069.06
LLVPFVQWFV
3832
env 338
A2





1147.16
HTLWKAGILYK
3833
pol 149
A3/A1





1083.01
STLPETTVVRR
3834
core 141
A3





1069.16
NVSIPWTHK
3835
pol 47
A3





1069.20
LVVDFSQFSR
3836
pol 388
A3





1090.10
QAFTFSPTYK
3837
pol 665
A3





1090.11
SAICSVVRR
3838
pol 531
A3





1142.05
KVGNFTGLY
3839
pol 629
A3/A1





1147.05
FPHCLAFSYM
3840
pol 530
B7





988.05
LPSDFFPSV
3841
core 19
B7





1145.04
IPIPSSWAF
3842
env 313
B7





1147.02
HPAAMPHLL
3843
pol 429
B7





26.0570
YPALMPLYACI
3844
pol 640
B7





1147.04
TPARVTGGVF
3845
pol 354
B7





1.0519
DLLDTASALY
3846
core 419
A1





2.0239
LSLDVSAAFY
3847
pol 1000
A1





1039.06
WMMWYWGPSLY
3848
env 359
A1





20.0269
RWMCLRRFII
3849
env 236
A24





20.0136
SWLSLLVPF
3850
env 334
A24





20.0137
SWWTSLNFL
3851
env 197
A24





13.0129
EYLVSFGVWI
3852
core 117
A24





1090.02
AYRPPNAPI
3853
core 131
A24





13.0073
WFHISCLTF
3854
core 102
A24





20.0271
SWPKFAVPNL
3855
pol 392
A24





1069.23
KYTSFPWLL
3856
pol 745
A24





2.0181
LYSHPIILGF
3857
pol 492
A24
















TABLE XXXVIIb







HBV Preferred HTL epitopes









Selection
Conservancy















Criteria
Peptide
Mol
1st Pos
Core
Total
SEQ ID NO:
Sequence


















DR supermotif
F107.03
POL
412
90
90
3838
LQSLTNLLSSNLSWL







1298.06
POL
664
95
60
3859
KQAFTFSPTYKAFLC






1280.06
ENV
180
80
80
3860
AGFFLLTRILTIPQS






1280.09
POL
774
90
80
3861
GTSFVYVPSALNPAD






CF-08
CORE
120

90
3862
VSFGVWIRTPPAYRPPNAPI






27.0281
POL
145
100
100
3863
RHYLHTLWKAGILYK






1186.15
ENV
339
95
95
3864
LVPFVQWFVGLSPTV






1280.15
POL
501
80
80
3865
LHLYSHPIILGFRKI






F107.04
POL
523
95
95
3866
PFLLAQFTSAICSVV






1298.04
POL
618
80
45
3867
KQCFRKLPVNRPIDW






1298.07
POL
767
80
70
3868
AANWILRGTSFVYVP






857.02
CORE
50

90
3869
PHHTALRQAILCWGELMTLA





DR3 motif
1280.14
POL
694
95
95
3870
LCQVFADATPTGWGL






35.0096
POL
385
100
45
3871
ESRLVVDFSQFSRGN






35.0093
POL
96
85
60
3872
VGPLTVNEKRRLKLI






1186.27
POL
420
100
85
3873
SSNLSWLSLDVSAAF
















TABLE XXXVIII







Estimated population coverage by a panel of HBV derived HTL epitopes













Population coverage



Representative
No. of
(phenotypic frequency)
















Antigen
Alleles
assay
epitopes2
Cauc.
Blk.
Jpn.
Chn.
Hisp.
Avg.



















DR1
DRB1*0101-03
DR1
12
18.5
8.4
10.7
4.5
10.1
10.4


DR2
DRB1*1501-03
DR2w2 β1
11
19.9
14.8
30.9
22.0
15.0
20.5


DR2
DRB5*0101
DR2w2 β2
8








DR3
DRB1*0301-2
DR3
4
17.7
19.5
0.40
7.3
14.4
11.9


DR4
DRB1*0401-12
DR4w4
11
23.6
6.1
40.4
21.9
29.8
24.4


DR4
DRB1*0401-12
DR4w15
9








DR7
DRB1*0701-02
DR7
9
26.2
11.1
1.0
15.0
16.6
14.0


DR8
DRB1*0801-5
DR8w2
7
5.5
10.9
25.0
10.7
23.3
15.1


DR9
DRB1*09011, 09012
DR9
10
3.6
4.7
24.5
19.9
6.7
11.9


DR11
DRB1*1101-05
DR5w11
11
17.0
18.0
4.9
19.4
18.1
15.5


DR13
DRB1*1301-06
DR6w19
7
21.7
16.5
14.6
12.2
10.5
15.1


Total1



98.5
95.1
97.1
91.3
94.3
95.1






1Total opulation coverage has been adjusted to acount for the presence of DRX in many ethnic populations. It has been assumed that the range of specificities represented by DRX alleles will mirror those of previously characterized HLA-DR alleles. The proportion of DRX incorporated under each motif is representative of the frequency of the motif in the remainder of the population. Total coverage has not been adjusted to account for unknown gene types.




2Number of epitopes represents a minimal estimate, considering only the epitopes shown in Table 12. Additional alleles possibly bound by nested epitopes have not been accounted.






Claims
  • 1. A peptide composition of less than 250 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 8 to about 13 amino acid residues that have at least 65% identity with a native amino acid sequence of HBV and, (b) binding to at least one HLA class I HLA allele with an IC50 of less than about 500 nM.
  • 2. The composition of claim 1, further wherein said peptide has at least 77% identity with a native HBV amino acid sequence.
  • 3. The composition of claim 1, further wherein said peptide has 100% identity with a native HBV amino acid sequence.
  • 4. A pharmaceutical composition comprising a peptide and a pharmaceutical carrier, wherein the peptide is a peptide of Table VII (A1 supermotif), Table VIII (A2 supermotif/A2.1 motif), Table IX (A3 supermotif), Table X (A24 supermotif), Table XI (B7 supermotif), Table XII (B27 supermotif), Table XIII (B58 supermotif), Table XIV (B62 supermotif), Table XV (A1 motif), Table XVI (A3 motif), Table XVII (A11 motif), or Table XVIII (A24 motif) comprising an IC50 of less than about 500 nM for at least one HLA class I molecule.
  • 5. The pharmaceutical composition of claim 4 wherein the composition comprises the peptide in a form of nucleic acids that encode the peptide.
  • 6. The pharmaceutical composition of claim 5 wherein the composition comprises the peptide in a form of nucleic acids that encode the epitope and one or more additional peptide(s).
  • 7. The composition of claim 4, wherein the peptide is comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 8. The pharmaceutical composition of claim 4 wherein the peptide is in a human dose form, and the carrier is in a human unit dose.
  • 9. A peptide composition of claim 1 comprising an analog of a peptide epitope, wherein the peptide epitope is an epitope of Table VII (A1 supermotif), Table VIII (A2 supermotif/A2.1 motif), Table IX (A3 supermotif), Table X (A24 supermotif), Table XI (B7 supermotif), Table XII (B27 supermotif), Table XIII (B58 supermotif), Table XIV (B62 supermotif), Table XV (A1 motif), Table XVI (A3 motif), Table XVII (A11 motif), or Table XVIII (A24 motif), said analog comprising a preferred or less preferred amino acid of Table II substituted in for a starting residue, or having a deleterious residue of Table II substituted out of the starting sequence and replaced by a non-deleterious residue.
  • 10. A peptide composition of claim 1 comprising a peptide of Table XXII.
  • 11. A method for inducing a cytotoxic T lymphocyte response, said method comprising steps of: providing a peptide that comprises an IC50 of less than about 500 nM for an HLA class I molecule, wherein the peptide is a peptide of Table VII (A1 supermotif), Table VIII (A2 supermotif/A2.1 motif), Table IX (A3 supermotif), Table X (A24 supermotif), Table XI (B7 supermotif), Table XII (B27 supermotif), Table XIII (B58 supermotif), Table XIV (B62 supermotif), Table XV (A1 motif), Table XVI (A3 motif), Table XVII (A11 motif), or Table XVIII (A24 motif); and,administering said peptide to a human.
  • 12. The method of claim 11, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide.
  • 13. The method of claim 12, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide and at least one additional peptide, with a proviso that an additional peptide is not an entire native antigen.
  • 14. The method of claim 11, wherein the providing step provides the peptide comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 15. A method for inducing a cytotoxic T lymphocyte response, said method comprising steps of: providing a peptide that induces a cytotoxic T cell response in vitro and/or in vivo, wherein the peptide is a peptide of Table VII (A1 supermotif), Table VIII (A2 supermotif/A2.1 motif), Table IX (A3 supermotif), Table X (A24 supermotif), Table XI (B7 supermotif), Table XII (B27 supermotif, Table XIII (B58 supermotif), Table XIV (B62 supermotif), Table XV (A1 motif), Table XVI (A3 motif), Table XVII (A11 motif), Table XVIII (A24 motif or Table XXIII; and,administering said pharmaceutical composition to a human.
  • 16. The method of claim 15, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide.
  • 17. The method of claim 16, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide and at least one additional peptide, with a proviso that an additional peptide is not an entire native antigen.
  • 18. The method of claim 15, wherein the providing step provides the peptide comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 19. The method of claim 15, wherein the providing step comprises a peptide that induces a cytotoxic T cell response when complexed with an HLA class I molecule and is presented to an HLA class I-restricted cytotoxic T cell.
  • 20. A peptide composition of less than 250 amino acid residues comprising a peptide epitope useful for inducing an immune response against hepatitis B virus (HBV) said epitope (a) having an amino acid sequence of about 6 to about 25 amino acid residues that have at least 65% identity with a native amino acid sequence of HBV and, (b) binding to at least one HLA class II HLA allele with an IC50 of less than about 1000 mM.
  • 21. The composition of claim 20, further wherein said peptide has at least 77% identity with a native HBV amino acid sequence.
  • 22. The composition of claim 20, further wherein said peptide has 100% identity with a native HBV amino acid sequence.
  • 23. A pharmaceutical composition comprising: a human dose form of a peptide of Table XIX or Table XX that comprises an IC50 of less than about 1,000 nM for at least one HLA DR molecule of an HLA DR supertype; and, a human dose of a pharmaceutically acceptable carrier.
  • 24. The pharmaceutical composition of claim 23 wherein the composition comprises the peptide in a form of nucleic acids that encode the peptide.
  • 25. The pharmaceutical composition of claim 24 wherein the composition comprises the peptide in a form of nucleic acids that encode the peptide and at least one additional peptide, with a proviso that an additional peptide is not an entire native antigen.
  • 26. The composition of claim 25, wherein the peptide is comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 27. A peptide composition of claim 20 comprising an analog of a peptide epitope of Table XIX or Table XX, said analog comprising a preferred or less preferred amino acid of Table III substituted in for a starting residue, and/or having a deleterious residue of Table III substituted out of the starting sequence and replaced by a non-deleterious residue.
  • 28. A method for inducing a helper T lymphocyte response, said method comprising steps of: providing a peptide that comprises an IC50 of less than about 1,000 nM for an HLA class II molecule, wherein the peptide is a peptide of Table XIX or Table XX; and,administering said peptide to a human.
  • 29. The method of claim 28, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide.
  • 30. The method of claim 29, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide and at least one additional peptide, with a proviso that an additional peptide is not an entire native antigen.
  • 31. The method of claim 28, wherein the providing step provides the peptide comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 32. A method for inducing a helper T lymphocyte response, said method comprising steps of: providing a peptide that induces a helper T cell response in vitro and/or in vivo, wherein the peptide is a peptide of Table XIX or Table XX; and,administering said pharmaceutical composition to a human.
  • 33. The method of claim 32, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide.
  • 34. The method of claim 33, wherein the providing step provides the peptide in a form of nucleic acids that encode the peptide and at least one additional peptide, with a proviso that an additional peptide is not an entire native antigen.
  • 35. The method of claim 32, wherein the providing step provides the peptide comprised by a longer peptide, with a proviso that the longer peptide is not an entire native antigen.
  • 36. The method of claim 32, wherein the providing step comprises a peptide that induces a helper T cell response when complexed with an HLA class II molecule and is presented to an HLA class I-restricted helper T cell.
  • 37. A vaccine for preventing or treating HBV infection that induces a protective or therapeutic immune response, wherein said vaccine comprises: at least one peptide selected from Table(s) VII-XX or Table XXII; and,a pharmaceutically acceptable carrier.
  • 38. A kit for a vaccine that induces a protective or therapeutic immune response to HBV, said vaccine comprising: at least one peptide selected from Table(s) VII-XX or Table XXII; a pharmaceutically acceptable carrier; and,instructions for administration to a patient.
  • 39. A method for monitoring or evaluating an immune response to HBV or an epitope thereof in a patient having a known HLA type, the method comprising: incubating a T lymphocyte sample from the patient with a peptide selected from Table(s) VII-XX or Table XXII, wherein that peptide bears a motif corresponding to at least one HLA allele present in said patient; and,detecting the presence of a T lymphocyte that recognizes the peptide.
  • 40. The method of claim 39, wherein the peptide is comprised by a tetrameric complex.
  • 41. The pharmaceutical composition of claim 4, wherein the peptide comprises a peptide of Table VII.
  • 42. The pharmaceutical composition of claim 41, wherein the peptide is an amino acid sequence selected from the group consisting of SEQ ID NOs: 3846, 3847 and 3848.
CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation-In-Part (“CIP”) of U.S. Ser. No. 08/820,360 filed Mar. 12, 1997, which claims the benefit of U.S. Provisional Application No. 60/013,363 filed Mar. 13, 1996 and now abandoned. The present application is also a CIP of U.S. Ser. No. 09/189,702 filed Nov. 10, 1998, which is a CIP of U.S. Ser. No. 08/205,713 filed Mar. 4, 1994, which is a CIP of Ser. No. 08/159,184 filed Nov. 29, 1993 and now abandoned, which is a CIP of Ser. No. 08/073,205 filed Jun. 4, 1993 and now abandoned, which is a CIP of Ser. No. 08/027,146 filed Mar. 5, 1993 and now abandoned. The present application is also related to U.S. Ser. No. 08/197,484, U.S. Ser. No. 08/464,234, U.S. Ser. No. 08/464,496, U.S. Ser. No. 08/464,031, abandoned U.S. Ser. No. 08/464,433, and U.S. Ser. No. 08/461,603, which is a continuation of abandoned U.S. Ser. No. 07/935,811, which is a CIP of abandoned U.S. Ser. No. 07/874,491, which is a CIP of abandoned U.S. Ser. No. 07/827,682, which is a CIP of abandoned Ser. No. 07/749,568. The present application is also related to U.S. patent application entitled “Peptides and Methods for Creating Synthetic Peptides with Modulated Binding Affinity for HLA Molecules”, Attorney Docket No. 018623-009520, filed Jan. 6, 1999, which is a CIP of U.S. Ser. No. 08/815,396, which is a CIP of abandoned U.S. Ser. No. 60/013,113. Furthermore, the present application is related to U.S. Ser. No. 09/017,735, which is a CIP of abandoned U.S. Ser. No. 08/589,108; U.S. Ser. No. 08/753,622, U.S. Ser. No. 08/822,382, abandoned U.S. Ser. No. 60/013,980, U.S. Ser. No. 08/454,033, U.S. Ser. No. 09/116,424, U.S. Ser. No. 08/205,713, and U.S. Ser. No. 08/349,177, which is a CIP of abandoned U.S. Ser. No. 08/159,184, which is a CIP of abandoned U.S. Ser. No. 08/073,205, which is a CIP of abandoned U.S. Ser. No. 08/027,146. The present application is also related to U.S. Ser. No. 09/017,524, U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No. 60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No. 08/589,107, U.S. Ser. No. 08/451,913, U.S. Ser. No. 08/186,266, U.S. Ser. No. 09/116,061, and U.S. Ser. No. 08/347,610, which is a CIP of U.S. Ser. No. 08/159,339, which is a CIP of abandoned U.S. Ser. No. 08/103,396, which is a CIP of abandoned U.S. Ser. No. 08/027,746, which is a CIP of abandoned U.S. Ser. No. 07/926,666. The present application is also related to U.S. Ser. No. 09/017,743, U.S. Ser. No. 08/753,615; U.S. Ser. No. 08/590,298, U.S. Ser. No. 09/115,400, and U.S. Ser. No. 08/452,843, which is a CIP of U.S. Ser. No. 08/344,824, which is a CIP of abandoned U.S. Ser. No. 08/278,634. The present application is also related to provisional U.S. Ser. No. 60/087,192 and U.S. Ser. No. 09/009,953, which is a CIP of abandoned U.S. Ser. No. 60/036,713 and abandoned U.S. Ser. No. 60/037,432. In addition, the present application is related to U.S. Ser. No. 09/098,584 and to Provisional U.S. Ser. No. 60/117,486. All of the above applications are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was funded, in part, by the United States government under grants with the National Institutes of Health. The U.S. government has certain rights in this invention.

Related Publications (1)
Number Date Country
20090311283 A1 Dec 2009 US
Provisional Applications (1)
Number Date Country
60013363 Mar 1996 US
Continuations (3)
Number Date Country
Parent 09350401 Jul 1999 US
Child 11976998 US
Parent 08461603 Jun 1995 US
Child 08978291 US
Parent 07935811 Aug 1992 US
Child 08461603 US
Continuation in Parts (20)
Number Date Country
Parent 09239043 Jan 1999 US
Child 09350401 US
Parent 08347610 Dec 1994 US
Child 09239043 US
Parent 08344824 Nov 1994 US
Child 08347610 US
Parent 08205713 Mar 1994 US
Child 08344824 US
Parent 09189702 Nov 1998 US
Child 08205713 US
Parent 08820360 Mar 1997 US
Child 09189702 US
Parent 08197484 Feb 1994 US
Child 08820360 US
Parent 07935811 Aug 1992 US
Child 08197484 US
Parent 07874491 Apr 1992 US
Child 07935811 US
Parent 07827682 Jan 1992 US
Child 07874491 US
Parent 08159339 Nov 1993 US
Child 08347610 US
Parent 08103396 Aug 1993 US
Child 08159339 US
Parent 08027746 Mar 1993 US
Child 08103396 US
Parent 07926666 Aug 1992 US
Child 08027746 US
Parent 08278634 Jul 1994 US
Child 08344824 US
Parent 08159184 Nov 1993 US
Child 08205713 US
Parent 08073205 Jun 1993 US
Child 08159184 US
Parent 08027146 Mar 1993 US
Child 08073205 US
Parent 08205713 Mar 1994 US
Child 09189702 US
Parent 08978291 Nov 1997 US
Child 08205713 US